WO2024040229A2

WO2024040229A2 - Combination therapies comprising myc modulators and checkpoint inhibitors

Info

Publication number: WO2024040229A2
Application number: PCT/US2023/072481
Authority: WO
Inventors: Abigail Elizabeth Witt; Jeremiah Dale FARELLI; Adam Walter SCHEIDEGGER; Jr. William Thomas Senapedis; Jodi Michelle KENNEDY; Houda BELAGHZAL; Defne YARAR; Eugine Lee; Sangeetha Sagar PALAKURTHI
Original assignee: Flagship Pioneering Innovations V Inc
Current assignee: Flagship Pioneering Innovations V Inc
Priority date: 2022-08-19
Filing date: 2023-08-18
Publication date: 2024-02-22
Anticipated expiration: 2025-02-19
Also published as: WO2024040229A3; TW202417012A; WO2024040229A8

Abstract

The present disclosure relates to combination therapies. The combination therapy may comprise administration of an expression repressor and an immune checkpoint inhibitor. In some embodiments, the expression repressor comprises a targeting moiety that binds a MYC promoter, anchor sequence, or super-enhancer. In some embodiments, the expression repressor comprises an effector moiety that represses transcription or methylates DNA. The compositions and methods can be used, for example, to treat cancers such as HCC and NSCLC.

Description

COMBINATION THERAPIES COMPRISING MYC MODULATORS AND CHECKPOINT INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application 63/371,970 filed on August 19, 2022, U.S. Provisional Application 63/376,386 filed on September 20, 2022, U.S. Provisional Application 63/481,073 filed on January 23, 2023, U.S. Provisional Application 63/489,633 filed on March 10, 2023, and U.S. Provisional Application 63/501,890 filed on May 12, 2023, the entire contents of which are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 15, 2023, is named O2057-7035WO_SL.xml and is 424,111 bytes in size.

BACKGROUND

Mis-regulation of gene expression is the underlying cause of many diseases (e.g., in mammals, e.g., humans) e.g., neoplasia, neurological disorders, metabolic disorders and obesity. The mis-regulation of the transcription factor MY C plays a central role in a variety of human tumors and chronic liver diseases. MYC protein is considered “undruggable” due to various factors, e.g., lack of a defined ligand binding site, physiological function essential to the maintenance of normal tissues. Techniques geared towards modulating the MY C gene expression provides a viable alternative approach in treating these diseases. There is a need for novel tools, systems, and methods to stably alter, e.g., decrease, expression of disease associated genes such as MYC.

SUMMARY

The disclosure provides, for instance, combination therapies comprising an expression repressor and an immune checkpoint inhibitor polypeptide (e.g., an antibody molecule or a peptide).

The disclosure provides, among other things, expression repressors and expression repressor systems that may be used to modulate, e.g., decrease, expression of a target gene, e.g., MYC.

In some aspects, the disclosure provides an expression repressor comprises a targeting moiety that binds to a target gene promoter, e.g., MYC promoter, and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of the target gene, e.g., MYC. In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety that binds a target gene locus, e.g., MYC, and an effector moiety comprising MQ1 or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety that binds to a regulatory element located in a super enhancer region of MYC, and optionally an effector moiety wherein the expression repressor is capable of decreasing expression of MYC.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety that binds to a regulatory element located in a super enhancer region of a target gene, e.g., MYC, and an effector moiety (e.g., KRAB, or MQ1, or a fragment or variant thereof) wherein the expression repressor is capable of decreasing expression of the target gene, e.g., MYC.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety that binds a regulatory element located in a super enhancer region of a target gene, e.g., MYC, wherein the targeting moiety comprises a zinc finger domain, wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety that binds a regulatory element located in a super enhancer region of MY C, wherein the targeting moiety comprises a zinc finger domain or a TAL effector domain, and an effector moiety, wherein the effector moiety comprises a transcription repressor (e.g., KRAB or a fragment or variant thereof) or a DNA methyltransferase (e.g., MQ1 or a fragment or variant thereof); wherein the expression repressor is capable of decreasing expression of MYC.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety that binds a target gene locus, e.g., MYC, wherein the targeting moiety comprises a zinc finger domain, wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.

In some aspects, the disclosure provides expression repressor comprising: a targeting moiety that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 1, 3, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, or 75, 76, 78, 79, 80, 81, 84, 85, 86, wherein the expression repressor is capable of decreasing expression of MY C.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety that bind a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2 or 77, 82, 83 and wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC. In some embodiments, the expression repressor comprises an effector moiety. In some aspects, the disclosure provides an expression repressor comprising a targeting moiety wherein the targeting moiety binds a genomic locus that is within 1400 nt upstream or downstream of SEQ ID NO: 4.

In some aspects, the disclosure provides an expression repressor comprising a targeting moiety wherein, the targeting moiety binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 4, 77, 82, or 83.

In some aspects, the disclosure provides an expression repressor comprising a targeting moiety wherein, the targeting moiety binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, 96, or 108.

In some aspects, the disclosure provides a system comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC or to a sequence proximal to the transcription regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MY C or to a sequence proximal to the anchor sequence.

In some aspects, the disclosure provides a system comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC, or to a sequence proximal to the transcription regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC.

In some embodiments, the first targeting moiety specifically binds a first DNA sequence and the second targeting moiety specifically binds a second DNA sequence different from the first DNA sequence. In some embodiments, the first effector moiety is different from the second effector moiety.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC or a sequence proximal to said transcription regulatory element; and an effector moiety comprising MQ 1 or a functional variant or fragment thereof.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein that binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC, and an effector moiety comprising KRAB, MQ1, or a functional variant or fragment thereof, wherein the expression repressor is capable of decreasing expression of target gene, e.g., MYC.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence; and an effector moiety comprising KRAB or a functional variant or fragment thereof.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety comprising a zinc finger molecule that binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC or a sequence proximal to said transcription regulatory element; and an effector moiety comprising MQ 1 or a functional variant or fragment thereof.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety comprising a zinc finger molecule that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence; and an effector moiety comprising KRAB or a functional variant or fragment thereof.

In some aspects, the disclosure provides an expression repressor comprising: a targeting moiety comprising a zinc finger molecule, that binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC, and an effector moiety comprising KRAB or a functional variant or fragment thereof.

In some aspects, the disclosure is directed to a nucleic acid encoding the first expression repressor, second expression repressor, both, or a component thereof (e.g., a gRNA, a mRNA). In some embodiments, the nucleic acid encoding the expression repressor system is a multi-cistronic sequence. In some embodiments, the multi-cistronic sequence is a bi-cistronic sequence.

In some aspects, the disclosure is directed to a vector comprising a nucleic acid, a system, or an expression repressor described herein. In another aspect, the disclosure is directed to a lipid nanoparticle comprising a vector, a nucleic acid, a system, or an expression repressor described herein. In another aspect, the disclosure is directed to a reaction mixture comprising an expression repressor, a system, a nucleic acid, a vector, or a lipid nanoparticle described herein. In another aspect, the disclosure is directed to a pharmaceutical composition comprising an expression repressor, a system, a nucleic acid, a vector, a lipid nanoparticle, or a reaction mixture described herein.

In some aspects, the disclosure is directed to a method of decreasing expression of a target gene comprising providing an expression repressor or an expression repression system described herein and contacting the target gene and/or one or more operably linked transcription control elements with the expression repressor or expression repression system, thereby decreasing expression of the target gene.

In some aspects, the disclosure is directed to a method of treating a condition associated with over-expression of a target gene e.g., MY C in a subject, comprising administering an expression repressor, or a system, nucleic acid, or vector described herein to the subject, thereby treating the condition.

In some aspects, the disclosure is directed to a method of treating a condition associated with misregulation of a target gene, e.g., MYC, in a subject, comprising administering an expression repressor, system, nucleic acid, or vector described herein to the subject, thereby treating the condition.

In some aspects, the disclosure provides, a method of decreasing expression of a target gene, e.g., MYC in a cell, the method comprising: contacting the cell with a system comprising: a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence thereby decreasing expression of the target gene, e.g., MYC in the cell.

In some aspects, the disclosure provides a method of decreasing expression of a target gene, e.g., MYC, in a cell, the method comprising: contacting the cell with a system comprising: a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC, thereby decreasing expression of the target gene, e.g., MYC, in the cell.

The present disclosure further provides, in part, a kit comprising: a) a container comprising a composition comprising an expression repressor comprising a targeting moiety that binds to a target gene, promoter, e.g., MYC, and an effector moiety capable of modulating, e.g., decreasing the expression of the target gene, e.g., MYC, and b) a set of instructions comprising at least one method for modulating the expression of a target gene, e.g., MYC within a cell with said composition.

The present disclosure further provides, in part, a kit comprising: a) a container comprising a composition comprising an expression repressor comprising a targeting moiety that binds to a locus located in a super enhancer region of a target gene, e.g., MYC, and an effector moiety capable of modulating, e.g., decreasing the expression of the target gene, e.g., MYC, and b) a set of instructions comprising at least one method for modulating the expression of a target gene, e.g., MYC within a cell with said composition.

In some aspects, the kit comprises a) a container comprising a composition comprising a system comprising two expression repressors, comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to target gene, e.g., MYC or to a sequence proximal to the transcription regulatory element and an expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising target gene, e.g., MYC or to a sequence proximal to the anchor sequence.

In some aspects, the kit comprises a) a container comprising a composition comprising a system comprising two expression repressors, comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to target gene, e.g., MYC, or to a sequence proximal to the transcription regulatory element and an expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC.

In some embodiments the kit further comprises b) a set of instructions comprising at least one method for treating a disease or modulating, e.g., decreasing the expression of target gene, e.g., MYC within a cell with said composition. In some embodiments, the kits can optionally include a delivery vehicle for said composition (e.g., a lipid nanoparticle). The reagents may be provided suspended in the excipient and/or delivery vehicle or may be provided as a separate component which can be later combined with the excipient and/or delivery vehicle. In some embodiments, the kits may optionally contain additional therapeutics to be co-administered with the compositions to affect the desired target gene expression, e.g., MYC gene expression modulation. While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. Additional features of any of the aforesaid methods or compositions include one or more of the following enumerated embodiments.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following enumerated embodiments.

All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referred to herein, e.g., in any Table herein, are incorporated by reference. Unless otherwise specified, the sequence accession numbers specified herein, including in any Table herein, refer to the database entries current as of December 15, 2020. When one gene or protein references a plurality of sequence accession numbers, all of the sequence variants are encompassed.

ENUMERATED EMBODIMENTS

1. An expression repressor comprising: a targeting moiety that binds to a MY C promoter, and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC.

2. The expression repressor of embodiment 1, wherein the targeting moiety binds a genomic locus that is within 1400, 1200, 1000, 800, 600, 400, or 200 nt upstream or downstream of SEQ ID NO: 4, 199, or 201.

3. The expression repressor of embodiment 1, wherein the targeting moiety binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 4, 77, 82, 83, 85, 199, or 201.

4. An expression repressor comprising: a targeting moiety that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 3, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 109, 110, 75, 76, 78, 79, 80, 81, 84, 85, 86, 190, 191 , 192, 200, or 202 and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC. 5. An expression repressor comprising: a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 77, 82, 83, 199, or 201 and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC.

6. An expression repressor comprising: a targeting moiety that binds a MY C locus, and an effector moiety comprising MQ 1 or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression of MYC.

7. An expression repressor comprising: a targeting moiety that binds a locus in MY C super enhancer region, optionally an effector moiety, e.g., an effector moiety comprising a DNA methyltransferase, wherein optionally the effector moiety comprises MQ 1 or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression of MYC.

8. An expression repressor comprising: a targeting moiety that binds a locus in MY C super enhancer region, an effector moiety comprising a transcription repressor, wherein optionally the effector moiety comprises KRAB or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression of MYC.

9. The expression repressor of embodiment 7 or 8, wherein the targeting moiety binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NO: 96-110, 83, 199, 201.

10. The expression repressor of any of embodiments 7-9, wherein the targeting moiety binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of GRCh37: chr8: 129162465-129212140, using the hgl9 reference genome.

11. The expression repressor of any of embodiments 7-10, wherein the targeting moiety binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 96 or 108.

12. The expression repressor of any of embodiments 7-11, wherein the targeting moiety comprises a zinc finger domain or a TAL effector domain.

13. An expression repressor comprising: a targeting moiety that binds a locus, e.g., a MYC locus, a first effector moiety comprising EZH2 or a fragment or variant thereof, and a second effector moiety comprising KRAB or a fragment or variant thereof, wherein the expression repressor is capable of decreasing expression at the locus, e.g., decreasing expression of MYC.

14. The expression repressor of embodiment 13, wherein the targeting moiety binds the MYC promoter, super enhancer region, or anchor sequence. 15. The expression repressor of embodiment 13 or 14, wherein the targeting moiety comprises a TAL effector domain, a CRISPR/Cas domain, or a zinc finger domain.

16. The expression repressor of any of embodiments 13-15, wherein the first effector moiety is N- terminal of the second effector, or wherein the first effector is C-terminal of the second effector moiety. 17. An expression repressor comprising: a targeting moiety that binds a MYC locus, wherein the targeting moiety comprises a zinc finger domain, and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC. 18. An expression repressor comprising: a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to a transcription regulatory element (e.g., a promoter, an enhancer, a super enhancer, or transcription start site (TSS)) operably linked to a MYC gene or a sequence proximal to said transcription regulatory element; and an effector moiety comprising MQ 1 or a functional variant or fragment thereof.

19. An expression repressor comprising: a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to a transcription regulatory element (e.g., a promoter, an enhancer, or transcription start site (TSS)) operably linked to a MYC gene or a sequence proximal to said transcription regulatory element; and an effector moiety comprising MQ 1 or a functional variant or fragment thereof. 0. An expression repressor comprising: a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to a transcription regulatory element (e.g., a promoter, an enhancer, or transcription start site (TSS)) operably linked to a MYC gene or a sequence proximal to said transcription regulatory element; and an effector moiety comprising KRAB or a functional variant or fragment thereof. 21. An expression repressor comprising: a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence; and an effector moiety comprising KRAB or a functional variant or fragment thereof.

22. An expression repressor comprising: a targeting moiety comprising a zinc finger domain that binds to a transcription regulatory' element (e.g., a promoter, an enhancer, or transcription start site (TSS)) operably linked to a MY C gene or a sequence proximal to said transcription regulatory' element; and an effector moiety comprising MQ 1 or a functional variant or fragment thereof.

23. An expression repressor comprising: a targeting moiety comprising a zinc finger domain that binds to a transcription regulatory' element (e.g., a promoter, an enhancer, or transcription start site (TSS)) operably linked to a MY C gene or a sequence proximal to said transcription regulatory' element; and an effector moiety comprising KRAB or a functional variant or fragment thereof.

24. An expression repressor comprising: a targeting moiety that binds a mouse genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 190-192 and optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC.

25. The expression repressor of claim 24, wherein the effector moiety comprises a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.

26. The expression repressor of embodiments 24 or 25, wherein the targeting moiety comprises a TAL effector domain, a CRISPR/Cas domain, a zinc finger domain, a tetR domain, a meganuclease domain, or an oligonucleotide.

27. The expression repressor of any of embodiments 24-26, wherein the targeting moiety comprises a zinc finger domain or a TAL effector domain.

28. The expression repressor of any of embodiments 24-27, wherein the expression repressor comprises an amino acid sequence chosen from any of SEQ ID NOs: 160-165, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

29. The expression repressor of any of embodiments 24-28, wherein the expression repressor is encoded by a nucleotide sequence chosen from any of SEQ ID NOs: 166-168, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

30. The expression repressor of any of embodiments 24-29, wherein the targeting moiety comprises an amino acid sequence according to any of SEQ ID NOs: 154-156, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

31. The expression repressor of any of embodiments 24-30, wherein the targeting moiety comprises a nucleic acid sequence according to any of SEQ ID NOs: 157-159, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

32. The expression repressor of any of embodiments 24-31, wherein the effector moiety is a durable effector moiety.

33. The expression repressor of any of embodiments 24-32, wherein the effector moiety is a transient effector moiety.

34. The expression repressor of any of embodiments 24-33, wherein the expression repressor is a fusion molecule.

35. The expression repressor of any of embodiments 24-34, wherein the targeting moiety comprises a zinc finger domain, and the effector moiety comprises an epigenetic modifying moiety, e.g., a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.

36. The expression repressor of any of embodiments 18-20, 22, or 23, wherein the regulatory element is part of a cluster of regulatory elements.

37. The expression repressor of any embodiments 18-20, 22, or 23, wherein the regulatory element is located in a non-coding region.

38. The expression repressor of any embodiments 18-20, 22, or 23, wherein the regulatory element is a distal enhancer e.g., located at least 1,000 nt away from a target gene promoter, e.g., MYC.

39. The expression repressor of any embodiments 18-20, 22, 23, or 36-38, wherein the regulatory element increases the expression of a target gene, e.g., MYC.

40. Hie expression repressor of any embodiments 18-20, 22, 23, or 36-39, wherein the regulatory element contains one or more mutations.

41. The expression repressor of any embodiments 18-20, 22, 23, or 36-40, wherein the regulatory element contains at least one disease-associated single nucleotide polymorphism (SNP).

42. The expression repressor of any of embodiments 18-20, 22, 23, or 36-41, wherein the transcription regulatory element interacts with the promoter of target gene, e.g., MYC through an enhancer docking site. 43. The expression repressor of embodiment 42, wherein the enhancer docking site comprises a nucleotide sequence of according to any of SEQ ID NOs: 71 or 72 or a nucleotide sequence of CCGCCATNTT (e.g., a YY 1 -binding motif) or AANATGGCGG (e.g., a YY 1 -binding motif in an opposite orientation).

44. An expression repressor comprising: a targeting moiety comprising a zinc finger domain that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MY C gene or to a sequence proximal to the anchor sequence; and an effector moiety comprising KRAB or a functional variant or fragment thereof.

45. The expression repressor of any of embodiments 1-23 or 36-43, wherein the expression repressor comprises an amino acid sequence chosen from any of SEQ ID NOs: 22-37, 129, 133, 134, 139- 149, or 177-186, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

46. The expression repressor of any of embodiments 1-23 or 36-45, wherein the expression repressor is encoded by a nucleotide sequence chosen from any of SEQ ID NOs: 55-70, 130, 189, or 193- 197, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

47. The expression repressor of any of embodiments 1-23 or 36-46, wherein the targeting moiety comprises an amino acid sequence according to any of SEQ ID NOs: 5-16, or 169-172, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

48. The expression repressor of any of the preceding embodiments, wherein the effector moiety comprises an amino acid sequence according to SEQ ID NO: 18, 19, or 87, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto

49. Hie expression repressor of any of embodiments 1-12, 17-19, 22, 36-42, or 44-47, wherein the effector moiety is a durable effector moiety.

50. The expression repressor of any of embodiments 1-23, or 36-48 wherein the effector moiety is a transient effector moiety.

51. The expression repressor of any of embodiments 1-12, 17-19, 22, 36-42, or 44-48, wherein the effector moiety comprises a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof. The expression repressor of any of embodiments 1-23, 36-47, or 49, wherein the effector moiety comprises a transcription repressor, e.g., comprises KRAB or a fragment or variant thereof. The expression repressor of any of the preceding embodiments, wherein the targeting moiety comprises a TAL effector domain, a CRISPR/Cas domain, a zinc finger domain, a tetR domain, a meganuclease domain, or an oligonucleotide. The expression repressor of embodiment 53, wherein the CRISPR/Cas domain binds a gRNA, e.g., a gRNA that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 1-4, e.g., wherein the gRNA comprises a sequence that comprises at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 1-4 The expression repressor of embodiment 53, wherein the CRISPR/Cas domain binds a gRNA, e.g., a gRNA that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 96-110, e.g., wherein the gRNA comprises a sequence that comprises at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 96-110. The expression repressor of any of embodiments 53-55, wherein the CRISPR/Cas domain comprises a Cas protein or Cpfl protein chosen from Table 1 or a variant (e.g., mutant) of any thereof. The expression repressor of any of embodiments 53-56, wherein the CRISPR/Cas domain comprises a catalytically inactive CRISPR/Cas protein, e.g., dCas9. The expression repressor of embodiment 53, wherein the zinc finger domain binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 96-110, e.g., wherein the gRNA comprises a sequence that comprises at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 96-110. The expression repressor of any of embodiments 17, 22, 26-53, or 57, wherein the zinc finger domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers (and optionally no more than 11, 10, 9, 8, 7, 6, or 5 zinc fingers). Tire expression repressor of any of embodiments 17, 22, 26-53, 57, or 58, wherein the zinc finger domain comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2- 3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6- 9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers. The expression repressor of any of embodiments 17, 22, 26-53, or 57-59, wherein the zinc finger domain comprises 3 or 9 zinc fingers. Tire expression repressor of any of the preceding embodiments, which is a fusion molecule. 63. The expression repressor of any of the preceding embodiments, which comprises a linker situated between the targeting domain and the effector domain, optionally wherein the linker comprises an amino sequence according to SEQ ID NO: 137 or SEQ ID NO: 138.

64. The expression repressor of any of embodiments 1-17, 20, 21, 23, 44-48, 50, or 52-57, wherein the targeting moiety comprises a catalytically inactive CRISPR/Cas domain (e.g., dCas9) and the effector moiety comprises a transcription repressor, e.g., KRAB or a fragment or variant thereof.

65. The expression repressor of any of embodiments 1-17, 20, 21, 23, 44-48, 50, 52, or 53-64, wherein the targeting moiety comprises a zinc finger domain, and the effector moiety comprises a transcription repressor, e.g., KRAB or a fragment or variant thereof.

66. The expression repressor of any of embodiments 17, 36-43, 45-47, 53, or 58-63, wherein the targeting moiety comprises a zinc finger domain, and the expression repressor does not comprise an effector moiety.

67. The expression repressor of any of embodiments 1-12, 18-19, 22, 36-43, 45-49, 51, or 53-57 wherein the targeting moiety comprises a catalytically inactive CRISPR/Cas domain (e.g., dCas9) and the effector moiety comprises an epigenetic modifying moiety, e.g., a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.

68. The expression repressor of any of embodiments 1-12, 17-19, 22, 36-43, 45-49, 51, 53, or 58-63, wherein the targeting moiety comprises a zinc finger domain, and the effector moiety comprises an epigenetic modifying moiety, e.g., a DNA methyltransferase, e.g., MQ1 or a fragment or variant thereof.

69. The expression repressor of any of the preceding embodiments, which comprises an amino acid sequence of any of SEQ ID NOS: 22-37, 129, 133, 134, 139-149, or 177-186, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

70. The expression repressor of any of the preceding embodiments, which: (i) comprises one or more nuclear localization signal sequences (NLS), or (ii) does not comprise an NLS.

71. The expression repressor of any of the preceding embodiments, comprising a first NLS at the N terminus, e.g., wherein the first NLS has a sequence of SEQ ID NO: 88.

72. Hie expression repressor of any of the preceding embodiments, comprising an NLS, e.g., a second NLS, at the C terminus, e.g., having a sequence of SEQ ID NO: 89.

73. The expression repressor of any of the preceding embodiments, wherein the first and the second NLS have the same sequence.

74. The expression repressor of any of embodiments 71-73, wherein the first and the second NLS have different sequences.

75. The expression repressor of any of the preceding embodiments, which comprises an epitope tag. The expression repressor of embodiment 75, wherein the epitope tag is an HA tag. The expression repressor of any of preceding embodiments, wherein the anchor sequence comprises the sequence of SEQ ID NO: 71 or 72, or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 alterations relative thereto. The expression repressor of any of embodiments 1-77, wherein the anchor sequence comprises a sequence of CCGCCATNTT (e.g., a YYl-binding motif) or AANATGGCGG (e.g., a YY1- binding motif in an opposite orientation), or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 alterations relative thereto. The expression repressor of any of preceding embodiments, wherein the anchor sequence is on the same chromosome as the MYC gene. The expression repressor of any of preceding embodiments, wherein the anchor sequence is upstream of the MYC gene (e.g., upstream of the TSS or upstream of the promoter). The expression repressor of any of preceding embodiments, wherein the anchor sequence is at least 1, 5, 10, 50, 100, or 1000 kilobases away from the MYC gene (e g., from the TSS or promoter of the MYC gene). The expression repressor of any of preceding embodiments, wherein the anchor sequence is 0. 1- 0.5, 0.1-1, 0.1-5, 0.1-10, 0.1-50, 0.1-100, 0.1-500, 0.1-1000, 0.5-1, 0.5-5, 0.5-10, 0.5-50, 0.5-100, 0.5-500, 0.5-1000, 1-5, 1-10, 1-50, 1-100, 1-500, 1-1000, 5-10, 5-50, 5-100, 5-500, 5-1000, 10- 50, 10-100, 10-500, 10-1000, 50-100, 50-500, 50-1000, 100-500, 100-1000, or 500-1000 kilobases away from the MYC gene (e.g., from the TSS or promoter of the MYC gene). The expression repressor of any of embodiments 1-79, 81, or 82, wherein the target sequence is downstream of the MYC gene (e.g., downstream of the TSS or downstream of the promoter). The expression repressor of any of preceding embodiments, wherein the targeting moiety binds to a sequence at chromosome coordinates 128746342-128746364, 128746321-128746343, 128746525-128746547, 128748014-128748036, 129188878-129188900, 129188958-129188980, 129188960-129188982, 129189067-129189089, 129189457-129189479, 129189554-129189576, 129189679-129189701, 129209511-129209533, 129209643-129209665, 129209658-129209680, 129209856-129209878, 129189452-129189474, 129189190-129189212, 129189274-129189296, 129189421-129189443, 128746405-128746425, 128748069-128748089, 129188825-129188845, or 129188822-129188842 or a sequence proximal thereto. The expression repressor of any of the preceding embodiments, wherein binding of the expression repressor to the target gene locus, e.g., MYC, increases methylation at a site in the target gene locus, e.g., MYC, by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to methylation in the absence of the expression repressor, e g., as measured by ELISA or as described in any of Examples 7 or 28 as described in the PCT publication WO/2022/132195, wherein optionally the site assayed for methylation is chr8: 129188693-129189048 according to hgl9 reference genome, e.g., comprises a sequence according to SEQ ID NO: 123. The expression repressor of any of the preceding embodiments, wherein binding of the expression repressor to the target gene locus, e.g., MYC increases methylation at a site in the target gene locus, e.g., MYC for a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as described in Example 28 as described in the PCT publication WO/2022/132195. The expression repressor of any of the preceding embodiments, wherein binding of the expression repressor to the MYC locus decreases expression of MYC in a cell by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to expression in the absence of the expression repressor, e.g., as measured by ELISA or as described in any of Examples 2-7 or 9 as described in the PCT publication WO/2022/132195. The expression repressor of any of the preceding embodiments, wherein binding of the expression repressor to the MYC locus appreciably decreases expression of MYC for a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by ELISA or as described in any of Examples 2-7 or 9 as described in the PCT publication WO/2022/132195. The expression repressor of any of the preceding embodiments, wherein binding of the expression repressor to the MYC locus appreciably decreases expression of MYC at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours post-transfection. The expression repressor of any of embodiments 1-23 or 36-89, wherein the targeting moiety binds to a human genomic locus. The expression repressor of any of embodiments 24-43, 49, 51,53, 56-57, 59-62, 66-68, or 70-89, wherein the targeting moiety binds to a mouse genomic locus. The expression repressor of any of the preceding embodiments, wherein binding of the expression repressor to the MYC locus decreases the viability of a cell comprising the MYC locus (e g., cancer cells). The expression repressor of any of the preceding embodiments, wherein contacting a plurality of cells with the expression repressor or a nucleic acid encoding the expression repressor decreases the viability of the plurality of cells. The expression repressor of any of the preceding embodiments, wherein viability is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to viability in the absence of the first expression repressor, e.g., as measured by CellTiter Gio or as described in any of Examples 2-7 as described in the PCT publication WO/2022/132195. The expression repressor of any of the preceding embodiments, wherein administration of the expression repressor results in apoptosis of at least 5%, 6%, 7%, 8%, 9% 10%, 12%, 15%, 17% 20%, 25% 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells). The expression repressor of any preceding embodiments, wherein the plurality of cells comprises a plurality of cancer cells and a plurality of non-cancer cells and/or a plurality of infected cells and a plurality of uninfected cells. The expression repressor of any of the preceding embodiments, wherein contacting the plurality of cells with the expression repressor or a nucleic acid encoding the expression repressor decreases the viability of the plurality of cancer cells more than it decreases the viability of the plurality of non-cancer cells. The expression repressor of any of the preceding embodiments, wherein contacting the plurality of cells with the expression repressor or a nucleic acid encoding the expression repressor decreases the viability of the plurality of cancer cells 1.05x (i.e., 1.05 times), l .lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx more than it decreases the viability of the plurality of non-cancer cells. The expression repressor of any of embodiments 92-98, wherein the cancer cells are lung cancer cells, gastric cancer cells, gastrointestinal cancer cells, colorectal cancer cells, pancreatic cancer cells, or hepatic cancer cells. The expression repressor of any of embodiments 92-99, wherein the cancer is hepatocellular carcinoma (HCC), fibrolamellar hepatocellular carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, small cell lung cancer (SCLC), large cell (undifferentiated) carcinoma, triple negative breast cancer, gastric adenocarcinoma, endometrial carcinoma, or pancreatic carcinoma. The expression repressor of any of the preceding embodiments, which, when contacted with a plurality of infected cells and a plurality of uninfected cells, decreases the viability of the plurality of infected cells more than it decreases the viability of tire plurality of uninfected cells. The expression repressor of any of preceding embodiments, wherein the infection is viral. The expression repressor of embodiment 102, wherein the viral infection is hepatitis, e.g., hepatitis B. The expression repressor of any of embodiments 92-103, wherein the infected cells are human hepatocytes. 105. The expression repressor of any of the preceding embodiments, which has an EC50 of 0.04 - 0.4, 0.04 - 0.1, 0.1 - 0.2, 0.2 - 0.3, or 0.3 - 0.4 pg/mL when tested in an assay for viability of cancer cells (e.g., HCC cells) using LNP delivery of mRNA encoding the expression repressor, e.g., in an assay according to Example 12 as described in the PCT publication WO/2022/132195.

106. The expression repressor of any of embodiments 1-104, which has an EC50 of 0.1- 2.5, 0.5-2.2, 1.0-1.5, 1 .2-2 pg/mL when tested in an assay for viability of cancer cells (e.g., lung cancer cells) using LNP delivery of mRNA encoding the expression repressor, e.g., in an assay according to Example 18 as described in the PCT publication WO/2022/132195.

107. The expression repressor of any of the preceding embodiments, which has an EC50 of 0.004 - 0.08, 0.004 - 0.01, 0.01 - 0.02, 0.02 - 0.04, or 0.04 - 0.08 pg/mL when tested in an assay for reducing MYC mRNA levels in cancer cells (e.g., HCC cells) using LNP delivery of mRNA encoding the expression repressor, e.g., in an assay according to Example 12 as described in the PCT publication WO/2022/132195.

108. The expression repressor of any of the preceding embodiments, which has an EC50 of 0.04 - 0.1, 0.04 - 0.09, 0.05 - 0.09, or 0.06 - 0.8 pg/mL when tested in an assay for reducing MYC mRNA levels in cancer cells (e.g., lung cancer cells) using LNP delivery of mRNA encoding the expression repressor, e.g., in an assay according to Example 18 as described in the PCT publication WO/2022/132195.

109. The expression repressor of any of the preceding embodiments, which reduces the level of a protein encoded by a target gene, e.g., MYC in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the protein level in an untreated cell.

110. The expression repressor of any of the preceding embodiments, which is capable of reducing tumor volume, e.g., in a human subject or in a mammalian model.

111. The expression repressor of any of preceding embodiments, wherein the expression repressor is capable of reducing tumor volume to a similar or greater degree compared to a chemotherapeutic agent, e.g., in a mammalian model, e.g., when measured at day 20 after initiation of treatment, e.g., wherein the expression repressor is administered every 5 days at a dose of 3mg/kg.

112. The expression repressor of any of preceding embodiments, wherein the expression repressor is capable of reducing tumor volume compared to a PBS control, e.g., in a mammalian model, e g., when measured at day 20 after initiation of treatment e.g., wherein the expression repressor is administered every 5 days for 4 doses followed by every 3 days for 3 doses at Img/kg, 1.5 mg/kg, or 3mg/kg. The expression repressor of any of preceding embodiments, wherein the tumor volume is reduced by at least about 10%, 20%, 30%, or 40% compared to a control treated with PBS, e.g., at day 20 after start of treatment. The expression repressor of any of embodiment 111-113, wherein the chemotherapeutic agent is sorafenib or cisplatin. The expression repressor of any of preceding embodiments, wherein the system is capable of reducing tumor volume to a similar or greater degree compared to a small molecule MY C inhibitor. The expression repressor of embodiment 115, wherein the small molecule MY C inhibitor is MYC1975 wherein optionally tumor volume is reduced by at least about 10%, 20%, 30%, or 40% compared to a control treated with the MYCi975, e.g., at day 20 after start of treatment. The expression repressor of any of preceding embodiments, which does not cause a decrease in body weight compared to at the start of treatment, or which causes a decrease in body weight of less than 3%, 2%, or 1%. A system comprising : a first expression repressor of any of the preceding embodiments, and a second expression repressor, e.g., a second expression repressor described herein, e.g., a second expression repressor of any of the preceding embodiments. A system comprising: a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory' element (e.g., a promoter, enhancer, or transcription start site (TSS)) operably linked to a MYC gene or to a sequence proximal to the transcription regulatory element, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence. Tire system of embodiment 118 or 119, wherein the transcription regulatory element comprises a promoter, and wherein the anchor sequence comprises a CTCF binding motif. The system of any of embodiments 118-120, wherein second expression repressor binds to a downstream region adjacent to the CTCF binding motif. The system of any of embodiments 118-120, wherein second expression repressor binds to an upstream region adjacent to the CTCF binding motif. The system of any of embodiments 118-122, wherein the first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 3, 4, 71, 72, 75-86, or 200-206, or a sequence of CCGCCATNTT (e.g., a YY 1-binding motif) or AANATGGCGG (e.g., a YY 1 -binding motif in an opposite orientation); and the second expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 2, 3, 4, 71, 72, 75-86, or 200-206, or a sequence of CCGCCATNTT (e.g., a YY1 -binding motif) or AANATGGCGG (e.g., a YY 1 -binding motif in an opposite orientation). The system of any of embodiments 118-123, wherein the first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NO: 96-110. The system of any of embodiments 118-124, wherein, the first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 83; and the second expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 77. A system comprising: a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a promoter operably linked to a MY C gene or to a sequence proximal to the promoter, and a second expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to an enhancer (e.g., a superenhancer) of the MYC gene. The system of embodiment 126, wherein, the first expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 204, and the second expression repressor comprises a targeting moiety that binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 199 or 201. A system for reducing MYC expression, the system comprising: a) a first expression repressor comprising: i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and b) a second expression repressor comprising: i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 7 169, or 171 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The system of embodiments 128, wherein the first expression repressor further comprises a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the first targeting moiety. The system of embodiment 128 or 129, wherein the first expression repressor further comprises a second nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the first effector moiety. The system of any of embodiments 128-130, wherein the second expression repressor further comprises a first nuclear localization signal, e g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the second targeting moiety. The system of any of embodiments 128-131, wherein the second expression repressor further comprises a second nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the second effector moiety. The system of any of embodiments 128-132, wherein the first expression repressor further comprises a first linker situated between the first targeting moiety and the first effector moiety, wherein optionally the first linker has an amino acid sequence according to SEQ ID NO: 137 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. 134. The system of any of embodiments 128-133, wherein the second expression repressor further comprises a second linker situated between the second targeting moiety and the second effector moiety, wherein optionally the second linker has an amino acid sequence according to SEQ ID NO: 138 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

135. The system of any of embodiments 128-134, wherein the first expression repressor further comprises an amino acid sequence C-terminal of the first effector moiety, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, e.g., a sequence according to SEQ ID NO: 126 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

136. The system of any of embodiments 128-132, wherein the second expression repressor further comprises an amino acid sequence N-terminal of the second targeting moiety, e g., a sequence of up to 30, 25, 20, or 18 amino acids, e.g., a sequence according to SEQ ID NO: 128 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

137. The system of any of embodiments 128-136, wherein the first expression repressor has an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

138. The system of any of embodiments 128-137, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 24, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

139. The system of any of embodiments 128-137, wherein the second targeting moiety comprises an amino acid sequence according to SEQ ID NO: 169, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

140. The system of any of embodiments 128-137, wherein the second targeting moiety comprises an amino acid sequence according to SEQ ID NO: 171, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

141. The system of any of embodiments 128-140, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 177 or 183 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

142. Hie system of any of embodiments 128-140, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 179, 185, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

143. A nucleic acid encoding the first expression repressor and second repressor of the system of any of embodiments 128-142. A nucleic acid encoding a system for reducing MYC expression, the nucleic acid comprising: a) a first region encoding a first expression repressor, the first expression repressor comprising: i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and b) a second region encoding a second expression repressor, the second expression repressor comprising: i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 7 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of embodiment 144, wherein the first region is 5’ of the second region. The nucleic acid of embodiment 144, wherein the first region is 3’ of the second region. The nucleic acid of embodiment 145 or 146, wherein the first region further comprises a nucleotide sequence encoding a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the first targeting moiety. The nucleic acid of any of embodiments 145-147, wherein the first region further comprises a nucleotide sequence encoding a second nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the first effector moiety. The nucleic acid of any of embodiments 145-148, wherein the second region further comprises a nucleotide sequence encoding a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the second targeting moiety. The nucleic acid of any of embodiments 145-149, wherein the second region further comprises a nucleotide sequence encoding a second nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of tire second effector moiety. The nucleic acid of any of embodiments 145-150, wherein the first region further comprises a nucleotide sequence encoding a first linker situated between the first targeting moiety and the first effector moiety, wherein optionally the first linker has an amino acid sequence according to SEQ ID NO: 137 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 145-151, wherein the second region further comprises a nucleotide sequence encoding a second linker situated between the second targeting moiety and the second effector moiety, wherein optionally the second linker has an amino acid sequence according to SEQ ID NO: 138 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 145-152, wherein the first region further comprises a nucleotide sequence encoding an amino acid sequence C-terminal of the first effector moiety, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, e.g., a sequence according to SEQ ID NO: 126 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 145-153, wherein the second region further comprises a nucleotide sequence encoding an amino acid sequence N-terminal of the second targeting moiety, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, or an amino acid P. The nucleic acid of any of embodiments 145-154, wherein the first expression repressor has an amino acid sequence according SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 145-155, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 24, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 145-156, wherein the first region comprises a nucleotide sequence encoding the first targeting moiety, wherein the nucleotide sequence encoding the first targeting moiety comprises a sequence according to SEQ ID NO: 46 or 131 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. Hie nucleic acid of any of embodiments 145-157, wherein the first region comprises a nucleotide sequence encoding the first effector moiety, wherein the nucleotide sequence encoding the first effector moiety comprises a sequence according to SEQ ID NO: 52 or 132, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of any of embodiments 145-158, wherein the second region comprises a nucleotide sequence encoding the second targeting moiety, wherein the nucleotide sequence encoding the second targeting moiety comprises a sequence according to SEQ ID NO: 40 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of any of embodiments 145-159, wherein the first region comprises a nucleotide sequence encoding the first effector moiety, wherein the nucleotide sequence encoding the first effector moiety comprises a sequence according to SEQ ID NO: 51, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of any of embodiments 145-160, wherein the first region comprises a nucleotide sequence according to SEQ ID NO: 63 or 130, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,

5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional. The nucleic acid of any of embodiments 145-161, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 57, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,

6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional. A nucleic acid encoding a system for reducing MYC expression, the nucleic acid comprising: a) a first region encoding a first expression repressor, the first expression repressor comprising: i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and b) a second region encoding a second expression repressor, the second expression repressor comprising: i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 169 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. A nucleic acid encoding a system for reducing MYC expression, the nucleic acid comprising: a) a first region encoding a first expression repressor, the first expression repressor comprising: i) a first targeting moiety having an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a first effector moiety having an amino acid sequence according to SEQ ID NO: 19 or 87 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and b) a second region encoding a second expression repressor, the second expression repressor comprising: i) a second targeting moiety having an amino acid sequence according to SEQ ID NO: 171 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and ii) a second effector moiety having an amino acid sequence according to SEQ ID NO: 18, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19,

18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of embodiment 163 or 164, wherein the first region is 5’ of the second region. The nucleic acid of embodiment 163 or 164, wherein the first region is 3’ of the second region. The nucleic acid of any of embodiments 163-166, wherein the first region further comprises a nucleotide sequence encoding a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the first targeting moiety. The nucleic acid of any of embodiments 163-167, wherein the first region further comprises a nucleotide sequence encoding a second nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated C-terminal of the first effector moiety. The nucleic acid of any of embodiments 163-168, wherein the second region further comprises a nucleotide sequence encoding a first nuclear localization signal, e.g., an SV40 NLS, e.g., a sequence according to SEQ ID NO: 135 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g., situated N-terminal of the second targeting moiety. The nucleic acid of any of embodiments 163-169, wherein the second region further comprises a nucleotide sequence encoding a second nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, e.g ., situated C-terminal of tire second effector moiety. The nucleic acid of any of embodiments 163-170, wherein the first region further comprises a nucleotide sequence encoding a first linker situated between the first targeting moiety and the first effector moiety, wherein optionally the first linker has an amino acid sequence according to SEQ ID NO: 137 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 163-171, wherein the second region further comprises a nucleotide sequence encoding a second linker situated between the second targeting moiety and the second effector moiety, wherein optionally the second linker has an amino acid sequence according to SEQ ID NO: 138 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 163-171, wherein the first region further comprises a nucleotide sequence encoding an amino acid sequence C-terminal of the first effector moiety, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, e.g., a sequence according to SEQ ID NO: 126 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 163-173, wherein the second region further comprises a nucleotide sequence encoding an amino acid sequence N-terminal of the second targeting moiety, e.g., a sequence of up to 30, 25, 20, or 18 amino acids, or an amino acid P. The nucleic acid of any of embodiments 163-174, wherein the first expression repressor has an amino acid sequence according SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 144-175, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 177, or 183 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 144-176, wherein the second expression repressor has an amino acid sequence according to SEQ ID NO: 179, or 185, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 144-177, wherein first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30, or 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto and the second expression repressor has an amino acid sequence according to SEQ ID NO: 24, 141, 177, 179, 183, or 185, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto. The nucleic acid of any of embodiments 144-178, wherein the first region comprises a nucleotide sequence encoding the first targeting moiety, wherein the nucleotide sequence encoding the first targeting moiety comprises a sequence according to SEQ ID NO: 46 or 131 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of any of embodiments 144-179, wherein the first region comprises a nucleotide sequence encoding the first effector moiety, wherein the nucleotide sequence encoding the first effector moiety comprises a sequence according to SEQ ID NO: 52 or 132, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of any of embodiments 144-180, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 173 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional. The nucleic acid of any of embodiments 144-181, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 175 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional. The nucleic acid of any of embodiments 144-182, wherein the second region comprises a nucleotide sequence encoding the second effector moiety, wherein the nucleotide sequence encoding the second effector moiety comprises a sequence according to SEQ ID NO: 51, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of any of embodiments 144-183, wherein the first region comprises a nucleotide sequence according to SEQ ID NO: 63 or 130, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,

5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional. The nucleic acid of any of embodiments 144-184, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 189, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,

6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional. The nucleic acid of any of embodiments 144-185, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 194, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, wherein a poly-A sequence is optional The nucleic acid of any of embodiments 144-186, wherein the first region comprises a nucleotide sequence encoding the first effector moiety, wherein the nucleotide sequence encoding tire first effector moiety comprises a sequence according to SEQ ID NO: 52 or 132 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

188. The nucleic acid of any of embodiments 144-187, wherein the first region comprises a nucleotide sequence encoding the first targeting moiety, wherein the nucleotide sequence encoding the first targeting moiety comprises a sequence according to SEQ ID NO: 46 or 131, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

189. The nucleic acid of any of embodiments 144-188, wherein the second region comprises a nucleotide sequence encoding the second effector moiety, wherein the nucleotide sequence encoding the second effector moiety comprises a sequence according to SEQ ID NO: 51 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

190. The nucleic acid of any of embodiments 144-189, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 189, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

191. The nucleic acid of any of embodiments 144-190, wherein the second region comprises a nucleotide sequence according to SEQ ID NO: 194, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

192. The nucleic acid of any of embodiments 144-191, which has a nucleotide sequence according to SEQ ID NO: 93, 112, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

193. The nucleic acid of any of embodiments 144-192, which has a nucleotide sequence according to SEQ ID NO: 196 or 197, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

194. The system or nucleic acid of any of embodiments 118-193, wherein the first expression repressor comprises the first effector moiety.

195. The system or nucleic acid of any of embodiments 118-194, wherein the second expression repressor comprises the second effector moiety. 196. The system or nucleic acid of any of embodiments 118-195, wherein the first effector moiety has a different amino acid sequence from the second effector moiety.

197. The system or nucleic acid of any of embodiments 118-196, wherein the first effector moiety is a durable effector moiety.

198. The system or nucleic acid of any of embodiments 118-125 or 144-197, wherein the first effector moiety is a transient effector moiety.

199. The system or nucleic acid of any of embodiments 118-198, wherein the first effector moiety is an epigenetic modifying moiety. 00. The system or nucleic acid of any of embodiments 118-143, 163-197 or 199, wherein the first effector moiety comprises a histone methyltransferase. 01. The system or nucleic acid of embodiment 200, wherein the first effector moiety comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any thereof, e.g., a SET domain of any thereof. 02. The system or nucleic acid of any of embodiments 118-143, 163-197, or 199, wherein the first effector moiety comprises a histone demethylase (e.g., a lysine demethylase). 03. The system or nucleic acid of embodiment 202, wherein the first effector moiety comprises a protein chosen from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or a functional variant or fragment of any thereof). 04. The system or nucleic acid of any of embodiments 118-143, 163-197, or 199, wherein the first effector moiety comprises a histone deacetylase. 05. The system or nucleic acid of embodiment 204, wherein the first effector moiety comprises a protein chosen from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any thereof. 06. The system or nucleic acid of any of embodiments 118-197 or 200, wherein the first effector moiety comprises a DNA methyltransferase. 07. The system or nucleic acid of embodiment 206, wherein the first effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof. 08. The system or nucleic acid of any of embodiments 118-143, 160-196, or 198, wherein the first effector moiety is a transcription repressor moiety, e.g., comprising a transcription repressor. The system or nucleic acid of embodiment 198 or 199, wherein the first effector moiety comprises aprotein chosen from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any thereof. The system or nucleic acid of any of embodiments 118-209, wherein the first effector moiety promotes epigenetic modification of the transcription regulatory element or a sequence proximal thereto. The system or nucleic acid of any of embodiments 118-210, wherein the first effector moiety catalyzes epigenetic modification of the transcription regulatory element or a sequence proximal thereto. The system or nucleic acid of any of embodiments 118-125, 194, or 197-211, wherein the second expression repressor does not comprise an effector moiety . The system or nucleic acid of any of embodiments 118-212, wherein the second effector moiety is a transient effector moiety. The system or nucleic acid of any of embodiments 118-125 or 194-211, wherein the second effector moiety is a durable effector moiety. The system or nucleic acid of any of embodiments 118-211 or 214, wherein the second effector moiety is an epigenetic modifying moiety. The system or nucleic acid of any of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a histone methyltransferase. The system or nucleic acid of embodiment 216, wherein the second effector moiety comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any thereof, e.g., a SET domain of any thereof. The system or nucleic acid of any of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a histone demethylase (e.g., a lysine demethylase). The system or nucleic acid of embodiment 218, wherein the second effector moiety comprises a protein chosen from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66 (or a functional variant or fragment of any thereof. The system or nucleic acid of any of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a histone deacetylase. The system or nucleic acid of embodiment 220, wherein the second effector moiety comprises a protein chosen from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any thereof. The system or nucleic acid of any of embodiments 118-125, 194-211, or 214-215, wherein the second effector moiety comprises a DNA methyltransferase. The system or nucleic acid of embodiment 222, wherein the second effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof. The system or nucleic acid of any of embodiments 118-211 or 213, wherein the second effector moiety is a transcription repressor moiety. The system or nucleic acid of embodiment 224, wherein the second effector moiety promotes epigenetic modification of the anchor sequence or a sequence proximal thereto. The system or nucleic acid of embodiment 223 or 224, wherein the second effector moiety binds to one or more endogenous epigenetic modifying proteins or one or more endogenous transcription modifying proteins. The system or nucleic acid of any of embodiments 223-226, wherein the second effector moiety comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any thereof. The system or nucleic acid of any of embodiments 118-197, 199-207, 210-211, 213, or 224-227, wherein: the first effector moiety is a durable effector moiety, and the second effector moiety is a transient effector moiety. The system or nucleic acid of embodiment 228, wherein the first effector moiety is an epigenetic modifying moiety. The system or nucleic acid of embodiment 227 or 228, wherein the second effector moiety is a transcription repressor moiety. The system or nucleic acid of any of embodiments 227-230, wherein: the first effector moiety comprises a histone methyltransferase, histone demethylase, histone deacetylase, DNA methyltransferase, a functional variant or fragment of any thereof, or a combination of any thereof, and the second effector moiety comprises a transcription repressor or a functional variant or fragment of any thereof. The system or nucleic acid of any of embodiments 118-125, 194, 197, 199-207, 210-212, or 229, wherein: the first effector moiety comprises a histone methyltransferase, histone demethylase, histone deacetylase, DNA methyltransferase, a functional variant or fragment of any thereof, or a combination of any thereof, and the second expression repressor does not comprise a second effector moiety. The system or nucleic acid of any of embodiments 118-125, 199-207, 210-211, 213 214, or 224-

231 wherein: the first effector moiety comprises a SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, a functional variant or fragment of any thereof, or a combination of any thereof, and the second effector moiety comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, a functional variant or fragment of any thereof, or a combination of any thereof. The system or nucleic acid of any of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, or 233, wherein: the first effector moiety comprises a DNA methyltransferase, and the second effector moiety comprises a transcription repressor. The system or nucleic acid of any of embodiments 118-125, 194, 197, 200, 206-207, 210-212, or

232 wherein: the first effector moiety comprises a DNA methyltransferase, and the second expression repressor does not comprise a second effector moiety. The system or nucleic acid of any of embodiments 118-125, 200, 206-207, 210-235 wherein the first effector moiety comprises MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof. The system or nucleic acid of any of embodiments 118-211, 214, 224-234, or 236, wherein the second effector moiety comprises KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any thereof. 238. The system or nucleic acid of any of embodiments 118-211, 199, 206-207, 210-211, 213, 224-

234, or 236-237, wherein: the first effector moiety comprises MQ 1 or a functional variant or fragment of any thereof, and the second effector comprises KRAB or a functional variant or fragment of any thereof.

239. The system or nucleic acid of any of embodiments 118-125, 194, 197, 199-207, or 210-212, 229, 232, 235, or 236, wherein: the first effector moiety comprises MQ 1 or a functional variant or fragment of any thereof, and the second expression repressor does not comprise a second effector moiety.

240. The system or nucleic acid of any of embodiments 118-200 wherein the first expression repressor comprises an amino acid sequence chosen from any of SEQ ID NOs: 22-37, 129, 133, 134, 139- 149, 177-180, or 183-186, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

241. The system or nucleic acid of any of embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240, wherein the second expression repressor comprises an amino acid sequence chosen from any of SEQ ID NOs: 22-37, 129, 133, 134, 139-149, 177-180, or 183-186, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

242. The system or nucleic acid of any of embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240-241, wherein the first expression repressor comprises an amino acid sequence of SEQ ID NO: 30, 129, 133, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the second expression repressor comprises an amino acid sequence of SEQ ID NO: 24, 134, 141, 177, 179, 183, or 185, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

243. The system or nucleic acid of any of embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240-242, wherein the first expression repressor is encoded by a first nucleotide sequence of SEQ ID NO: 63 or 130, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the second expression repressor are encoded by a second nucleotide sequence of SEQ ID NO: 57, 189, or 194, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. 244. The system or nucleic acid of any of embodiments 118-198, 200, 206-211, 213-216, 222-223, 236-237, or 240-243, wherein the first and the second repressor are encoded by a nucleic acid sequence of SEQ ID NO: 93, 94, 112, 113, 196, or 197, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

245. The system or nucleic acid of embodiment 244 comprising an amino acid sequence of SEQ ID NO: 91, 92, 121, 122, 181, 182, 187, or 188, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

246. The system or nucleic acid of any of embodiments 118-197 , 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, wherein: the first expression repressor comprises from N-terminus to C-terminus:

(i) a first nuclear localization signal, e.g., a SV40 NLS; e.g., a sequence according to SEQ ID NO: 135;

(ii) a first targeting moiety, e.g., a zinc finger binding domain, e.g., ZF9; e.g., a sequence according to SEQ ID NO: 13;

(iii) a first effector moiety, e.g., a DNA methyltransferase, e.g., MQ1; e.g., a sequence according to SEQ ID NO: 19 or 87;

(iv) a second nuclear localization signal, e.g., a nucleoplasmin NLS; e.g., a sequence according to SEQ ID NO: 136; and the second expression repressor comprises, from N-terminus to C-terminus:

(v) a third nuclear localization signal, e.g., a SV40NLS; e.g., a sequence according to SEQ ID NO: 135;

(vi) a second targeting moiety, e.g., a zinc finger binding domain, e.g., ZF3; e.g., a sequence according to SEQ ID NO: 7;

(vii) a second effector moiety, e.g., KRAB, e.g., a sequence according to SEQ ID NO: 18; and

(viii) a fourth nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136.

247. The system or nucleic acid of any of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, wherein: the first expression repressor comprises from N-terminus to C-terminus:

(i) a first nuclear localization signal, e.g., a SV40 NLS; e.g., a sequence according to SEQ ID NO:

(iii) a first effector moiety, e.g., a DNA methyltransferase, e.g., MQ1; e g., a sequence according to SEQ ID NO: 19 or 87;

(vi) a second targeting moiety, e.g., a zinc finger binding domain, e.g., ZF54; e.g., a sequence according to SEQ ID NO: 169;

248. The system or nucleic acid of any of embodiments 118-197, 199, 206-207, 210-211, 213, 215, 224-231, 233-234, 236-237, or 240-244, wherein: the first expression repressor comprises from N-terminus to C-terminus:

(vi) a second targeting moiety, e.g., a zinc finger binding domain, e.g., ZF67; e.g., a sequence according to SEQ ID NO: 171;

(viii) a fourth nuclear localization signal, e.g., a nucleoplasmin NLS, e.g., a sequence according to SEQ ID NO: 136. The system of any of embodiment 118-248, wherein the system is capable of the decreasing expression of MY C to a greater degree compared to the first expression repressor alone or the second expression repressor alone. The system of any of embodiments 128-194, or 242-249, wherein the system is capable of decreasing expression of MYC to a greater degree compared to any of the expression repressors of SEQ ID: 22, 23, 25-29, 31-37 alone or in combination. The system of any of embodiments 118-250, which is capable of reducing tumor volume, e.g., in a human subject or in a mammalian model. The system of any of embodiments 128-193 or 242-251, wherein the system is capable of reducing tumor volume to a similar or greater degree compared to a chemotherapeutic agent, e.g., in a mammalian model, e.g., when measured at day 20 after initiation of treatment, e.g., wherein the expression repressor is administered every 5 days at a dose of 3 mg/kg, e.g., in a model system as described in Example 15 as described in the PCT publication WO/2022/132195. The system of any of embodiments 128-193 or 242-252, wherein the system is capable of reducing tumor volume to a greater degree compared to a chemotherapeutic agent, e.g., in a mammalian model, e.g., when measured at day 15 after initiation of treatment, e.g., wherein the expression repressor is administered every 5 days at a dose of 6 mg/kg, e.g., in a model system as described in Example 14 as described in the PCT publication WO/2022/132195. The system of any of embodiments 128-193 or 242-253, wherein the tumor volume is reduced by at least about 10%, 20%, 30%, 40%, 50%, or 60% compared to a control treated with PBS, e.g., at day 20 after start of treatment. The system of embodiment 254 wherein the chemotherapeutic agent is sorafenib or cisplatin. The system of any of embodiments 128-193 or 242-253, wherein the system is capable of reducing tumor volume to a similar or greater degree compared to a small molecule MY C inhibitor. The system of embodiment 256 wherein the small molecule MYC inhibitor is MYCi975 wherein optionally tumor volume is reduced by at least about 10%, 20%, 30%, or 40% compared to a control treated with tire MY Ci975, e.g., at day 20 after start of treatment. The system of any of embodiments 118-257, which does not cause a decrease in body weight compared to at the start of treatment, or which causes a decrease in body weight of less than 3%, 2%, or 1%. The system or nucleic acid of any of embodiments 118-258, wherein the first targeting moiety is selected from a TAL effector domain, a CRISPR/Cas domain, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide. 260. The system or nucleic acid of any of embodiments 118-259, wherein the second targeting moiety is selected from a TAL effector domain, a CRISPR/Cas domain, a zinc finger domain, a tetR domain, a meganuclease, or an oligonucleotide.

261. The system or nucleic acid of any of embodiments 118-260, wherein the first targeting moiety comprises a CRISPR/Cas domain (e.g., a first CRISPR/Cas domain).

262. The system or nucleic acid of any of embodiments 118-261, wherein the second targeting moiety comprises a second CRISPR/Cas domain (e g., a second CRISPR/Cas domain).

263. The system or nucleic acid of embodiment 262, wherein: i) the first CRISPR/Cas domain binds a first guide RNA, ii) the second CRISPR/Cas domain binds a second guide RNA, or iii) both (i) and (ii).

264. The system or nucleic acid of embodiment 262 or 263, wherein the first CRISPR/Cas domain does not bind the second guide RNA or binds with a KD of at least 10, 20, 50, 100, 1000, or 10,000 nM, and the second CRISPR/Cas domain does not bind the first guide RNA, or binds with a K_D of at least 10, 20, 50, 100, 1000, or 10,000 nM.

265. The system or nucleic acid of any of embodiments 260-264, wherein the first CRISPR/Cas domain comprises a different amino acid sequence than the second CRISPR/Cas domain.

266. The system or nucleic acid of any of embodiments 260-265, wherein the first or second CRISPR/Cas domain comprises an amino acid sequence of a Cas protein or Cpfl protein chosen from Table 1 or a variant (e.g., mutant) of any thereof.

267. The system or nucleic acid of any of embodiments 260-266, wherein the first CRISPR/Cas domain comprises an amino acid sequence of a Cas protein or Cpfl protein chosen from Table 1 or a variant (e g., mutant) of any thereof, and tire second CRISPR/Cas domain comprises an amino acid sequence of a different Cas protein or Cpfl protein chosen from Table 1 or a variant (e.g., mutant) of any thereof.

268. The system or nucleic acid of any of embodiments 118-260, wherein the first targeting moiety comprises a zinc finger domain (e.g., a first zinc finger domain).

269. The system or nucleic acid of any of embodiments 118-260 or 268, wherein the second targeting moiety comprises a zinc finger domain (e.g., a second zinc finger domain).

270. The system or nucleic acid of any of embodiments 118-261 or 268-269, wherein the first targeting moiety comprises a first zinc finger domain and the second targeting moiety comprises a second zinc finger domain.

271. The system or nucleic acid of any of embodiments 268-270, wherein the first zinc finger domain and the second zinc finger domain bind the same genomic locus, e.g., have the same amino acid sequence. The system or nucleic acid of any of embodiments 268-271, wherein the first zinc finger domain and the second zinc finger domain have different amino acid sequences or bind different genomic loci. The system or nucleic acid of any of embodiments 118-261 or 267-272, wherein the first zinc finger molecule comprises at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally no more than 11, 10, 9, 8, 7, 6, or 5 zinc fingers). The system or nucleic acid of any of embodiments 267-273, wherein the first zinc finger molecule comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers. The system or nucleic acid of any of embodiments 268-274, wherein the first zinc finger domain comprises 3 or 9 zinc fingers. The system or nucleic acid of any of embodiments 268-275, wherein the second zinc finger domain comprises at least 1, 2, 3, 4, 5, 7, 8, 9, or 10 zinc fingers (and optionally no more than 11, 10, 9, 8, 7, 6, or 5 zinc fingers). The system or nucleic acid of any of embodiments 268-276, wherein the second zinc finger domain comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2- 3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6- 9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers. The system or nucleic acid of any of embodiments 268-277, wherein the second zinc finger domain comprises 3 or 9 zinc fingers. The system or nucleic acid of any of embodiments 118-278, wherein the first targeting moiety comprises a TAL effector domain (e.g., a first TAL effector domain). The system or nucleic acid of any of embodiments 118-260 or 279 wherein the second targeting moiety comprises a TAL effector domain (e.g., a second TAL effector domain). The system or nucleic acid of any of embodiments 279 or 280, wherein the first TAL effector domain comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 central repeats (and optionally, no more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats). The system or nucleic acid of any of embodiments 279-281, wherein the first TAL effector domain compnses 2-40, 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 2-35, 5-35, 10-35, 15- 35, 20-35, 25-35, 30-35, 2-30, 5-30, 10-30, 15-30, 20-30, 25-30, 2-25, 5-25, 10-25, 15-25, 20-25, 2-20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2-10, 5-10, or 2-5 central repeats. The system or nucleic acid of any of embodiments 279-282, wherein the second TAL effector domain comprises at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 central repeats (and optionally, no more than 45, 40, 35, 30, 25, 20, 15, or 10 central repeats). The system or nucleic acid of any of embodiments 279-283, wherein the second TAL effector domain comprises 2-40, 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 2-35, 5-35, 10-35, 15- 35, 20-35, 25-35, 30-35, 2-30, 5-30, 10-30, 15-30, 20-30, 25-30, 2-25, 5-25, 10-25, 15-25, 20-25, 2-20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2-10, 5-10, or 2-5 central repeats. The system or nucleic acid of any of embodiments 118-284, wherein the first targeting moiety comprises a nucleic acid (e.g., a first nucleic acid). The system of any of embodiments 129-285, wherein the second targeting moiety comprises a nucleic acid (e.g., a second nucleic acid). The system or nucleic acid of any of embodiments 129-286, wherein the first targeting moiety comprises a polypeptide (e.g., a first polypeptide). The system or nucleic acid of any of embodiments 129-287, wherein the second targeting moiety comprises a polypeptide (e.g., a second polypeptide). The system of embodiment 287 or 288, wherein the nucleic acid is covalently attached to the polypeptide. The system of embodiment 288 or 289, wherein the nucleic acid is non-covalently associated with the polypeptide. The system or nucleic acid of any of embodiments 275-290, wherein the nucleic acid comprises a sequence that is complementary to the transcriptional regulatory element or a sequence proximal thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches relative to the transcriptional regulatory element or a sequence proximal thereto. The system or nucleic acid of any of embodiments 275-291, wherein the nucleic acid comprises a sequence that is complementary to the anchor sequence or a sequence proximal thereto, or comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches relative to the anchor sequence or a sequence proximal thereto. Tire system of any of embodiments 275-292, wherein the nucleic acid comprises DNA, a peptide nucleic acid (PNA), a peptide-oligonucleotide conjugate, a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a polyamide, a triplex-forming oligonucleotide, an antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or other RNAi molecule. The system of any of embodiments 275-293, wherein the nucleic acid comprises a gRNA. The system of any of embodiments 275-294, wherein the nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 1-4, or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto. The system of any of embodiments 275-295, wherein the first nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 1-4 or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto, and the second nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 1-4 or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto. The system of any of embodiments 275-295, wherein the first nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 96-110 or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto, and the second nucleic acid comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity to any of SEQ ID NOs: 96-110 or has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions of difference thereto. The system of any of embodiments 118-297, wherein the transcriptional regulatory element comprises a promoter. The system of any of embodiments 118-298, wherein the transcriptional regulatory element comprises an enhancer; e.g., a super enhancer. The system of any of embodiments 118-299, wherein the anchor sequence comprises a CTCF binding motif. The system of any of embodiments 118-300, wherein the anchor sequence comprises a YY1 binding motif. The system of any of embodiments 118-301, wherein the anchor sequence comprises the sequence of SEQ ID NO: 71 or 72, or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 alterations relative thereto. The system of any of embodiments 118-302, wherein the anchor sequence comprises a sequence of CCGCCATNTT G.g , a YYl-binding motif) or AANATGGCGG (e.g., a YYl-binding motif in an opposite orientation), or a sequence with no more than 8, 7, 6, 5, 4, 3, 2, or 1 alterations relative thereto. The system of any of embodiments 118-303, wherein the anchor sequence is on the same chromosome as the MYC gene. The system of any of embodiments 118-304, wherein the anchor sequence is upstream of the MYC gene (e.g., upstream of the TSS or upstream of the promoter). The system of any of embodiments 118-305, wherein the anchor sequence is at least 1, 5, 10, 50, 100, or 1000 kilobases away from the MYC gene (e.g., from the TSS or promoter of the MYC gene). The system of any of embodiments 118-306, wherein the anchor sequence is 0. 1-0.5, 0.1-1, 0.1-5, 0.1-10, 0.1-50, 0.1-100, 0.1-500, 0.1-1000, 0.5-1, 0.5-5, 0.5-10, 0.5-50, 0.5-100, 0.5-500, 0.5- 1000, 1-5, 1-10, 1-50, 1-100, 1-500, 1-1000, 5-10, 5-50, 5-100, 5-500, 5-1000, 10-50, 10-100, 10- 500, 10-1000, 50-100, 50-500, 50-1000, 100-500, 100-1000, or 500-1000 kilobases away from the MY C gene (e.g., from the TSS or promoter of the MY C gene). The system of any of embodiments 118-303 or 305-307, wherein the anchor sequence is on a different chromosome than the MYC gene. The system of any of embodiments 118-308, wherein the second targeting moiety binds to the anchor sequence or a sequence proximal to the anchor sequence with affinity sufficient to compete for binding with an endogenous polypeptide (e.g., CTCF or YY1). The system of any of embodiments 118-309, wherein the first targeting moiety binds to a sequence at chromosome coordinates 128746342-128746364, 128746321-128746343, or 128746525-128746547, or a sequence proximal thereto. The system of any of embodiments 118-309, wherein the first targeting moiety binds to a sequence at chromosome coordinates 128746405-128746425, 128748069-128748089, 129188825-129188845, or 129188822-129188842 or a sequence proximal thereto. The system of any of embodiments 118-311, wherein the second targeting moiety binds to a sequence at chromosome coordinates 128748014-128748036, or a sequence proximal thereto. The system of any of embodiments 118-311, wherein the second targeting moiety binds to a sequence at chromosome coordinates 128746405-128746425, 128748069-128748089, 129188825-129188845, or 129188822-129188842, or a sequence proximal thereto. The system of any of embodiments 118-313, wherein the first expression repressor is a fusion molecule. The system of any of embodiments 118-314, wherein the second expression repressor is a fusion molecule. The system of any of embodiments 118-315, wherein the first expression repressor comprises a linker. The system of any of embodiments 118-316, wherein the second expression repressor comprises a linker. 318. The system of any of embodiments 118-267 or 285-317, wherein: the first expression repressor comprises a targeting moiety comprising a first CRISPR/Cas molecule, e.g., comprising a first catalytically inactive CRISPR/Cas protein, and an effector moiety comprising an epigenetic modifying moiety; and the second expression repressor comprises a targeting moiety comprising a second CRISPR/Cas molecule, e.g., comprising a second catalytically inactive CRISPR/Cas protein, and an optionally an effector moiety comprising a transcription repressor.

319. The system of any of embodiments 118-260, 268-278, or 285-317 wherein: the first expression repressor comprises a targeting moiety comprising a first zinc finger domain, and an effector moiety comprising an epigenetic modifying moiety; and the second expression repressor comprises a targeting moiety comprising a second zinc finger domain, and optionally an effector moiety comprising a transcription repressor.

320. The system of any of embodiments 118-120, 262, 268, or 275-318, wherein: the first expression repressor comprises a targeting moiety comprising a CRISPR/Cas molecule, e.g., comprising a catalytically inactive CRISPR/Cas protein, and an effector moiety comprising an epigenetic modifying moiety; and the second expression repressor comprises a targeting moiety comprising a zinc finger domain, and optionally an effector moiety comprising a transcription repressor.

321. The system of any of embodiments 118-260, 268, or 275-318, wherein: the first expression repressor comprises a targeting moiety' comprising a zinc finger domain, and an effector moiety comprising an epigenetic modifying moiety; and the second expression repressor comprises a targeting moiety comprising a CRISPR/Cas domain, e.g., comprising a catalytically inactive CRISPR/Cas protein, and optionally an effector moiety comprising a transcription repressor.

322. The system of any of embodiments 260, 268-278, or 275-318, wherein the zinc finger domain

(e g., the first or second zinc finger domain) comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4- 5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 zinc fingers, e.g., 3 or 9 zinc fingers.

323. The system of any of embodiments 322, wherein the epigenetic modifying moiety comprises a DNA methyltransferase.

324. The system of any of embodiments 118-323, wherein the epigenetic modifying moiety comprises MQ1 or a functional variant or fragment thereof. 325. The system of any of embodiments 118-324, wherein the second expression repressor comprises an effector moiety comprising a transcription repressor.

326. The system of any of embodiments 118-323, wherein the transcription repressor comprises KRAB or a functional variant or fragment thereof.

327. The system of any of embodiments 118-326, wherein the first expression repressor comprises an amino acid sequence of any of SEQ ID NOS: 28-33 or 35-37, 145-149, 151, 152, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

328. The system of any of embodiments 118-327, wherein the second expression repressor comprises an amino acid sequence of any of SEQ ID NOS: 22-27, 34, 139-144, 150, 177-180, 183-186, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

329. The system of any of embodiments 118-328, wherein binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto decreases expression of MYC in a cell.

330. The system of embodiment 327, wherein expression is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to expression in the absence of the first expression repressor, e.g., as measured by QPCRor ELISA.

331. The system of embodiment 326 or 327, wherein binding of the first expression repressor to the transcription regulatory element appreciably decreases expression of MY C for a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA.

332. The system of any of embodiments 329-331, wherein binding of the first expression repressor to the transcription regulatory element appreciably decreases expression of MYC at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours posttransfection.

333. The system of any of embodiments 328-332, wherein binding of the second expression repressor to tire anchor sequence or a sequence proximal thereto decreases expression of MY C in a cell.

334. The system of embodiment 333, wherein expression is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to expression in the absence of the second expression repressor, e.g., as measured by QPCRor ELISA.

335. The system of embodiment 333 or 334, wherein binding of the second expression repressor to the anchor sequence or a sequence proximal thereto appreciably decreases expression of MY C for a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA. The system of any of embodiments 334-335, wherein binding of the second expression repressor to the anchor sequence or a sequence proximal thereto appreciably decreases expression of MY C at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours post-transfection. The system of any of embodiments 329-336, wherein binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to the anchor sequence or a sequence proximal thereto decreases expression of MYC in a cell. The system of any of embodiments 329-337, wherein binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and binding of the second expression repressor to the anchor sequence or a sequence proximal thereto appreciably decreases expression of MYC at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, or 96 hours post-transfection. The system of any of embodiments 337 or 338, wherein expression is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to expression in the absence of the first and second expression repressors, e g., as measured by QPCR or ELISA. The system of any of embodiments 329-339, wherein binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to the anchor sequence or a sequence proximal thereto appreciably decreases expression of MYC for a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA. The system of any of embodiments 329-340, wherein the decrease in expression resulting from the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to the anchor sequence or a sequence proximal thereto is greater than the decrease in expression resulting from tire binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto or the binding of the second expression repressor to the anchor sequence or a sequence proximal thereto individually. The system of embodiment 341, wherein the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to tire anchor sequence or a sequence proximal thereto decreases expression 1.05x (i.e., 1.05 times), l.lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx more than either the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto or the binding of the second expression repressor to the anchor sequence or a sequence proximal thereto individually, e.g., as measured by QPCR or ELISA. The system of any of embodiments 329-342, wherein the decrease in expression resulting from the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to the anchor sequence or a sequence proximal thereto persists for a longer time (e.g., more hours, days, or cell divisions) than the decrease in expression resulting from the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto or the binding of the second expression repressor to the anchor sequence or a sequence proximal thereto individually. The system of embodiment 343, wherein the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to the anchor sequence or a sequence proximal thereto decreases expression 1.05x (i.e.,

I.05 times), l.lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx longer (e.g., as measured in hours, days, or cell divisions) than either the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto or the binding of the second expression repressor to the anchor sequence or a sequence proximal thereto individually, e.g., as measured by QPCR or ELISA. The system of any of embodiments 329-344, wherein binding of the first expression repressor to the promoter or a sequence proximal thereto and the second expression repressor to the superenhancer or a sequence proximal thereto appreciably decreases expression of MY C for a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by QPCR or ELISA. Hie system of any of embodiments 329-345, wherein the decrease in expression resulting from the binding of the first expression repressor to the promoter or a sequence proximal thereto and the second expression repressor to the super-enhancer or a sequence proximal thereto is greater than the decrease in expression resulting from the binding of the first expression repressor to the promoter or a sequence proximal thereto or the binding of the second expression repressor to the super-enhancer or a sequence proximal thereto individually. The system of embodiment 346, wherein the binding of the first expression repressor to the promoter or a sequence proximal thereto and the second expression repressor to the superenhancer or a sequence proximal thereto decreases expression 1.05x (i.e., 1.05 times), l. lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx more than either the binding of the first expression repressor to the promoter or a sequence proximal thereto or the binding of the second expression repressor to the super-enhancer or a sequence proximal thereto individually, e.g., as measured by QPCR or ELISA. The system of any of embodiments 329-347, wherein the decrease in expression resulting from the binding of the first expression repressor to the promoter or a sequence proximal thereto and the second expression repressor to the super-enhancer or a sequence proximal thereto persists for a longer time (e.g., more hours, days, or cell divisions) than the decrease in expression resulting from the binding of the first expression repressor to the promoter or a sequence proximal thereto or the binding of the second expression repressor to the super-enhancer or a sequence proximal thereto individually. The system of embodiment 348, wherein the binding of the first expression repressor to the promoter or a sequence proximal thereto and the second expression repressor to the superenhancer or a sequence proximal thereto decreases expression 1 ,05x (i.e., 1 .05 times), 1. lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx longer (e.g., as measured in hours, days, or cell divisions) than either the binding of the first expression repressor to the promoter or a sequence proximal thereto or the binding of the second expression repressor to the super-enhancer or a sequence proximal thereto individually, e.g., as measured by QPCR or ELISA. The system of any of embodiments 329-349, wherein expression is appreciably decreased indefinitely (e.g., for a time period greater than can be experimentally measured). The system of any of embodiments 329-350, wherein binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto decreases the viability of a cell comprising the transcription regulatory element or a sequence proximal thereto. The system of any of embodiments 329-351, wherein contacting a plurality of cells with the first expression repressor or a nucleic acid encoding the first expression repressor decreases the viability of the plurality of cells, optionally wherein the plurality of cells comprise cancerous and non-cancerous cells and/or infected cells and uninfected cells. 353. The system of embodiment 352, wherein viability is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to viability in the absence of the first expression repressor, e.g., as measured by CellTiter Gio.

354. The system of any of embodiments 329-353, wherein, administration of the first expression repressor results in apoptosis of at least 5%, 6%, 7%, 8%, 9% 10%, 12%, 15%, 17% 20%, 25% 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells).

355. The system of any of embodiments 329-354, wherein binding of the second expression repressor to the anchor sequence or a sequence proximal thereto decreases the viability of a cell comprising the anchor sequence or a sequence proximal thereto.

356. The system of any of embodiments 329-355, wherein contacting a plurality of cells with the second expression repressor or a nucleic acid encoding the second expression repressor decreases the viability of the plurality of cells.

357. The system of any of embodiments 329-356, wherein binding of the second expression repressor to the super-enhancer or a sequence proximal thereto decreases the viability of a cell comprising the transcription regulatory element or a sequence proximal thereto.

358. The system of any of embodiments 329-357, wherein contacting a plurality of cells with the second expression repressor or a nucleic acid encoding the first expression repressor decreases the viability of the plurality of cells, optionally wherein the plurality of cells comprise cancerous and non-cancerous cells and/or infected cells and uninfected cells.

359. The system of embodiment 358, wherein viability is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to viability in the absence of the second expression repressor, e.g., as measured by CellTiter Gio.

360. The system of any of embodiments 329-359, wherein, administration of the second expression repressor results in apoptosis of at least 5%, 6%, 7%, 8%, 9% 10%, 12%, 15%, 17% 20%, 25% 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells).

361. The system of any of embodiments 329-360, wherein binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to tire anchor sequence or a sequence proximal thereto decreases the viability of a cell comprising the anchor sequence or a sequence proximal thereto.

362. The system of any of embodiments 329-361, wherein binding of the first expression repressor to the promoter or a sequence proximal thereto and the second expression repressor to the superenhancer or a sequence proximal thereto decreases the viability of a cell

363. The system of any of embodiments 329-362, wherein contacting a plurality of cells with the system or a nucleic acid encoding tire system decreases tire viability of the plurality of cells. The system of embodiments 329-363, wherein viability is decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to viability in the absence of the system, e.g., as measured by CellTiter Gio. The system of any of embodiments 329-364, wherein the decrease in viability resulting from the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to the anchor sequence or a sequence proximal thereto is greater than the decrease in viability resulting from the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto or the binding of the second expression repressor to the anchor sequence or a sequence proximal thereto individually. The system of any of embodiments 329-365, wherein the decrease in viability resulting from the binding of the first expression repressor to the promoter or a sequence proximal thereto and the second expression repressor to the super-enhancer or a sequence proximal thereto is greater than the decrease in viability resulting from the binding of the first expression repressor to the promoter or a sequence proximal thereto or the binding of the second expression repressor to the super-enhancer or a sequence proximal thereto individually. The system of embodiment 366, wherein the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto and the second expression repressor to the anchor sequence or a sequence proximal thereto decreases viability 1 .05x (i.e., 1.05 times), l.lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx more than either the binding of the first expression repressor to the transcription regulatory element or a sequence proximal thereto or the binding of the second expression repressor to the anchor sequence or a sequence proximal thereto individually, e.g., as measured by CellTiter Gio. The system of embodiment 366 or 367 , wherein the binding of the first expression repressor to the promoter or a sequence proximal thereto and the second expression repressor to the superenhancer or a sequence proximal thereto decreases viability 1.05x (i.e., 1.05 times), l. lx, 1 .15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx more than either the binding of the first expression repressor to the promoter or a sequence proximal thereto or the binding of the second expression repressor to the super-enhancer or a sequence proximal thereto individually, e g., as measured by CellTiter Gio. The system of any of embodiments 329-368, wherein, administration of the first expression repressor and the second expression repressor result in apoptosis of at least 5%, 6%, 7%, 8%, 9% 10%, 12%, 15%, 17% 20%, 25% 30%, 40%, 45%, 50%, 55%, 60%, 65%, 75% of target cells (e.g., cancer cells).

370. The system of embodiments 329-369, wherein the plurality of cells comprises a plurality of cancer cells and a plurality of non-cancer cells.

371. The system of embodiment 370, wherein contacting the plurality of cells with the system or a nucleic acid encoding the system decreases the viability of the plurality of cancer cells more than it decreases the viability of the plurality of non-cancer cells.

372. The system of embodiment 370 or 371, wherein contacting the plurality of cells with the system or a nucleic acid encoding the system decreases the viability of the plurality of cancer cells 1 ,05x (i.e., 1.05 times), l. lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx more than it decreases the viability of the plurality of non-cancer cells.

373. The expression repressor or system of any preceding embodiments, which does not reduce viability of non-cancer cells (e.g., primary hepatocytes) by more than 5, 10, 15, or 20%, e.g., when assayed according to Example 29 as described in the PCT publication WO/2022/132195.

374. The expression repressor or system of embodiment 373, wherein viability is assayed 72 hours after contacting the cells with the expression repressor or system.

375. The expression repressor or system of embodiment 374, wherein the assay comprises contacting the non-cancer cells with 2.5, 2, 1.25, 1, 0.6, or 0.5 pg/ml of the expression repressor or system.

376. The system of any of embodiments 352-375, which, when contacted with a plurality of infected cells and a plurality of uninfected cells, decreases the viability of the plurality of infected cells more than it decreases the viability of the plurality of uninfected cells and/or decreases the viability of the plurality of cancerous cells more than it decreases the viability of the plurality of non-cancerous cells.

377. The system of any of embodiments 352-376, wherein the cancer is hepatocellular carcinoma (HCC), Fibrolamellar Hepatocellular Carcinoma (FHCC), Cholangiocarcinoma, Angiosarcoma, secondary liver cancer, Non-small cell lung cancer (NSCEC), Adenocarcinoma, Small cell lung cancer (SCEC), Earge cell (undifferentiated) carcinoma, triple negative breast cancer, gastric adenocarcinoma, endometrial carcinoma, or pancreatic carcinoma.

378. The system of any of embodiments 352-377, wherein the cancer cells are lung cancer cells, gastric cancer cells, gastrointestinal cancer cells, colorectal cancer cells, pancreatic cancer cells, or hepatic cancer cells.

379. The system of any of embodiments 352-378, wherein the cells are human lung epithelial cells or human lung fibroblast cells. 380. The system of any of embodiments 352-379, wherein the infection is viral.

381. The system of embodiment 380, wherein the viral infection is hepatitis, e.g., hepatitis B.

382. The system of any of embodiments 378-381, wherein the infected cells are human hepatocytes.

383. The system of any of embodiments 352-382, wherein the viral infection is a chronic infection.

384. A fusion protein comprising: a first amino acid region comprising a sequence encoding the first expression repressor of a system of any of embodiments 118-383; and a second amino acid region comprising a sequence encoding the second expression repressor of a system of any of embodiments 118-383.

385. The fusion protein of embodiment 384 which comprises a third amino acid region, wherein the third amino acid region is situated between the first amino acid region and the second amino acid region.

386. The fusion protein of embodiment 385, wherein the third amino acid region comprises a protease cleavage peptide sequence, e.g., a self-cleaving peptide sequence, e.g., a T2A self-cleaving peptide sequence, e.g., a sequence according to SEQ ID NO: 120.

387. The fusion protein of embodiment 386, wherein the third amino acid region comprises a protease cleavage peptide sequence, e.g., a self-cleaving peptide sequence, e.g., a tandem 2A peptide sequence, e.g., atPT2A sequence, e.g., a sequence according to SEQ ID NO: 124.

388. The fusion protein of embodiment 385, wherein the peptide sequence comprises a T2A peptide sequence and a P2A peptide sequence.

389. The fusion protein of any of embodiments 384-388, wherein: the first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto; and the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 24 or 142, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto.

390. The fusion protein of any of embodiments 384-388, wherein: the first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto; and the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 177 or 183, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto.

391. The fusion protein of any of embodiments 384-388, wherein: the first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30 or 129, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto; and the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 179 or 185, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto. The fusion protein of any of embodiments 384-391, which comprises an amino acid sequence of SEQ ID NO: 91, 92, 121, or 122, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The fusion protein of any of embodiments 384-392, which comprises an amino acid sequence of SEQ ID NO: 181, 182, 187, or 188, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. A nucleic acid comprising a sequence encoding the system of any of embodiments 118-393. A nucleic acid comprising a sequence encoding the system of embodiment 394. The nucleic acid of embodiment 394 or 395, which comprises: a first region comprising a sequence encoding the first expression repressor of a system of any of embodiments 118-3 3; and a second region comprising a sequence encoding the second expression repressor of a system of any of embodiments 118-393. The nucleic acid of any of embodiments 394-396, which comprises a third region, wherein the third region is situated between the first region and the second region. The nucleic acid of any of embodiments 394-397, wherein the third region encodes a ribosomeskipping sequence. The nucleic acid of embodiment 397 or 398, wherein the third region encodes a tPT2A peptide sequence, e.g., a sequence according to SEQ ID NO: 124. The nucleic acid of any of embodiments 397-399, wherein the third region encodes a protease cleavage peptide sequence, e.g., a self-cleaving peptide sequence, e.g., a T2A self-cleaving peptide sequence, e.g., a sequence according to SEQ ID NO: 95. The nucleic acid of any of embodiments 397-400, wherein the third region encodes a protease cleavage peptide sequence, e.g., a self-cleaving peptide sequence, e.g., a tandem 2A peptide sequence, e.g., atPT2A peptide sequence, e.g., a sequence according to SEQ ID NO: 124. The nucleic acid of any of embodiments 394-401, wherein the first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30, 129 or a sequence with at least 80, 85, 90, 95, or 99% identity thereto: and the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 24, 142, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto. The nucleic acid of any of embodiments 394-401, wherein the first expression repressor comprises an amino acid sequence according to SEQ ID NO: 30, 129 or a sequence with at least 80, 85, 90, 95, or 99% identity thereto: and the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 177, 179, 183, or 185 or a sequence with at least 80, 85, 90, 95, or 99% identity thereto. The nucleic acid of any of embodiments 394-403, which encodes an amino acid sequence of SEQ ID NO: 91, 92, 121, 122 or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of any of embodiments 394-404, which encodes an amino acid sequence of SEQ ID NO: 181, 182, 187, 188, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of any of embodiments 394-405, which comprises a nucleotide sequence of SEQ ID NO: 93, 94, 112, or 113 or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid of any of embodiments 394-406, which comprises a nucleotide sequence of SEQ ID NO: 196, 197, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. A nucleic acid comprising a sequence encoding the expression repressor or the expression repressor system of any of embodiments 1-407. The nucleic acid of any of embodiments 394-408, which is an RNA, e.g., an mRNA. The nucleic acid of any of embodiments 394-409, which comprises an N7-methylated guanosine, e.g., linked to the 5’ end of the RNA, e.g., via a reverse 5' to 5' triphosphate linkage. The nucleic acid of any of embodiments 394-410, which comprises a 5 ’ UTR. Tire nucleic acid of any of embodiments 394-411, which comprises a Kozak sequence, e.g., between the 5’ UTR and the sequence encoding the expression repressor. A system comprising : a first nucleic acid comprising a sequence encoding the first expression repressor of a system of any of embodiments 118-393; and a second nucleic acid comprising a sequence encoding a second expression repressor, e.g., the second expression repressor of a system of any of embodiments 118-393. The system of embodiment 413, wherein the first nucleic acid has a nucleotide sequence of SEQ ID NO: 63, 130, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the second nucleic acid having a nucleotide sequence of SEQ ID NO: 57, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The system of embodiment 414, wherein the first nucleic acid has a nucleotide sequence of SEQ ID NO: 63, 130, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the second nucleic acid having a nucleotide sequence of SEQ ID NO: 189, or 194, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The system of embodiment 415, wherein the first nucleic acid has a nucleotide sequence of SEQ ID NO: 189, 194, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the second nucleic acid having a nucleotide sequence of SEQ ID NO: 63, 130, or a sequence with at least 80, 85, 90, 95, or 99% identity thereto, or a sequence with no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid or system of any of embodiments 394-416, wherein the nucleic acid comprises mRNA. A vector comprising the nucleic acid encoding the system, or expression repressor of any of the preceding embodiments. A lipid nanoparticle comprising the system, nucleic acid, mRNA, or vector of any of the preceding embodiments. Hie lipid nanoparticle of embodiment 419 comprising an ionizable lipid, e.g., a cationic lipid, e.g., MC3, SSOP. The lipid nanoparticle of embodiment 419 or 420, further comprising one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids, polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, or polymer conjugated lipids. A reaction mixture comprising the expression repressor, system, nucleic acid, vector, or lipid nanoparticle of any of the preceding embodiments. The reaction mixture of embodiment 422, further comprising a cell. A pharmaceutical composition comprising the expression repressor, system, nucleic acid, vector, lipid nanoparticle or the reaction mixture of any preceding embodiments. A method of decreasing expression of a MYC gene in a cell, the method comprising: contacting the cell (e.g., a cancer cell) with an expression repressor, a system, one or more nucleic acids encoding said system or expression repressor, a vector, a lipid nanoparticle, or a pharmaceutical composition of any of embodiments 1-424, thereby decreasing expression of the MYC gene in the cell. A method of treating cancer in a subject in need thereof, the method comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition of any of embodiments 1-424 to the subject, thereby treating the cancer in the subject. A method of reducing tumor growth in a subject in need thereof, the method comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition of any of embodiments 1-424 to the subject, thereby reducing the tumor size in the subject. The method of embodiment 427, wherein the reduction in tumor growth comprises reduction of tumor volume compared to tumor volume at the start of treatment. The method of embodiment 428, wherein the reduction in tumor growth in the subject is greater compared to an untreated subject. A method of increasing or restoring sensitivity of a cancer to a kinase inhibitor, e.g., sorafenib, the method comprising administering an expression repressor or system described herein to a subject having the cancer. The method of embodiment 430, wherein administration of the expression repressor or system lowers the IC50 of the kinase inhibitor by 10%, 20%, 30%, or 40%, e.g., in a cancer cell viability assay, e.g., an assay according to Example 38 as described in tire PCT publication WO/2022/132195. The method of embodiment 430 or 431, wherein the kinase inhibitor inhibits one or more of (e.g., all of) VEGFR, PDGFR, or RAF kinase. A method of increasing or restoring sensitivity of a cancer to a bromodomain inhibitor, e.g., a BET inhibitor, e.g., JQ1, the method comprising administering an expression repressor, system, or nucleic acid described herein (e.g., of any of embodiments 1-423) to a subject having the cancer, wherein optionally administration of the expression repressor or system lowers the IC50 of the bromodomain inhibitor by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, e.g., in a cancer cell viability assay, e.g., an assay according to Example 39 as described in the PCT publication WO/2022/132195.

434. The method of embodiment 433 wherein the bromodomain inhibitor is or comprises JQ1, BET672, or birabresib.

435. A method of increasing or restoring sensitivity of a cancer to a MEK inhibitor, e.g., Trametinib, the method comprising administering an expression repressor, system, or nucleic acid described herein (e.g., of any of embodiments 1-423) to a subject having the cancer, wherein optionally administration of the expression repressor or system lowers the IC50 of the MEK inhibitor by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, e.g., in a cancer cell viability assay, e.g., an assay according to Example 51 as described in the PCT publication WO/2022/132195.

436. The method of any of embodiments 427-435, wherein the reduction in tumor growth in the subject is greater than or similar to a tumor size reduction when the subject is treated with a chemotherapeutic agent or small molecule MY C inhibitor.

437. The method of embodiment 436, wherein the chemotherapeutic agent is sorafenib or cisplatin.

438. The method of embodiment 437, wherein the small molecule MYC inhibitor is MYCi975.

439. The method of reducing tumor size in a subject in need thereof, the method comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition of 1-424 to the subject, wherein the reduction in tumor size is greater than or similar to a tumor size reduction when the subject is treated with a chemotherapeutic agent.

440. The method of 439 wherein the chemotherapeutic agent is sorafenib or cisplatin.

441. The method of any of the preceding embodiments wherein the subject does not experience any significant side effects compared to when treated with a chemotherapeutic agent or a small molecule MYC inhibitor.

442. The method of any of embodiments 436-441, wherein the chemotherapeutic agent is sorafenib or cisplatin.

443. The method of embodiment 442, wherein the small molecule MY C inhibitor is MY Ci975.

444. The method of any of embodiments 426-443, wherein the cancer is stage I, stage II, stage III, or stage IV cancer.

445. The method of any of preceding embodiments wherein the subject’s body weight remains about the same before treatment and post-treatment. The method of any of preceding embodiments, wherein the subject does not experience a decrease in body weight, or wherein the subject experiences a decrease in body weight of less than 3%, 2%, or 1% compared to at the start of treatment. The method of any of the preceding embodiments wherein the subject does not experience a reduction or gain in body weight post-treatment compared to the subject’s body weight before the treatment. A method of treating a liver disease in a subject in need thereof, the method comprising: administering an expression repressor to the subject, wherein the expression repressor comprises targeting moiety that binds a MYC locus (e.g., a transcribed region of MYC, a MYC promoter, or an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MY C gene or to a sequence proximal to the anchor sequence), and optionally, an effector moiety, e.g., an effector moiety described herein; thereby treating the liver disease in the subject. The method of embodiment 448, which further comprises administering to the subject a second expression repressor, the second expression repressor comprising a targeting moiety that binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC, and optionally, a second effector moiety, e.g., an effector moiety described herein; e.g., KRAB; thereby treating the liver disease in the subject. A method of treating a liver disease in a subject in need thereof, the method comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition, of any of embodiments 1-424 to the subject, thereby treating the liver disease in the subject. The method of embodiment 450, wherein the liver disease is a chronic liver disease. The method of embodiment 450 or 451 wherein the liver disease is viral or alcohol related. The method of any of embodiments 450-452, wherein the liver disease is hepatitis or hepatocellular carcinoma. Hie method of embodiment 453, wherein the hepatocellular carcinoma is selected from HCC subtype SI, HCC subtype S2, or HCC subtype S3. The method of embodiment 453 or 454, wherein the hepatocellular carcinoma is HCC S 1 . The method of embodiment 453 or 454, wherein the hepatocellular carcinoma is HCC S2. The method of any of embodiments 450-456 where the liver disease is caused by a hepatitis B virus or hepatitis C vims. A method of treating a pulmonary disease in a subject in need thereof, the method comprising: administering an expression repressor to the subject, wherein the expression repressor comprises targeting moiety that binds a MYC locus (e.g., a transcribed region of MYC, a MYC promoter, or an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MY C gene or to a sequence proximal to the anchor sequence), and optionally, an effector moiety, e.g., an effector moiety described herein; thereby treating the pulmonary disease in the subject. The method of embodiment 458, which further comprises administering to the subject a second expression repressor, the second expression repressor comprising a targeting moiety that binds a genomic locus located in a super enhancer region of a target gene, e.g., MYC, and optionally, a second effector moiety, e.g., an effector moiety described herein; e.g., KRAB; thereby treating the pulmonary disease in the subject. A method of treating a pulmonary disease in a subject in need thereof, the method comprising: administering the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition, of any of embodiments 1-424 to the subject, thereby treating the pulmonary disease in the subject. The method of embodiment 459 or 460 where the pulmonary disease is a cancer, e.g., a lung cancer, e.g., a lung carcinoma, e.g., non-small cell lung carcinoma or small cell lung carcinoma. The method of any of embodiments 425-461, wherein contacting or administering comprises intravenous administration to a subject. The method of any of embodiments 425-462, wherein contacting or administering comprises intra-tumoral delivery (e.g., injection). The method of any of embodiments 425-463, wherein the cancer is characterized by increased MYC expression relative to a reference level (e.g., relative to a reference cell’s MYC expression, e.g., an otherwise similar non-cancerous cell of the subject). The method of any of embodiments 426-464, wherein the cancer is characterized by duplication of a portion of or all of a MYC gene. Hie method of any of embodiments 426-465, wherein the cancer is selected from colorectal cancer, breast cancer, AML, prostate cancer, neuroblastoma, lung cancer, endometrial cancer, liver cancer, a lymphoma (e.g., Burkitt lymphoma), carcinoma of the cervix, or stomach cancer. The method of any of embodiments 426-466, wherein the cancer is a human chorionic gonadotropin (hCG) secreting cancer. The method of any of embodiments 426-467, wherein the cancer is hepatocarcinoma. 469. The method of any of embodiments 426-468, wherein the cancer is a non-responsive cancer, e.g., a non-responsive hepatocarcinoma.

470. The method of any of embodiments 426-469, wherein the cancer is non-small cell lung carcinoma or small cell lung carcinoma.

471. The method of any of embodiments 426-470, wherein the cancer over-expresses alpha-fetoprotein (AFP) (e.g., relative to a reference cell’s AFP expression, e.g., an otherwise similar non- cancerous cell of the subject).

472. The method of any of embodiments 431-471, wherein cells of the cancer are characterized by the presence of a super enhancer, e.g., comprising the MYC gene or comprising the anchor-sequence mediated conjunction comprising the MYC gene, wherein optionally the cancer is selected from liver cancer, colorectal cancer, breast cancer, AML, prostate cancer, neuroblastoma, lung cancer, or endometrial cancer.

473. The method of embodiment 471, wherein the expression repressor (e.g., the second expression repressor) binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a MYC gene or to a sequence proximal to the anchor sequence.

474. The method of any of embodiments 426-473, wherein cells of the cancer are characterized by the absence of a super enhancer comprising the MY C gene or comprising the anchor-sequence mediated conjunction comprising the MYC gene.

475. The method of embodiment 474, wherein the expression repressor (e.g., the first expression repressor) binds the MYC promoter.

476. The method of any of embodiments 426-475, wherein the cancer comprises cells comprising a super enhancer comprising the MY C gene or comprising the anchor-sequence mediated conjunction comprising the MY C gene, and cells not comprising a super enhancer comprising the MYC gene or comprising the anchor-sequence mediated conjunction comprising the MYC gene.

477. The method of any of embodiments 426-476, wherein the cancer comprises cells characterized by increased MYC expression relative to a reference level (e.g., relative to a reference cell’s MYC expression, e.g., an otherwise similar non-cancerous cell of the subject), and cells not characterized by increased MYC expression relative to a reference level (e.g., relative to a reference cell’s MYC expression, e.g., an otherwise similar non-cancerous cell of the subject), e.g., having normal MYC expression.

478. The method of any of embodiments 426-477, wherein the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition is administered a monotherapy. 479. The method of any of embodiments 426-478, which comprises administering a plurality of doses of the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition to the subject, e.g., at least 2, 3, 4, 5, or 6 doses.

480. The method of any of embodiments 426-477, which comprises administering a plurality of doses of the expression repressor, system, nucleic acid, vector, lipid nanoparticle, or a pharmaceutical composition to the subject in 5 day intervals.

481. The method of any of embodiments 426-480, comprising: a) first, administering to the subject a first plurality of doses of an expression repressor or system described herein (e.g., of any of embodiments 1-424), wherein optionally each subsequent dose in the first plurality is administered 5 days after the previous dose in the first plurality; b) second, withdrawing the expression repressor or system for a period of time (a “drug holiday”), e.g., for about 2 weeks), and c) third, administering to the subject a second plurality of doses of the expression repressor or system, wherein optionally a subsequent dose of the second plurality is administered 5 days after the previous dose in the second plurality.

482. The method of embodiment 481, wherein the first plurality of doses comprises 4 doses.

483. The method of embodiment 480 or 481, wherein the second plurality of doses comprises 2 doses.

484. The method of any of embodiments 481-483, wherein the subject receives no therapeutic at all during the drug holiday.

485. The method of any of embodiments 481-484, wherein the subject receives a second therapeutic agent during the drug holiday.

486. The method of any of embodiments 481-485, wherein the drug holiday is at least twice as long as the time between administration of doses in the first plurality of doses.

487. The method of any of embodiments 481-486, wherein the drug holiday is at least twice as long as the time between administration of doses in the second plurality of doses.

488. The method of any of embodiments 426-487, wherein volume of the tumor declines to undetectable levels following treatment with the expression repressor or system.

489. Hie method of any of embodiments 426-488, tumor volume declines (e.g., to undetectable levels) after cessation of treatment with the expression repressor or system.

490. The method of any of embodiments 425-489, wherein the cancer does not become resistant to the expression repressor or system, or does not become resistant to the expression repressor or system within a period of 10, 20, 30, 40, 50, or 60 days.

491. The method of any of embodiments 425-490, wherein the cancer cells have a functional apoptotic pathway. 492. The method of any of embodiments 425-491, wherein the cancer cells have functional Caspase 3.

493. The method of embodiment 492, wherein Caspase 3 is up regulated in cancer cells upon administration of the expression repressor or system to the subject.

494. The method of any of embodiments 425-493, wherein Ki67 is downregulated in cancer cells upon administration of the expression repressor or system to the subject.

495. The method of any of embodiments 425-494, wherein cancer cell proliferation declines upon administration of the expression repressor or system to the subject.

496. The method of any of embodiments 425-495, wherein the method further comprises a. contacting the cell with a second therapeutic agent or b. administering a second therapeutic agent to the subject.

497. The method of embodiment 496, wherein the second therapeutic agent is not an expression repressor binding to MYC promoter.

498. The method of embodiment 496 or 497 wherein the second therapeutic agent is not an expression repressor, system, fusion protein, nucleic acid, vector, reaction mixture, pharmaceutical composition, or lipid nanoparticle of any of embodiments 1-424.

499. The method of any of embodiments 495-497, wherein the second therapeutic agent is the expression repressor, system, fusion protein, nucleic acid, vector, reaction mixture, pharmaceutical composition, or lipid nanoparticle, of any of embodiments 1-424..

500. The method of any of embodiments 496-498, wherein the second therapeutic agent is an immunotherapy, one or both of immune checkpoint and anti-vascular-endothelial-growth-factor therapy, systemic chemotherapy, a tyrosine kinase inhibitor, e.g., sorafenib, a mitogen-activated protein kinase kinase inhibitor (MEK inhibitor), e.g., trametinib, or a bromodomain inhibitor, e.g., a BET inhibitor, e.g., JQ1 or birabresib.

501. The method of any of embodiments 496-500, wherein the second therapeutic agent is a tyrosine kinase inhibitor, e.g., sorafenib.

502. The method of any of embodiments 496-500, wherein the second therapeutic agent is a bromodomain inhibitor, e.g., a BET inhibitor, e.g., JQ1, birabresib, or BET 672.

503. Hie method of any of embodiments 496-500, wherein the second therapeutic agent is a mitogen- activated protein kinase kinase inhibitor (MEK inhibitor), e.g., trametinib.

504. The method of any of embodiments 496-503, wherein the method further comprises administering an additional therapy to the subject.

505. The method of embodiment 504, wherein the additional therapy comprises surgical resection orthotopic liver transplantation, radiofrequency ablation, photodynamic therapy (PDT), laser therapy, brachytherapy, radiation therapy, trans-catheter arterial chemo- or radio-embolization, or stereotactic radiation therapy.

506. The method of any of embodiments 496-505, wherein the second therapeutic agent is selected from a checkpoint inhibitor or a small molecule.

507. The method of any of embodiments 496-506, wherein the second therapeutic agent is a chemotherapeutic agent, e.g., a kinase inhibitor or a bromodomain inhibitor, e.g., a BET inhibitor.

508. The method of embodiments 506 or 507 wherein the second therapeutic agent is selected from sorafenib, JQ1, BET672, birabresib, or trametinib.

509. The method of any of embodiments 496-507, wherein the expression repressor, system, or nucleic acid and the second therapeutic agent are administered concurrently.

510. The method of any of embodiments 496-509, wherein the expression repressor, system, or nucleic acid and the second therapeutic agent are administered sequentially.

511. The method of any of embodiments 504-509, wherein the additional therapy is administered concurrently.

512. The method of any of embodiments 504-510, wherein the additional therapy is administered sequentially.

513. The method of any of embodiments 496-512, wherein second therapeutic agent is administered simultaneously with the expression repressor, system, nucleic acid, vector, lipid nanoparticle, pharmaceutical composition, or reaction mixture of any of embodiments 1-424.

514. The method of any of embodiments 496-513, wherein second therapeutic agent is administered consecutively with the expression repressor, system, nucleic acid, vector, lipid nanoparticle, pharmaceutical composition, or reaction mixture of any of embodiments 1-424.

515. The method of any of embodiments 496-514, wherein the expression repressor, system, or nucleic acid is administered intravenously, and the second therapy is administered orally.

516. The method of any of the preceding embodiments, wherein the cancer is a resistant or refractory cancer.

517. The method of any of the preceding embodiments, wherein the cancer is resistant or refractory to a kinase inhibitor, e.g., a kinase inhibitor that inhibits one or more of VEGFR, PDGFR, or RAF kinase, e.g., sorafenib.

518. The method of any of the preceding embodiments, wherein the subject has an amplification in the MY C super-enhancer.

519. A kit comprising a container comprising a composition comprising an expression repressor, a system, one or more nucleic acids encoding said system or expression repressor, a vector, a lipid nanoparticle, reaction mixture, or a phannaceutical composition of any of embodiments 1 -424 and a set of instructions comprising at least one method for modulating, e.g., decreasing the expression of a MYC gene within a cell with said composition. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject:

(1) the expression repressor of any one of embodiments 1-117 or 373-375, the system of any one of any one of embodiments 118-142, 194-383, 413-417, the fusion protein of any one of embodiments 384-393 or a nucleic acid (e.g., RNA, e.g., mRNA) encoding the same, or the nucleic acid of any one of embodiments 143-248, 259-288, 291, 292, 394-412, or 417, the vector of embodiment 418, the lipid nanoparticle of any one of embodiments 419-421, or the reaction mixture of embodiment 422 or 423; and

(2) an immune checkpoint inhibitor polypeptide (e.g., an antibody molecule or peptide). A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject:

(1) a nucleic acid (e g., RNA, e.g., mRNA) encoding an expression repressor, wherein the expression repressor comprises:

(a) a targeting moiety that binds to a MY C locus, and

(b) optionally, an effector moiety, wherein the expression repressor is capable of decreasing expression of MYC; and

(2) an immune checkpoint inhibitor polypeptide (e.g., an antibody molecule or peptide). The method of claim 521, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-PD-1 antibody molecule, an anti-PD-Ll antibody molecule, an anti- CTLA4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, an anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule. The method of embodiment 521, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-PD-1 antibody molecule. The method of embodiment 523, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 18. The method of embodiment 523 or 524, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region listed in Table 18, e.g., SEQ ID Nos.: 208, 211, 214, or 217; ii) an amino acid sequence having at least one, two or three modifications but not more than 30,

20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 18, e.g., SEQ ID Nos.: 208, 211, 214, or 217; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 18, e.g., SEQ ID Nos.: 208, 211, 214, or 217. The method of any one of embodiments 523-525, wherein the immune checkpoint inhibitor antibody molecule comprises a light chain variable region comprising: i) the amino acid sequence of any light chain variable region listed in Table 18, e.g., SEQ ID Nos.: 209, 212, 215, or 218; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 18, e.g., SEQ ID Nos.: 209, 212, 215, or 218; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 18, e.g., SEQ ID Nos.: 209, 212, 215, or 218. The method of any one of embodiments 523-526, wherein the anti-PD-1 antibody molecule comprises aHC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of cemiplimab according to Kabat or Chothia. The method of any one of embodiments 523-526, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of dostarlimab according to Kabat or Chothia. The method of any one of embodiments 523-526, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of nivolumab according to Kabat or Chothia. The method of any one of embodiments 523-526, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of pembrolizumab according to Kabat or Chothia. The method of any one of embodiments 523-526, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 19 or 20. The method of embodiment 523, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of MIH4, according to Kabat or Chothia. The method of embodiment 521, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-PD-Ll antibody molecule. The method of embodiment 533, wherein the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 21 or 22. The method of embodiment 533 or 534, wherein the anti-PD-Ll antibody molecule comprises a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region listed in Table 21, e.g., SEQ ID Nos.: 238, 241, or 244; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 21, e.g., SEQ ID Nos.: 238, 241, or 244; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 21, e.g., SEQ ID Nos.: 238, 241, or 244. The method of any one of embodiments 533-535, wherein the anti-PD-Ll antibody molecule comprises a light chain variable region comprising: i) the amino acid sequence of any light chain variable region listed in Table 21, e.g., SEQ ID Nos.: 239, 242, or 245; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 21, e.g., SEQ ID Nos.: 239, 242, or 245; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 21, e.g., SEQ ID Nos.: 239, 242, or 245. The method of embodiment 533, where the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of mAb 10F.9G2, according to Kabat or Chothia. The method of embodiment 533, wherein the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of atezolizumab according to Kabat or Chothia, e.g., CDRs of a sequence of Table 22. Tire method of embodiment 533 or 538, wherein the anti-PD-Ll antibody molecule comprising a HC CDR1 comprising an amino acid sequence GFTFSDSWIH (SEQ ID NO: 259), a HC CDR2 comprising an ammo acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 260), and a HC CDR3 comprising an amino acid sequence RHWPGGFDY (SEQ ID NO: 261); and/or a LC CDR1 comprising an amino acid sequence RASQDVSTAVA (SEQ ID NO: 262), a LC CDR2 comprising an amino acid sequence SASFLYS (SEQ ID NO: 263), and a LC CDR3 comprising an amino acid sequence QQYLYHPAT (SEQ ID NO: 264). The method of embodiment 533, wherein the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of avelumab according to Kabat or Chothia. The method of embodiment 533, wherein the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of durvalumab according to Kabat or Chothia. The method of embodiment 521, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-CTLA-4 antibody molecule. The method of embodiment 542, wherein the anti-CTLA-4 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 23. The method of embodiment 542 or 543, wherein the anti-CTLA-4 antibody molecule comprises a hcaw chain variable region comprising: i) the amino acid sequence of any heavy chain variable region listed in Table 23, e.g., SEQ ID No: 247; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 23, e.g., SEQ ID No: 247; or lii) an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 23, e.g., SEQ ID No: 247. The method of any one of embodiments 542-544, wherein the anti-CTLA-4 antibody molecule comprises a light chain variable region comprising: i) the amino acid sequence of any light chain variable region listed in Table 23, e.g., SEQ ID No: 248; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 23, e.g., SEQ ID No: 248; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 23, e.g., SEQ ID No: 248. The method of embodiment 543, wherein the anti-CTLA-4 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of ipilimumab, according to Kabat or Chothia. The method of embodiment 521, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-LAG3 antibody molecule. The method of embodiment 547, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 24. The method of embodiment 547 or 548, wherein the anti-LAG3 antibody molecule comprises a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region listed in Table 24, e.g., SEQ ID No: 250 or 280; ii) an amino acid sequence having at least one, two or three modifications but not more than 30,

20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 24, e.g., SEQ ID No: 250 or 280; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 24, e.g., SEQ ID No: 250 or 280. The method of any one of embodiments 547-549, wherein the anti-LAG3 antibody molecule comprises a light chain variable region comprising: i) the amino acid sequence of any light chain variable region listed in Table 24, e.g., SEQ ID No: 251 or 281; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 24, e.g., SEQ ID No: 251 or 281; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 24, e.g., SEQ ID No: 251 or 281. The method of embodiment 547, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of relatlimab, according to Kabat or Chothia. The method of embodiment 547, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of relatlimab, according to table 25. The method of embodiment 547, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of favezelimab, according to Kabat or Chothia. The method of embodiment 548, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of favezelimab, according to table 25. The method of embodiment 521, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-PD-L2 antibody molecule. The method of embodiment 555, wherein the anti-PD-L2 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of rHIgM12B7 according to Kabat or Chothia. The method of embodiment 555 or 556, wherein the anti-PD-L2 antibody molecule competes for binding to PD-L2 with an antibody molecule comprising a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of rHIgM12B7 according to Kabat or Chothia. The method of embodiment 521, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-Tim3 antibody molecule. The method of embodiment 558, wherein the anti-Tim3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of MBG 453 according to Kabat or Chothia. The method of embodiment 558 or 559, wherein the anti-Tim3 antibody molecule competes for binding to Tim3 with an antibody molecule comprising a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of MBG453 according to Kabat or Chothia. The method of embodiment 521, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti- killer IgG-like receptor (KIR) antibody molecule. The method of embodiment 561, wherein the anti-KIR antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 26. The method of embodiment 561 or 562, wherein the anti-KIR antibody molecule comprises a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region listed in Table 26, e.g., SEQ ID No: 277 or 279; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 26, e.g., SEQ ID No: 277 or 279; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 26, e.g., SEQ ID No: 277 or 279. Hie method of any one of embodiments 561 or 562, wherein the anti-KIR antibody molecule comprises a light chain variable region comprising: i) the amino acid sequence of any light chain variable region listed in Table 26, e.g., SEQ ID No: 278 or 279; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 26, e.g., SEQ ID No: 278 or 279; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 26, e.g., SEQ ID No: 278 or 279.

565. The method of embodiment 561, wherein the anti-KIR antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of lirilumab, according to Kabat or Chothia.

566. The method of embodiment 561, wherein the anti-KIR antibody molecule competes for binding to BTLA with an antibody molecule comprising a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of lirilumab according to Kabat or Chothia.

567. The method of embodiment 521, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti -BTLA antibody molecule.

568. The method of embodiment 567, wherein the anti-BTLA antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of TAB004, according to Kabat or Chothia.

569. The method of embodiment 567 or 568, wherein the anti-BTLA antibody molecule competes for binding to BTLA with an antibody molecule comprising a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of TAB004 according to Kabat or Chothia.

570. The method of any one of embodiments 567-569, wherein the anti-BTLA antibody molecule competes for binding to BTLA with an antibody molecule disclosed in US Pat. 8,518,405 or 9,845,362.

571. The method of any of embodiments 521-570, wherein the immune checkpoint inhibitor antibody molecule is administered by injection (e.g., subcutaneously or intravenously.

572. The method of any of embodiments 521-571, wherein the immune checkpoint inhibitor antibody molecule is administered at a dose of about 1 mg/kg to 30 mg/kg or about 100 mg to about 2000 mg, e.g., administered every 1, 2, 3, 4, 5, 6 or 7 weeks.

573. The method of any of embodiments 521, 522, 533-536, 538, 571, or 572, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising atezolizumab and is administered (e.g., at 840 mg) as an intravenous infusion (e.g., over 30 minutes or over 60 minutes) every 2 weeks.

574. The method of any of embodiments 521, 522, 533-536, 538, 539, 571, or 572, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising atezolizumab and is administered (e.g., at 1200 mg) as an intravenous infusion (e.g., over 30 minutes or over 60 minutes) every 3 weeks.

575. The method of any of embodiments 521, 522, 533-536, 538, 539, 571, or 572, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising atezolizumab and is administered (e.g., at 1680 mg) as an intravenous infusion (e.g., over 30 minutes or over 60 minutes) every 4 weeks.

576. The method of any of embodiments 521-575, wherein after administration, tumor volume decreases in the subject.

577. The method of any of embodiments 521-576, wherein after administration, MYC mRNA levels decrease in the subject.

578. The method of any of embodiments 521-577, wherein after administration, surface PD-L1 protein levels decrease in the subject.

579. The method of any of embodiments 521-578, wherein the targeting moiety binds to a MY C promoter.

580. The method of any of embodiments 521-579, wherein the targeting moiety binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 83,

2, 3, 75-86, 97-107, 109, 110, 190-192, or 199-202.

581. The method of any of embodiments 521-580, wherein the cancer is a hepatocellular carcinoma (HCC), Fibrolamellar Hepatocellular Carcinoma (FHCC), Cholangiocarcinoma, Angiosarcoma, or secondary liver cancer.

582. The method of any of embodiments 521-581, wherein the nucleic acid comprises an RNA, e.g., an mRNA.

583. The method of any of embodiments 521-582, wherein: the targeting moiety binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, and the expression repressor comprises the first effector moiety, wherein the effector moiety comprises a DNA methyltransferase.

584. The method of embodiment 583, wherein the targeting moiety comprises a zinc finger domain.

585. The method of embodiment 583 or 584, wherein the targeting moiety comprises an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,

3, 2, or 1 positions of difference thereto.

586. The method of any of embodiments 583 or 584, wherein the effector moiety comprises MQ1 or a functional variant or fragment thereof.

587. The method of any of embodiments 583-586, wherein the effector moiety comprises a sequence of SEQ ID NO: 19 or 87, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. 588. The method of any of embodiments 583-587, wherein the effector moiety comprises a sequence of SEQ ID NO: 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

589. The method of any of embodiments 583-588, wherein the RNA comprises a nucleotide sequence encoding the targeting moiety, wherein the nucleotide sequence encoding the targeting moiety comprises a sequence according to SEQ ID NO: 131 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

590. The method of any of embodiments 583-589, wherein the RNA comprises a nucleotide sequence encoding the effector moiety, wherein the nucleotide sequence encoding the effector moiety comprises a sequence according to SEQ ID NO: 132, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity' thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

591. The method of any of embodiments 583-589, wherein the RNA comprises a nucleotide sequence according to SEQ ID NO: 130, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

592. The method of any of embodiments 583-591, wherein the RNA further encodes a second expression repressor, wherein the second expression repressor comprises: a second targeting moiety that binds a second genomic locus, and a second effector moiety.

593. The method of embodiment 592, wherein the second targeting moiety binds a second genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 77.

594. The method of embodiments 592 or 593, wherein the second targeting moiety comprises a zinc finger domain.

595. Hie method of any of embodiments 592-594, wherein the second targeting moiety comprises an amino acid sequence according to SEQ ID NO: 7, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

596. The method of any of embodiments 592-595, wherein the second effector moiety comprises KRAB or a functional variant or fragment thereof. 597. The method of any of embodiments 592-596, wherein the second effector moiety comprises an amino acid sequence according to SEQ ID NO: 18, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

598. The method of any of embodiments 592-597, wherein the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 24 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

599. The method of any of embodiments 592-598, wherein the RNA comprises a nucleotide sequence according to SEQ ID NO: 113.

600. The method of any of embodiments 521-599, wherein the nucleic acid is formulated in lipid nanoparticles (LNPs), wherein optionally the nucleic acid is encapsulated inside of the LNPs.

601. The method of any of embodiments 521-600, wherein the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor is administered as an IV infusion, e.g., over 80-120 minutes, e.g., every 2 weeks.

602. The method of any of embodiments 521-601, wherein the nucleic acid is administered at a dose of about 0.001 mg/kg to 1.5 mg/kg or about 0.002 mg/kg to 1.5 mg/kg, e.g., administered every 1, 2, 3, 4, 5, 6 or 7 weeks.

603. The method of any of embodiments 521-602, wherein the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor and the checkpoint inhibitor polypeptide are administered on different days.

604. The method of any of embodiments 521-603, wherein the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor and the checkpoint inhibitor polypeptide are administered on the same day.

605. The method of claim 604, wherein the of the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor is administered prior to administration of the checkpoint inhibitor polypeptide.

606. Hie method of claim 604 or 605, wherein the checkpoint inhibitor polypeptide is administered between 1-24 hours after completion of administration of the nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor.

607. The method of any of the preceding claims, which results in a reduction of tumor-infiltrating Treg cells (e.g., a reduction by about 10%, 20%, 30%, 40%, or 50%) compared to administration of the immune checkpoint inhibitor polypeptide alone. The method of any of the preceding claims, which results in a reduction of CD3+CD4+CD25+CD127Lo cells (e.g., a reduction by about 10%, 20%, 30%, 40%, or 50%) in the tumor compared to administration of the immune checkpoint inhibitor polypeptide alone. The method of any of the preceding claims, which results in no increase in tumor-infiltrating Treg cells relative to levels before the administration. The method of any of the preceding claims, which results in no increase in CD3+CD4+CD25+CD127Lo cells in the tumor relative to levels before the administration. The method of any of the preceding claims, which results in an increase in tumor-infiltrating T cells relative to levels before the administration. The method of any of the preceding claims, which results in an increase in CD45+CD3+ cells in the tumor relative to levels before the administration. The method of any of the preceding claims, which results in an increase in tumor-infiltrating CD8+ T cells relative to levels before the administration. The method of any of the preceding claims, which results in an increase in CD45+CD3+CD8+ cells in the tumor relative to levels before the administration. The method of any of the preceding claims, which results in an increase in activated tumorinfiltrating CD8+ T cells relative to levels before the administration. The method of any of the preceding claims, which results in an increase in CD45+CD3+CD8+CD69+ cells in the tumor relative to levels before the administration. The method of any of the preceding claims, which results in an increase in the ratio of tumorinfiltrating CD8+ T cells to tumor-infiltrating Treg cells relative to the ratio before the administration. The method of any of the preceding claims, which results in an increase in the ratio of activated tumor-infiltrating CD 8+ T cells to tumor-infiltrating Treg cells relative to the ratio before the administration. The method of any of the preceding claims, which results in a complete remission, e.g., for at least 10, 20, 30, 40, 50, 60, or 70 days. Hie method of claim 619, wherein tire subject does not receive one of both of tire nucleic acid encoding the expression repressor or immune checkpoint inhibitor polypeptide during the period of complete remission. DEFINITIONS

A, an, the. As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

Agent: As used herein, the term “agent”, may be used to refer to a compound or entity of any chemical class including, for example, a polypeptide, nucleic acid, saccharide, lipid, small molecule, metal, or combination or complex thereof. As will be clear from context to those skilled in the art, in some embodiments, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively, or additionally, as those skilled in the art will understand in light of context, in some embodiments, the term may be used to refer to a natural product in that it is found in and/or is obtained from nature. In some embodiments, again as will be understood by those skilled in the art in light of context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some embodiments, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

Anchor Sequence: The term “anchor sequence” as used herein, refers to a nucleic acid sequence recognized by a nucleating agent that binds sufficiently to form an anchor sequence-mediated conjunction, e g., a complex. In some embodiments, an anchor sequence comprises one or more CTCF binding motifs. In some embodiments, an anchor sequence is not located within a gene coding region. In some embodiments, an anchor sequence is located within an intergenic region. In some embodiments, an anchor sequence is not located within either of an enhancer or a promoter. In some embodiments, an anchor sequence is located at least 400 bp, at least 450 bp, at least 500 bp, at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, or at least Ikb away from any transcription start site. In some embodiments, an anchor sequence is located within a region that is not associated with genomic imprinting, monoallelic expression, and/or monoallelic epigenetic marks. In some embodiments, the anchor sequence has one or more functions selected from binding an endogenous nucleating polypeptide (e.g., CTCF), interacting with a second anchor sequence to fonn an anchor sequence mediated conjunction, or insulating against an enhancer that is outside the anchor sequence mediated conjunction. In some embodiments of the present disclosure, technologies are provided that may specifically target a particular anchor sequence or anchor sequences, without targeting other anchor sequences (e.g., sequences that may contain a nucleating agent (e.g., CTCF) binding motif in a different context); such targeted anchor sequences may be referred to as the “target anchor sequence”. In some embodiments, sequence and/or activity of a target anchor sequence is modulated while sequence and/or activity of one or more other anchor sequences that may be present in the same system (e.g., in the same cell and/or in some embodiments on the same nucleic acid molecule - e.g., the same chromosome) as the targeted anchor sequence is not modulated. In some embodiments, the anchor sequence comprises or is a nucleating polypeptide binding motif. In some embodiments, the anchor sequence is adjacent to a nucleating polypeptide binding motif.

Anchor Sequence-Mediated Conjunction: The term “anchor sequence-mediated conjunction” as used herein, refers to a DNA structure, in some cases, a complex, that occurs and/or is maintained via physical interaction or binding of at least two anchor sequences in the DNA by one or more polypeptides, such as nucleating polypeptides, or one or more proteins and/or a nucleic acid entity (such as RNA or DNA), that bind the anchor sequences to enable spatial proximity and functional linkage between the anchor sequences (see, e.g. Figure 1).

Antibody molecule: As used herein, the term “antibody” or “antibody molecule” refers to a molecule that specifically binds to, or is immunologically reactive with, a particular antigen and includes at least the variable domain of a heavy chain and may also include the variable domain of a light chain of an immunoglobulin. An antibody or antibody molecule generally comprises an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term “antibody molecule” includes, for example, a monoclonal antibody (including a full-length antibody which has an immunoglobulin Fc region). In an embodiment, an antibody molecule comprises a full- length antibody, or a full-length immunoglobulin chain. In an embodiment, an antibody molecule comprises an antigen binding fragment or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. Antibodies and antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), single-domain antibodies (sdAb), epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), rlgG, single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing either a VL or VH domain, fragments produced by an Fab expression library, and anti-idiotypic (anti-Id) antibodies. Antibody molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. Moreover, unless otherwise indicated, the term “monoclonal antibody” (mAb) is meant to include both intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) that are capable of specifically binding to a target protein. Fab and F(ab')2 fragments lack the Fc fragment of an intact antibody.

Associated with: Two events or entities are “associated” with one another, as that term is used herein, if presence, level, form and/or function of one is correlated with that of the other. For example, in some embodiments, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level, form and/or function correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof. In some embodiments, a DNA sequence is “associated with” a target genomic or transcription complex when the nucleic acid is at least partially within the target genomic or transcription complex, and expression of a gene in the DNA sequence is affected by formation or disruption of the target genomic or transcription complex.

Domain: As used herein, the term “domain” refers to a section or portion of an entity . In some embodiments, a “domain” is associated with a particular structural and/or functional feature of the entity so that, when the domain is physically separated from the rest of its parent entity, it substantially or entirely retains the particular structural and/or functional feature. Alternatively or additionally, in some embodiments, a domain may be or include a portion of an entity that, when separated from that (parent) entity and linked with a different (recipient) entity, substantially retains and/or imparts on the recipient entity one or more structural and/or functional features that characterized it in the parent entity. In some embodiments, a domain is or comprises a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, polypeptide, etc.). In some embodiments, a domain is or comprises a section of a polypeptide, hi some such embodiments, a domain is characterized by a particular structural element (e.g., a particular amino acid sequence or sequence motif, alpha-helix character, beta-sheet character, coiled-coil character, random coil character, etc.), and/or by a particular functional feature (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

Effector moiety: As used herein, the term “effector moiety” refers to a domain that is capable of altering the expression of a target gene when localized to an appropriate site in the nucleus of a cell. In some embodiments, an effector moiety recruits components of the transcription machinery. In some embodiments, an effector moiety inhibits recruitment of components of transcription factors or expression repressing factors. In some embodiments, an effector moiety comprises an epigenetic modifying moiety (e.g., epigenetically modifies a target DNA sequence).

Epigenetic modifying moiety: As used herein, “epigenetic modifying moiety” refers to a domain that alters: i) the structure, e g., two dimensional structure, of chromatin; and/or ii) an epigenetic marker (e.g., one or more of DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing), when the epigenetic modifying moiety is appropriately localized to a nucleic acid (e.g., by a targeting moiety). In some embodiments, an epigenetic modifying moiety comprises an enzyme, or a functional fragment or variant thereof, that affects (e.g., increases or decreases the level of) one or more epigenetic markers. In some embodiments, an epigenetic modifying moiety comprises a DNA methyltransferase, a histone methyltransferase, CREB-binding protein (CBP), or a functional fragment of any thereof.

Expression control sequence: As used herein, the term “expression control sequence” refers to a nucleic acid sequence that increases or decreases transcription of a gene and includes (but is not limited to) a promoter and an enhancer. An “enhancing sequence” refers to a subtype of expression control sequence and increases the likelihood of gene transcription. A “silencing or repressor sequence” refers to a subtype of expression control sequence and decreases the likelihood of gene transcription.

Expression repressor: As used herein, the term “expression repressor” refers to an agent or entity with one or more functionalities that decreases expression of a target gene in a cell and that specifically binds to a DNA sequence (e.g., a DNA sequence associated with a target gene or a transcription control element operably linked to a target gene). An expression repressor comprises at least one targeting moiety and optionally one effector moiety.

Expression repression system: As used herein, the term “expression repression system” refers to a plurality of expression repressors which decrease expression of a target gene in a cell. In some embodiments, an expression repression system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor and second expression repressor (or nucleic acids encoding the first expression repressor and second expression repressor) are present together in a single composition, mixture, or phannaceutical composition, hi some embodiments, an expression repression system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor and second expression repressor (or nucleic acids encoding the first expression repressor and second expression repressor) are present in separate compositions or pharmaceutical compositions. In some embodiments, the first expression repressor and the second expression repressor are present in the same cell at the same time. In some embodiments, the first expression repressor and the second expression repressor are not present in the same cell at the same time, e.g., they are present sequentially. For example, the first expression repressor may be present in a cell for a first time period, and then the second expression repressor may be present in the cell for a second time period, wherein the first and second time periods may be overlapping or non-overlapping.

Fusion Molecule-. As used herein, the term “fusion molecule” refers to a compound comprising two or more moieties, e.g., a targeting moiety and an effector moiety, that are covalently linked. A fusion molecule and its moieties may comprise any combination of polypeptide, nucleic acid, glycan, small molecule, or other components described herein (e.g., a targeting moiety may comprise a nucleic acid and an effector moiety may comprise a polypeptide). In some embodiments, a fusion molecule is a fusion protein, e.g., comprising one or more polypeptide domains covalently linked via peptide bonds. In some embodiments, a fusion molecule is a conjugate molecule that comprises a targeting moiety and effector moiety' that are linked by a covalent bond other than a peptide bond or phosphodiester bond (e.g., a targeting moiety that comprises a nucleic acid and an effector moiety comprising a polypeptide linked by a covalent bond other than a peptide bond or phosphodiester bond). In some embodiments, an expression repressor is or comprises a fusion molecule.

Genomic complex. As used herein, the term “genomic complex” is a complex that brings together two genomic sequence elements that are spaced apart from one another on one or more chromosomes, via interactions between and among a plurality of protein and/or other components (potentially including, the genomic sequence elements). In some embodiments, the genomic sequence elements are anchor sequences to which one or more protein components of the complex binds. In some embodiments, a genomic complex may comprise an anchor sequence-mediated conjunction. In some embodiments, a genomic sequence element may be or comprise a CTCF binding motif, a promoter and/or an enhancer. In some embodiments, a genomic sequence element includes at least one or both of a promoter and/or regulatory site (e.g., an enhancer). In some embodiments, complex formation is nucleated at the genomic sequence element(s) and/or by binding of one or more of the protein component(s) to the genomic sequence element(s). As will be understood by those skilled in the art, in some embodiments, co-localization (e.g., conjunction) of the genomic sites via formation of the complex alters DNA topology at or near the genomic sequence element(s), including, in some embodiments, between them. In some embodiments, a genomic complex comprises an anchor sequence-mediated conjunction, which comprises one or more loops. In some embodiments, a genomic complex as described herein is nucleated by a nucleating polypeptide such as, for example, CTCF and/or Cohesin. In some embodiments, a genomic complex as described herein may include, for example, one or more of CTCF, Cohesin, non-coding RNA (e.g., eRNA), transcriptional machinery proteins (e.g., RNA polymerase, one or more transcription factors, for example selected from the group consisting of TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, etc.), transcriptional regulators (e.g., Mediator, P300, enhancer- binding proteins, repressor-binding proteins, histone modifiers, etc.), etc. In some embodiments, a genomic complex as described herein includes one or more polypeptide components and/or one or more nucleic acid components (e.g., one or more RNA components), which may, in some embodiments, be interacting with one another and/or with one or more genomic sequence elements (e.g., anchor sequences, promoter sequences, regulatory sequences (e.g., enhancer sequences)) so as to constrain a stretch of genomic DNA into a topological configuration (e.g., a loop) that it does not adopt when the complex is not formed.

Immune checkpoint inhibitor polypeptide: As used herein, the term “immune checkpoint inhibitor polypeptide” refers to a polypeptide that inhibits an immune checkpoint molecule. Immune checkpoint molecules can typically down-modulate or inhibit an anti-tumor immune response. Immune checkpoint molecules include, but are not limited to, Programmed Cell Death Protein 1 (PD-1), Cvtotoxic T-Lymphocyte Antigen 4 (CTLA-4), Programmed Cell Death Ligand 1 (PD-L1, also called B7H1), PD-L2, KIR, BTLA, LAG-3 and TIM-3, which directly inhibit immune cells. In some embodiments, the immune checkpoint inhibitor polypeptide comprises an antibody molecule. In some embodiments, the immune checkpoint inhibitor polypeptide comprises a plurality of polypeptide chains, for example wherein the chains are covalently linked together through disulfide bridges. In some embodiments, the immune checkpoint inhibitor polypeptide comprises a peptide.

Moiety . As used herein, the term “moiety” refers to a defined chemical group or entity with a particular structure and/or or activity, as described herein.

Modulating agent: As used herein, the term “modulating agent” refers to an agent comprising one or more targeting moieties and one or more effector moieties that is capable of altering (e.g., increasing or decreasing) expression of a target gene, e.g., MYC.

MYC: As used herein, the terms “MYC locus” refer to the portion of the human genome that encodes a MYC polypeptide (e.g., the polypeptide disclosed in NCBI Accession Number NP002458.2, or a mutant thereof), the promoter operably linked to MY C (“MY C promoter”), and the anchor sequences that form an ASMC comprising the MYC gene. In some embodiments, the MYC locus encodes a nucleic acid having NCBI Accession Number NM — 002467. In some embodiments, the MYC gene is a proto-oncogene, and in some embodiments the MYC gene is an oncogene. In certain instances, a MYC gene is found on chromosome 8, at 8q24.21 . In certain instances, a MYC gene begins at 128,816,862 bp from pter and ends at 128,822,856 bp from pter. In certain instances, a MYC gene is about 6 kb. In certain instances, a MYC gene encodes at least eight separate mRNA sequences — 5 alternatively spliced variants and 3 un-spliced variants.

Nucleic acid. As used herein, in its broadest sense, the term “nucleic acid” refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain, hi some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present disclosure. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosme, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5 -bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl- uridine, C5 -propynyl-cytidine, C5 -methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

Nucleating polypeptide. As used herein, the term “nucleating polypeptide” or “conjunction nucleating polypeptide” as used herein, refers to a protein that associates with an anchor sequence directly or indirectly and may interact with one or more conjunction nucleating polypeptides (that may interact with an anchor sequence or other nucleic acids) to form a dimer (or higher order structure) comprised of two or more such conjunction nucleating polypeptides, which may or may not be identical to one another. When conjunction nucleating polypeptides associated with different anchor sequences associate with each other so that the different anchor sequences are maintained in physical proximity with one another, the structure generated thereby is an anchor-sequence-mediated conjunction. That is, the close physical proximity of a nucleating polypeptide-anchor sequence interacting with another nucleating polypeptide-anchor sequence generates an anchor sequence-mediated conjunction (e.g., in some cases, a DNA loop), that begins and ends at the anchor sequence. As those skilled in the art, reading the present specification will immediately appreciate, terms such as “nucleating polypeptide”, “nucleating molecule”, “nucleating protein”, “conjunction nucleating protein”, may sometimes be used to refer to a conjunction nucleating polypeptide. As will similarly be immediately appreciated by those skilled in the art reading the present specification, an assembles collection of two or more conjunction nucleating polypeptides (which may, in some embodiments, include multiple copies of the same agent and/or in some embodiments one or more of each of a plurality of different agents) may be referred to as a “complex”, a “dimer” a “multimer”, etc.

Operably linked. As used herein, the phrase “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A transcription control element "operably linked" to a functional element, e.g., gene, is associated in such a way that expression and/or activity of the functional element, e.g., gene, is achieved under conditions compatible with the transcription control element. In some embodiments, "operably linked" transcription control elements are contiguous (e.g., covalently linked) with coding elements, e.g., genes, of interest: in some embodiments, operably linked transcription control elements act in trans to or otherwise at a distance from tire functional element, e.g., gene, of interest, hr some embodiments, operably linked means two nucleic acid sequences are comprised on the same nucleic acid molecule. In a further embodiment, operably linked may further mean that the two nucleic acid sequences are proximal to one another on the same nucleic acid molecule, e.g., within 1000, 500, 100, 50, or 10 base pairs of each other or directly adjacent to each other.

Peptide, Polypeptide, Protein: As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to a compound comprised of amino acid residues covalently linked by peptide bonds, or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.

Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent (e.g., a modulating agent, e.g., a disrupting agent), formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; trans-dermally; or nasally, pulmonary, and/or to other mucosal surfaces.

Proximal. As used herein, “proximal” refers to a closeness of two sites, e.g., nucleic acid sites, such that binding of an expression repressor at the first site and/or modification of the first site by an expression repressor will produce the same or substantially the same effect as binding and/or modification of the other site. For example, a targeting moiety may bind to a first site that is proximal to an enhancer (the second site), and the effector moiety associated with said targeting moiety may epigenetically modify the first site such that the enhancer’s effect on expression of a target gene is modified, substantially the same as if the second site (the enhancer sequence) had been bound and/or modified. In some embodiments, a site proximal to a target gene (e.g., an exon, intron, or splice site within the target gene), proximal to a transcription control element operably linked to the target gene, or proximal to an anchor sequence is less than 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 25 base pairs from the target gene (e.g., an exon, intron, or splice site within the target gene), transcription control element, or anchor sequence (and optionally at least 20, 25, 50, 100, 200, or 300 base pairs from the target gene (e.g., an exon, intron, or splice site within the target gene), transcription control element, or anchor sequence).

Specific: As used herein, the term “specific”, referring to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities or states. For example, an in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In some embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of the binding agent for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding agent. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding agent. In some embodiments, the agent or entity does not detectably bind to the competing alternative target under conditions of binding to its target entity. In some embodiments, binding agent binds with higher on-rate, lower off-rate, increased affinity, decreased dissociation, and/or increased stability to its target entity as compared with the competing alternative target(s).

Specific binding: As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. In some embodiments, a binding agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agent-partner complex. In some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete with an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.

Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” may therefore be used in some embodiments herein to capture potential lack of completeness inherent in many biological and chemical phenomena.

Symptoms are reduced: As used herein, the phrase “symptoms are reduced” may be used when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and/or frequency. In some embodiments, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.

Target. An agent or entity is considered to “target” another agent or entity, in accordance with the present disclosure, if it binds specifically to the targeted agent or entity under conditions in which they come into contact with one another. In some embodiments, for example, an antibody (or antigenbinding fragment thereof) targets its cognate epitope or antigen. In some embodiments, a nucleic acid having a particular sequence targets a nucleic acid of substantially complementary sequence.

Target gene: As used herein, the term “target gene” means a gene that is targeted for modulation, e.g., of expression. In some embodiments, a target gene is part of a targeted genomic complex (e.g. a gene that has at least part of its genomic sequence as part of a target genomic complex, e.g. inside an anchor sequence-mediated conjunction), which genomic complex is targeted by one or more modulating agents as described herein. In some embodiments, modulation comprises inhibition of expression of the target gene. In some embodiments, a target gene is modulated by contacting the target gene or a transcription control element operably linked to the target gene with an expression repression system, e.g., expression repressor(s), described herein. In some embodiments, a target gene is aberrantly expressed (e.g., over-expressed) in a cell, e.g., a cell in a subject (e.g., patient).

Targeting moiety. As used herein, the term “targeting moiety” means an agent or entity that specifically targets, e.g., binds, a genomic sequence element (e.g., an expression control sequence or anchor sequence). In some embodiments, the genomic sequence element is proximal to and/or operably linked to a target gene (e.g., MYC).

Therapeutic agent: As used herein, the phrase “therapeutic agent” refers to an agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a therapeutic agent comprises an expression repression system, e.g., an expression repressor, described herein. In some embodiments, a therapeutic agent comprises a nucleic acid encoding an expression repression system, e.g., an expression repressor, described herein, hi some embodiments, a therapeutic agent comprises a pharmaceutical composition described herein.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, an effective amount of a substance may vary depending on such factors as desired biological endpoint(s), substance to be delivered, target cell(s) or tissue(s), etc. For example, in some embodiments, an effective amount of compound in a formulation to treat a disease, disorder, and/or condition is an amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

Cationic lipid: As used herein, the term “cationic lipid” refers to a lipid capable of being positively charged. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Exemplary cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid may affect the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S. C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic non-bilayer structures (Hafez, I. M., et 35 al., Gene Ther 8: 1188-1196 (2001)) for the intracellular delivery of nucleic acids.

Neutral lipid: As used herein, the term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dimyristoyl-sn- glycero-3-phosphocholine (DMPC), l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidylethano lan lines such as 1,2-Dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), sphingomy-elins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

Charged lipid: As used herein, the term “charged lipid” refers to any of a number of lipid species that exist in either a positively charged or negatively charged form independent of the pH within a useful physiological range e.g., pH -3 to pH -9. Charged lipids may be synthetic or naturally derived. Examples of charged lipids include phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates, dialkyl trimethylammonium-propanes, (e.g., DOTAP, DOTMA), dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylanimoethane carbamoyl sterols (e.g. DCChol).

Lipid nanoparticle: As used herein, the term “lipid nanoparticle” refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include specified lipids. A lipid nanoparticle may also include a nucleic acid (e.g., mRNA). hi some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles comprise a nucleic acid. Such lipid nanoparticles typically comprise a nucleic acid (e g., mRNA) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The following detailed description of the embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, there are shown in the drawing embodiments, which are presently exemplified. It should be understood, however, that the disclosure is not limited to the precise arrangement and instrumentalities of the embodiments shown in the drawings.

Fig. 1 depicts a schematic representation of a dual target approach based on a durable block of the MYC promoter using a DBD fused to a DNA methyltransferase, and a transient (48/72 Hours) block of CTCF/TF sites using a DBD or a DBD fused to a short-term effector.

Fig. 2A is a bar graph showing PD-L1 mRNA expression of IFNy stimulated HCC cell line, (Hep 3B) relative to untreated control, and shows a ZF9-MQ1_ZF3-KRAB (drug substance (DS)) induced reduction in PD-L1 mRNA expression. This study was performed as described in Example 1 .

Fig. 2B is a bar graph showing PD-L1 mRNA expression of IFNy stimulated HCC cell line, (SK- HEP-1) relative to untreated control, and shows a ZF9-MQ1 ZF3-KRAB (drug substance (DS)) induced reduction in PD-L1 mRNA expression. This study was performed as described in Example 1.

Fig. 2C is a bar graph showing PD-L1 surface expression of IFNy stimulated HCC cell line, (Hep 3B) relative to untreated control, and shows a ZF9-MQ1_ZF3-KRAB (drug substance (DS)) induced reduction in PD-L1 surface expression. This study was performed as described in Example 1 . Fig. 2D is a bar graph showing PD-L1 surface expression of IFNy stimulated HCC cell line, (SK- HEP-1) relative to untreated control, and shows a ZF9-MQ1 ZF3-KRAB (drug substance (DS)) induced reduction in PD-L1 surface expression. This study was performed as described in Example 1 .

Fig. 3 A is a graph showing MY C mRNA expression in H2009 cells after 48 hour after no treatment (untreated), control (scrambled), or expression repressor (ZF9-MQ1_ZF54-KRAB). This study was performed as described in Example 2.

Fig. 3B is a graph showing PD-L1 expression in H2009 cells after 48 hour after no treatment (untreated), control (scrambled), or expression repressor (ZF9-MQ1 ZF54-KRAB). This study was performed as described in Example 2.

Fig. 4A is a graph showing MY C mRNA expression in H460 cells after 48 hour after no treatment (untreated), control (scrambled), or expression repressor (ZF9-MQ1_ZF54-KRAB). This study was performed as described in Example 2.

Fig. 4B is a graph showing PD-L1 expression in H460 cells after 48 hour after no treatment (untreated), control (scrambled), or expression repressor (ZF9-MQ1 ZF54-KRAB). This study was performed as described in Example 2.

Fig. 5 A is a bar graph showing a significant reduction in MYC mRNA expression in EIepal.6 cells at 24 and 48 hours after treatment with an expression repressor, ZF17-MQ1. This study was performed as described in Example 3.

Fig. 5B is a bar graph showing a significant increase in Hepal.6 DNA methylation 24 and 48 hours after treatment with an expression repressor, ZF-MQ 1. This study was performed as described in Example 3.

Fig. 6A is a graph showing Hepal-6 tumor volume in subcutaneous xenograft tumor model. The graph shows the expression repressor (ZF17-MQ1) reduces in vivo tumor burden in combination with immune checkpoint inhibitor polypeptides (anti-PD-1 and anti-PD-Ll antibody molecules). This study was performed as described in Example 5.

Fig. 6B is a graph showing change in body weight (BW) percent from baseline in Hepal-6 in syngeneic mouse tumor model. This study was performed as described in Example 5.

Fig. 7A is a graph showing MC38 tumor volume in subcutaneous xenograft tumor model. The graph shows the expression repressor reduces in vivo tumor burden in combination with immune checkpoint inhibitor polypeptides (anti-PD-1 and anti-PD-Ll antibody molecules). This study was performed as described in Example 6.

Fig. 7B is a graph showing change in body weight (BW) percent from baseline in MC38 subcutaneous xenograft model. This study was performed as described in Example 6. Fig. 8 is a graph showing tumor volume in LLC1 subcutaneous tumor model. The graph shows expression repressors (ZF17-MQ1) significantly inhibits tumor growth in combination with an immune checkpoint inhibitor polypeptide, an anti-PD-1 antibody molecule. This study was performed as described in Example 7.

Fig. 9 is a graph showing tumor volume in LLC1 subcutaneous tumor model. The graph shows expression repressors (ZF17-MQ1) significantly inhibits tumor growth in combination with an immune checkpoint inhibitor polypeptide, an anti-PD-Ll antibody molecule. This study was performed as described in Example 7.

Fig. 10 is a graph showing change in body weight (BW) percent from baseline in LLC1 subcutaneous tumor model. This study was performed as described in Example 7.

Fig. 11 is a graph showing tumor volume in CT26 subcutaneous tumor model. The graph shows expression repressors (ZF17-MQ1) significantly inhibits tumor growth in combination with an immune checkpoint inhibitor polypeptide, an anti-PD-1 antibody molecule. This study was performed as described in Example 8.

Fig. 12 is a graph showing tumor volume in CT26 subcutaneous tumor model. The graph shows expression repressors (ZF17-MQ1) significantly inhibits tumor growth in combination with an immune checkpoint inhibitor polypeptide, an anti-PD-Ll antibody molecule. This study was performed as described in Example 8.

Fig. 13 is a graph showing change in body weight (BW) percent from baseline in CT26 subcutaneous tumor model. This study was performed as described in Example 8.

Fig. 14 is a graph showing the decreased survival probability with treatment on Pembrolizumab. Univariate Kaplan-Meier analysis of MYC over-expression and time to progression. Time to progression entries that are greater than 99th percentile and less than 30 days have been omitted.

Fig. 15A is a graph showing tumor volume in Hepal-6 subcutaneous tumor model. The graph shows expression repressor (ZF17-MQ1) inhibits tumor growth alone or in combination with an immune checkpoint inhibitor polypeptide, an anti-PD-1 antibody molecule. This study was performed as described in Example 12.

Fig. 15B is a graph showing change in body weight (BW) percent from baseline in Hepal-6 subcutaneous tumor model. This study was performed as described in Example 12.

Fig. 16A is a plot showing the % CD45 cell population in total live cells within a Hepal-6 subcutaneous tumor. This example was performed as described in Example 12.

Fig. 16B is a plot showing the % of CD3+ cells within the CD45+ cell population within a Hepal-6 subcutaneous tumor. This example was performed as described in Example 12. Fig. 16C is a plot showing the % of CD3+CD4+ cells within the CD45+ cell population within a Hepal-6 subcutaneous tumor. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. This example was performed as described in Example 12.

Fig. 16D is a plot showing the % of CD3+CD8+ cells within the CD45+ cell population within a Hepal-6 subcutaneous tumor. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. This example was performed as described in Example 12.

Fig. 16E is a plot showing the % of CD3+CD4+CD25+CD127Lo (Treg) cells within the CD45+ cell population within a Hepal-6 subcutaneous tumor. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. This example was performed as described in Example 12.

Fig. 16F is a plot showing the % of Activated CD8 T cells (CD3+CD8+CD69+) within the CD45+ cell population within a Hepal-6 subcutaneous tumor. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. This example was performed as described in Example 12.

Fig. 16G is a plot showing the ratio of CD8+ T cells to Treg (Regulatory T cell population) within a Hepal-6 subcutaneous tumor. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. This example was performed as described in Example 12.

Fig. 16H is a plot showing the ratio of activated CD8 T cells (population of CD3+CD8+CD69+) to Treg (Regulatory T cell population) within a Hepal-6 subcutaneous tumor. *p<0.05, **p<0.005, ***p<0 001. ****p<0.0001. This example was performed as described in Example 12.

Fig. 17A is a graph showing tumor volume in Hepal-6 subcutaneous tumor model. The graph shows the effects of T-cell depletion and NK cell depletion on the efficacy of expression repressors (ZF17-MQ1). The graph shows tumor growth, in descending order at day 17, for group GFP, ZF17-MQ1 + anti-CD4, ZF17-MQ1 + anti-CD8a, ZF17-MQ1, ZF17-MQ1 + anti-NKl.l. The star indicates one outlier mouse data for ZF17-MQ1 group data were not included in the graph. This study was performed as described in Example 13.

Fig. 17B is a graph showing tumor volume in Hepal-6 subcutaneous tumor model. The graph shows the effects of T-cell depletion and NK cell depletion on the efficacy of expression repressors (ZF17-MQ1) in combination with an immune checkpoint inhibitor polypeptide, an anti-PD-1 antibody molecule. Hie star indicates one outlier mouse data for ZF17-MQ1 group data were not included in the graph. This study was performed as described in Example 13.

Fig. 18A is a graph showing tumor volume in Hepal-6 subcutaneous tumor model upon treatment with expression repressor (ZF17-MQ1), anti-PD-1 antibody molecule, or a combination thereof. This study was performed as described in Example 14.

Fig. 18B is a graph showing change in body weight (BW) percent from baseline in Hepal-6 subcutaneous tumor model. Uris study was performed as described in Example 14. Fig. 19A is a graph showing tumor volume in Hepal-6 subcutaneous tumor model upon (re)challenge with Hepal-6 cells. This study was performed as described in Example 14.

Fig. 19B is a graph showing tumor volume in Hepal-6 subcutaneous tumor model upon challenge with LL/2 cells as a control. This study was performed as described in Example 14.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present disclosure provides technologies for modulating, e.g., decreasing, expression of a target gene e.g., MYC in cell, e.g., in a subject or patient, through the use of an expression repressor or a system described herein.

Many different diseases and syndromes, including cancer, autoimmunity, cardiovascular disease, and obesity, can be caused by mis-regulation of gene expression. Particularly, overexpression of transcription factors has long been known to known to contribute to tumorigenesis, and recent studies indicate that overexpressed oncogenic transcription factors can alter the core autoregulatory circuitry of the cell.

MYC, a transcription factor and master cell regulator, is frequently dysregulated in over 50% of human cancer and plays a central role in nearly every aspect of the tumorigenic process. Except for early response genes, MYC typically upregulates gene expression. MYC is the most frequently amplified oncogene, and the elevated expression of its gene product is associated with tumor aggression and poor clinical outcome. Elevated levels of c-MYC can promote tumorigenesis in a wide range of tissues. Most tumor cells depend on the transcription factor c-MYC for their growth and proliferation. MYC overexpression is also associated in chronic liver disease e.g., viral and alcohol related liver disease. MYC overexpression level varies in specific cancer subtypes. Without wishing to be bound by theory, it is thought that modulating e g., decreasing the levels of MYC in a subject (e.g., overall, or in a specific target tissue or tissues) suffering from MYC mis-regulation disorder may lessen or eliminate the symptoms of the MY C mis-regulation disorder.

The present disclosure provides, in part, an expression repressor comprising a targeting moiety that binds to a target gene promoter, e.g., MYC promoter or operably linked to the target gene, e.g., MYC gene and an effector moiety capable of modulating (e.g., decreasing) expression of the target gene, e.g., MYC when localized by the targeting moiety. In some embodiments, the expression repressors disclosed herein specifically bind to an expression control element (e.g., a promoter or enhancer, repressor or silencer) operably linked to the target gene, e.g., MYC via the targeting moiety and the effector moiety modulates expression of the target gene, e g., MYC. In some embodiments, the expression repressors disclosed herein specifically bind to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC or to a sequence proximal to the anchor sequence via the targeting moiety and the effector moiety modulates expression of the target gene, e.g., MYC. In some embodiments, the expression repressors disclosed herein specifically bind to a genomic locus located in a super enhancer region of a target gene, e.g., MYC and the effector moiety modulates expression of the target gene, e.g., MYC.

The disclosure further provides in part, an expression repression system comprising two or more expression repressors, each comprising a targeting moiety and optionally an effector moiety. In some embodiments, the targeting moieties target two or more different sequences (e.g., each expression repressor may target a different sequence). In some embodiments, the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MYC and the second expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MYC. In some embodiments, the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to a target gene, e.g., MY C and the second expression repressor binds to an expression control element (e.g., an enhancer, a super-enhancer, a repressor, or a silencer) operably linked to a target gene, e.g., MY C. In some embodiments, the first expression repressor binds to an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene, e.g., MY C and the second expression repressor binds to an expression control element (e.g., an enhancer, a superenhancer, a repressor, or a silencer) operably linked to a target gene. Generally, modulation of expression of a target gene, e.g., MYC by an expression repression system involves the binding of the first expression repressor and second expression repressor to the first and second DNA sequences, respectively. Binding of the first and second DNA sequences localizes the functionalities of the first and second effector moieties to those sites. Without wishing to be bound by theory, in some embodiments employing the functionalities of both the first and second repressor moieties stably represses expression of a target gene associated with or comprising the first and/or second DNA sequences, e.g., wherein the first and/or second DNA sequences are or comprise sequences of the target gene or one or more operably linked transcription control elements. In some embodiments, the expression repressor system is encoded by a bi-cistronic nucleic acid sequence.

The disclosure further provides nucleic acids encoding said expression repressors and/or expression repressor systems, compositions comprising expression repressors and/or expression repressor systems, and methods for delivering said nucleic acids. Further provided are methods for increasing target gene expression, e.g., MYC gene expression in a cell using the expression repressors or expression repressor systems described herein. Expression repressors

As described herein, the present disclosure in part provides expression repressors for modulating, e.g., decreasing the expression of a target gene, e.g., MYC. In some embodiments, an expression repressor may comprise a targeting moiety that binds to a target gene promoter, e.g., MY C promoter and optionally an effector moiety. In some embodiments, the targeting moiety specifically binds a target DNA sequence, e.g., MYC DNA sequence, thereby localizing the expression repressor’s functionality to the DNA sequence. In some embodiments, an expression repressor comprises a targeting moiety and one effector moiety. In some embodiments an expression repressor comprises a targeting moiety and a plurality of effector moieties (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more effector domains (and optionally, less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 effector domains)).

An expression repressor may comprise a plurality of effector moieties, where each effector moiety comprises a different functionality than the other effector moieties. For example, an expression repressor may comprise two effector moieties, where the first effector moiety comprises DNA methylase functionality and the second effector moiety comprises a transcriptional repressor functionality. In some embodiments, an expression repressor comprises effector moieties whose functionalities are complementary to one another with regard to decreasing expression of a target gene, e.g., MYC, where the functionalities together enable inhibition of expression and, optionally, do not inhibit or negligibly inhibit expression when present individually. In some embodiments, an expression repressor comprises a plurality of effector moieties, wherein each effector moiety complements each other effector moiety, each effector moiety decreases expression of a target gene, e.g., MYC.

In some embodiments, an expression repressor comprises a combination of effector moieties whose functionalities synergize with one another with regard to decreasing expression of a target gene, e.g., MYC. Without wishing to be bound by theory, in some embodiments, epigenetic modifications to a genomic locus are cumulative, in that multiple transcription activating epigenetic markers (e.g., multiple different types of epigenetic markers and/or more extensive marking of a given type) individually together inhibit expression more effectively than individual modifications alone (e.g., producing a greater decrease in expression and/or a longer-lasting decrease in expression), hi some embodiments, an expression repressor comprises a plurality of effector moieties, wherein each effector moiety synergizes with each other effector moiety, e.g., each effector moiety decreases expression of a target gene, e.g., MYC. In some embodiments, an expression repressor (comprising a plurality of effector moieties which synergize with one another) is more effective at inhibiting expression of a target gene, e.g., MYC than an expression repressor comprising an individual effector moiety. In some embodiments, an expression repressor comprising said plurality of effector moieties is at least 1.05x (i.e., 1.05 times), l. lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 8Ox, 90x, or lOOx as effective at decreasing expression of a target gene, e.g., MYC than an expression repressor comprising an individual effector moiety.

In some embodiments, an expression repressor comprises one or more targeting moieties e.g., a Cas domain, TAL effector domain, or Zn Finger domain. In an embodiment, when an expression repressor system comprises two or more targeting moieties of the same type, e.g., two or more Cas domains, the targeting moieties specifically bind two or more different sequences. For example, in an expression repressor system comprising two or more Cas domains, the two or more Cas domains may be chosen or altered such that they only appreciably bind the gRNA corresponding to their target sequence (e.g., and do not appreciably bind the gRNA corresponding to the target of another Cas domain).

In some embodiments, an expression repressor comprises a targeting moiety and an effector moiety that are covalently linked, e.g., by a peptide bond. In some embodiments, the targeting moiety and the effector moiety are situated on the same polypeptide chain, e.g., connected by one or more peptide bonds and/or a linker. In some embodiments, the expression repressor is or comprises a fusion molecule, e.g., comprising the targeting moiety and the effector moiety linked by a peptide bond and/or a linker. In some embodiments, the expression repressor comprises a targeting moiety that is disposed N-terminal of an effector moiety on the same polypeptide chain. In some embodiments, the expression repressor comprises a targeting moiety that is disposed C-terminal of an effector moiety on the same polypeptide chain. In some embodiments, an expression repressor comprises a targeting moiety and an effector moiety that are covalently linked by a non-peptide bond. In some embodiments, a targeting moiety is conjugated to an effector moiety by a non-peptide bond. In some embodiments, an expression repressor comprises a targeting moiety and a plurality of effector moieties, wherein the targeting moiety and the plurality of effector moieties are covalently linked, e.g., by peptide bonds (e.g., the targeting moiety and plurality of effector moieties are all connected by a series of covalent bonds, although each individual moiety may not share a covalent bond with every other effector moiety).

In other embodiments, an expression repressor comprises a targeting moiety and an effector moiety that are not covalently linked, e.g., that are non-covalently associated with one another, hi some embodiments, an expression repressor comprises a targeting moiety that non-covalently binds to an effector moiety or vice versa. In some embodiments, an expression repressor comprises a targeting moiety and a plurality of effector moieties, wherein the targeting moiety and at least one effector moiety are not covalently linked, e.g., are non-covalently associated with one another, and wherein the targeting moiety and at least one other effector moiety are covalently linked, e.g., by a peptide bond. In general, an expression repressor as described herein binds (e.g., via a targeting moiety) a genomic sequence element proximal to and/or operably linked to a target gene, e.g., MYC. In some embodiments, binding of the expression repressor to the genomic sequence element modulates (e.g., decreases) expression of the target gene, e.g., MYC. For example, binding of an expression repressor comprising an effector moiety that recruits or inhibits recruitment of components of the transcription machinery to the genomic sequence element may modulate (e.g., decrease) expression of the target gene, e.g., MYC. As a further example, binding of an expression repressor comprising an effector moiety with an enzymatic activity (e.g., an epigenetic modifying moiety) may modulate (e g., decrease) expression of the target gene, e.g., MYC) through the localized enzymatic activity of the effector moiety. As a further example, both binding of an expression repressor to a genomic sequence element and the localized enzymatic activity of an expression repressor may contribute to the resulting modulation (e.g., decrease) in expression of the target gene, e.g., MYC.

In some embodiments, an expression repressor comprises an effector moiety wherein the effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB (e.g., a KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 or a functional variant or fragment thereof.

In some embodiments, an expression repressor comprises a first effector moiety and a second effector moiety , wherein the first effector moiety comprises a protein chosen from MQ 1 , DNMT1 , DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB (e g., a KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 or a functional variant or fragment thereof, and the second effector moiety comprises a different protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB (e.g., a KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 , or a functional variant or fragment thereof.

In some embodiments, the disclosure provides nucleic acid sequences encoding an expression repressor, an expression repressor system, a targeting moiety and/or an effector moiety as described herein. A skilled artisan is aware that the nucleic acid sequences of RNA are identical to the corresponding DNA sequences, except that typically thymine (T) is replaced by uracil (U). It will be understood that when a nucleotide sequence is represented by a DNA sequence (e.g., comprising, A, T, G, C), this disclosure also provides the corresponding RNA sequence (e.g., comprising, A, U, G, C) in which “U” replaces “T.” Conventional notation is used herein to describe polynucleotide sequences: the lefthand end of a single-stranded polynucleotide sequence is the 5 '-end; the left-hand direction of a doublestranded polynucleotide sequence is referred to as the 5 '-direction.

It will be appreciated by those skilled in the art that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding an expression repressor comprising targeting moiety and/or an effector moiety as described herein may be produced, some of which have similarity, e.g., 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequences disclosed herein. For instance, codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acid molecules of the invention where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide.

In some embodiments a nucleic acid cohesion encoding an expression repressor comprising a targeting moiety and/or an effector moiety may be part or all of a codon-optimized coding region, optimized according to codon usage in mammals, e.g., humans. In some embodiments, a nucleic acid cohesion encoding a targeting moiety and/or an effector moiety is codon optimized for increasing the protein expression and/or increasing the duration of protein expression. In some embodiments, a protein produced by the codon optimized nucleic acid sequence is at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% higher compared to levels of tire protein when encoded by a nucleic acid sequence that is not codon optimized.

Expression Repression Systems

Expression repression systems of the present disclosure may comprise two or more expression repressors. In some embodiments, an expression repression system comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more expression repressors (and optionally no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2). hi some embodiments, an expression repression system targets two or more different sequences (e.g., a 1^st and 2^nd, 3^rd, 4^th, 5^th, 6^th, 7^th, 8^th, 9^th, 10^th, 11^th, 12^th, and/or further DNA sequence, and optionally no more than a 20^th, 19^th, 18^th, 17^th, 16^th, 15^th. 14^th, 13^th, 12^th, l l¹¹¹, 10^th, 9^th, 8^th, 6^th, 5^th, 4^th, 3^rd, or 2^nd sequence). In some embodiments, an expression repression system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors does not detectably bind, e.g., does not bind, to another member of the plurality of expression repressors. In some embodiments, an expression repression system comprises a first expression repressor and a second expression repressor, wherein the first expression repressor does not detectably bind, e.g., does not bind, to the second expression repressor.

In some embodiments, an expression repression system of the present disclosure comprises two or more expression repressors, wherein the expression repressors are present together in a composition, pharmaceutical composition, or mixture. In some embodiments, an expression repression system of the present disclosure comprises two or more expression repressors, wherein one or more expression repressors is not admixed with at least one other expression repressor. For example, an expression repression system may comprise a first expression repressor and a second expression repressor, wherein the presence of the first expression repressor in the nucleus of a cell does not overlap with the presence of the second expression repressor in the nucleus of the same cell, wherein the expression repression system achieves a decrease in expression of a MYC gene via the non-overlapping presences of the first and second expression repressors. In some embodiments, the expression repression system achieves a greater decrease in expression of a MYC gene in comparison to the decrease in expression of a MYC gene achieved by the first or the second expression repressor alone.

In some embodiments, the expression repressors of an expression repressor system each comprise a different targeting moiety (e.g., the first, second, third, or further expression repressors each comprise different targeting moieties from one another). For example, an expression repression system may comprise a first expression repressor and a second expression repressor wherein the first expression repressor comprises a first targeting moiety (e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain), and the second expression repressor comprises a second targeting moiety (e.g., a Cas9 domain, TAL effector domain, or Zn Finger domain) different from the first targeting moiety. In some embodiments, different can mean comprising distinct types of targeting moiety, e.g., the first targeting moiety comprises a Cas9 domain, and the second DNA-targeting moiety comprises a Zn finger domain. In other embodiments, different can mean comprising distinct variants of the same type of targeting moiety, e.g., the first targeting moiety comprises a first Cas9 domain (e.g., from a first species) and the second targeting moiety comprises a second Cas9 domain (e.g., from a second species). In an embodiment, when an expression repressor system comprises two or more targeting moieties of the same type, e.g., two or more Cas9 or ZF domains, tire targeting moieties specifically bind two or more different sequences. For example, in an expression repressor system comprising two or more Cas9 domains, the two or more Cas9 domains may be chosen or altered such that they only appreciably bind the gRNA corresponding to their target sequence (e.g., and do not appreciably bind the gRNA corresponding to the target of another Cas9 domain). In a further example, in an expression repressor system comprising two or more effector moieties, the two or more effector moieties may be chosen or altered such that they only appreciably bind to their target sequence (e.g., and do not appreciably bind the target sequence of another effector moiety).

In some embodiments, an expression repressor system comprises three or more expression repressors and two or more expression repressors comprise the same targeting moiety. For example, an expression repressor system may comprise three expression repressors, wherein the first and second expression repressors both comprise a first targeting moiety and the third expression repressor comprises a second different targeting moiety. For a further example, an expression repressor system may comprise four expression repressors, wherein the first and second expression repressors both comprise a first targeting moiety and the third and fourth expression repressors comprises a second different targeting moiety'. For a further example, an expression repressor system may comprise five expression repressors, wherein the first and second expression repressors both comprise a first targeting moiety, the third and fourth expression repressors both comprise a second different targeting moiety, and the fifth expression repressor comprises a third different targeting moiety. As described above, different can mean comprising different types of -targeting moieties or comprising distinct variants of the same type of targeting moiety.

In some embodiments, the expression repressors of an expression repressor system each bind to a different DNA sequence (e.g., the first, second, third, or further expression repressors each bind DNA sequences that are different from one another). For example, an expression repression system may comprise a first expression repressor and a second expression repressor wherein the first expression repressor binds to a first DNA sequence, and the second expression repressor binds to a second DNA sequence. In some embodiments, different can mean that: there is at least one position that is not identical between the DNA sequence bound by one expression repressor and the DNA sequence bound by another expression repressor, or that there is at least one position present in the DNA sequence bound by one expression repressor that is not present in the DNA sequence bound by another expression repressor.

In some embodiments, the first DNA sequence may be situated on a first genomic DNA strand and the second DNA sequence may be situated on a second genomic DNA strand. In some embodiments, the first DNA sequence may be situated on the same genomic DNA strand as the second DNA sequence.

In some embodiments, an expression repressor system comprises three or more expression repressors and two or more expression repressors bind the same DNA sequence. For example, an expression repressor system may comprise three expression repressors, wherein the first and second expression repressors both bind a first DNA sequence, and the third expression repressor binds a second different DNA sequence. For a further example, an expression repressor system may comprise four expression repressors, wherein the first and second expression repressors both bind a first DNA sequence and the third and fourth expression repressors both bind a second DNA sequence. For a further example, an expression repressor system may comprise five expression repressors, wherein the first and second expression repressors both bind a first DNA sequence, the third and fourth expression repressors both bind a second DNA sequence, and the fifth expression repressor binds a third DNA sequence. As described above, different can mean that there is at least one position that is not identical between the DNA sequence bound by one expression repressor and the DNA sequence bound by another expression repressor, or that there is at least one position present in the DNA sequence bound by one expression repressor that is not present in the DNA sequence bound by another expression repressor.

In some embodiments, an expression repression system comprises two or more (e.g., two) expression repressors and a plurality (e.g., two) of the expression repressors comprise targeting moieties that bind to different DNA sequences. In such embodiments, a first targeting moiety may bind to a first DNA sequence and a second DNA-targeting moiety may bind to a second DNA sequence, wherein the first and the second DNA sequences are different and do not overlap. In some such embodiments, the first DNA sequence is separated from the second DNA sequence by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs (and optionally, no more than 500, 400, 300, 200, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50 base pairs). In some such embodiments, the first DNA sequence is separated from the second DNA sequence by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs (and optionally, no base pairs, e g., the first and second sequence are directly adjacent one another).

In some embodiments, the expression repressors of an expression repressor system each comprise a different effector moiety (e.g., the first, second, third, or further expression repressors each comprise a different effector moiety from one another). For example, an expression repression system may comprise a first expression repressor and a second expression repressor wherein the first expression repressor comprises a first effector moiety (e.g., comprising a DNA methyltransferase or functional fragment thereof), and the second expression repressor comprises a second effector moiety (e.g., comprising a transcription repressor (e.g., KRAB) or functional fragment thereof) different from the first effector moiety'. In some embodiments, different can mean comprising distinct types of effector moiety. In other embodiments, different can mean comprising distinct variants of the same type of effector moiety, e.g., the first effector moiety comprises a first DNA methyltransferase (e g., having a first site specificity or amino acid sequence) and the second effector moiety comprises a second DNA methyltransferase (e.g., having a second site specificity or amino acid sequence).

In some embodiments, an expression repressor system comprises a first expression repressor comprising a first effector moiety and a second expression repressor comprising a second effector moiety, wherein the first effector moiety comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 or a functional variant or fragment thereof, and the second effector moiety comprises a different protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12 , or a functional variant or fragment thereof.

In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity (e.g, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L , or a functional variant or fragment of any thereof, and the other effector moiety comprises a transcription repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any thereof), the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a histone deacetylase activity (e.g, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any thereof). In some embodiments, the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a DNA methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof). In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a transcription repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, F0G1, SUZ12, or a functional variant or fragment of any thereof). In some embodiments, the first or second effector moiety comprises a transcription repressor activity and the other effector moiety comprises a different transcription repressor activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises the same DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a histone deacetylase activity. In some embodiments, the first or second effector moiety' comprises a histone demethylase activity and the other effector moiety comprises a DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a histone methyltransferase activity and the other effector moiety comprises a DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments, the first or second effector moiety' comprises a histone demethylase activity and the other effector moiety comprises a different histone demethylase activity. In some embodiments, the first or second effector moiety comprises a histone demethylase activity and the other effector moiety comprises the same histone demethylase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a DNA demethylase activity. In some embodiments, the first or second effector moiety' comprises a histone deacetylase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises a different histone deacetylase activity. In some embodiments, the first or second effector moiety comprises a histone deacetylase activity and the other effector moiety comprises the same histone deacetylase activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises a transcription repressor activity, hi some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises a different DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a DNA methyltransferase activity and the other effector moiety comprises the same DNA methyltransferase activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises a transcription repressor activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises a different DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a DNA demethylase activity and the other effector moiety comprises the same DNA demethylase activity. In some embodiments, the first or second effector moiety comprises a transcription repressor activity and the other effector moiety comprises a different transcription repressor activity. In some embodiments, the first or second effector moiety comprises a transcription repressor activity and the other effector moiety comprises the same transcription repressor activity.

In some embodiments, an expression repressor system comprises three or more expression repressors and two or more expression repressors comprise the same DNA-targeting moiety. For example, an expression repressor system may comprise three expression repressors, wherein the first and second expression repressors both comprise a first effector moiety and the third expression repressor comprises a second different effector moiety. For a further example, an expression repressor system may comprise four expression repressors, wherein the first and second expression repressors both comprise a first effector moiety and the third and fourth expression repressors comprises a second different effector moiety. For a further example, an expression repressor system may comprise five expression repressors, wherein the first and second expression repressors both comprise a first effector moiety, the third and fourth expression repressors both comprise a second different effector moiety, and the fifth expression repressor comprises a third different effector moiety. As described above, different can mean comprising different types of effector moiety or comprising distinct variants of the same type of effector moiety.

In some embodiments, two or more (e.g., all) expression repressors of an expression repressor system are not covalently associated with each other, e.g., each expression repressor is not covalently associated with any other expression repressor. In another embodiment, two or more expression repressors of an expression repressor system are covalently associated with one another. In an embodiment, an expression repression system comprises a first expression repressor and a second expression repressor disposed on the same polypeptide, e.g., as a fusion molecule, e g., connected by a peptide bond and optionally a linker. In some embodiments, the peptide is a self-cleaving peptide, e.g., a T2A self-cleaving peptide. In an embodiment, an expression repression system comprises a first expression repressor and a second expression repressor that are connected by a non-peptide bond, e.g., are conjugated to one another.

Linkers

An expression repressor or an expression repressor system as disclosed herein may comprise one or more linkers. A linker may connect a targeting moiety to an effector moiety, an effector moiety to another effector moiety, or a targeting moiety to another targeting moiety. A linker may be a chemical bond, e.g., one or more covalent bonds or non-covalent bonds, hi some embodiments, a linker is covalent. In some embodiments, a linker is non-covalent. In some embodiments, a linker is a peptide linker. Such a linker may be between 2-30, 5-30, 10-30, 15-30, 20-30, 25-30, 2-25, 5-25, 10-25, 15-25, 20-25, 2-20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2-10, 5-10, or 2-5 amino acids in length, or greater than or equal to 2, 5, 10, 15, 20, 25, or 30 amino acids in length (and optionally up to 50, 40, 30, 25, 20, 15, 10, or 5 amino acids in length). In some embodiments, a linker can be used to space a first domain or moiety from a second domain or moiety, e.g., a DNA-targeting moiety from an effector moiety. In some embodiments, for example, a linker can be positioned between a DNA-targeting moiety and an effector moiety, e.g., to provide molecular flexibility of secondary and tertiary structures. A linker may comprise flexible, rigid, and/or cleavable linkers described herein. In some embodiments, a linker includes at least one glycine, alanine, and serine amino acids to provide for flexibility. In some embodiments, a linker is a hydrophobic linker, such as including a negatively charged sulfonate group, polyethylene glycol (PEG) group, or pyrophosphate diester group. In some embodiments, a linker is cleavable to selectively release a moiety (e.g., polypeptide) from a modulating agent, but sufficiently stable to prevent premature cleavage.

In some embodiments, one or more moieties and/or domains of an expression repressor described herein are linked with one or more linkers. In some embodiments, an expression repression may comprise a linker situated between the targeting moiety and the effector moiety. In some embodiments, the linker may have a sequence of ASGSGGGSGGARD (SEQ ID NO: 137), or ASGSGGGSGG (SEQ ID NO: 138). In some embodiments, a system comprising a first and second repressor may comprise a first linker situated between the first targeting moiety and the first effector moiety, and a second linker situated between the second targeting moiety and the second effector moiety. In some embodiments, the first and the second linker may be identical. In some embodiments, the first and the second linker may be different. In some embodiments, the first linker may comprise an amino acid sequence according to SEQ ID NO: 137 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto and the second linker may comprise an amino acid sequence according to SEQ ID NO: 138 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto.

As will be known by one of skill in the art, commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). Flexible linkers may be useful for joining domains/moieties that require a certain degree of movement or interaction and may include small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain the stability of a linker in aqueous solutions by forming hydrogen bonds with water molecules, and therefore reduce unfavorable interactions between a linker and moieties/domains. In some embodiments, the linker is a GS linker or a variant thereof e.g., G4S (SEQ ID NO: 207). Rigid linkers are useful to keep a fixed distance between domains/moieties and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of domains is critical to preserve the stability or bioactivity of one or more components in the fusion. Rigid linkers may have an alpha helix-structure or Pro-rich sequence. (XP)_n, with X designating any amino acid, preferably Ala, Lys, or Glu.

Cleavable linkers may release free functional domains in vivo. In some embodiments, linkers may be cleaved under specific conditions, such as presence of reducing reagents or proteases. In vivo cleavable linkers may utilize reversible nature of a disulfide bond. One example includes a thrombinsensitive sequence (e.g., PRS) between the two Cys residues. In vitro thrombin treatment of CPRSC results in the cleavage of a thrombin-sensitive sequence, while a reversible disulfide linkage remains intact. Such linkers are known and described, e.g., in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369. In vivo cleavage of linkers in fusions may also be carried out by proteases that are expressed in vivo under certain conditions, in specific cells or tissues, or constrained within certain cellular compartments. Specificity of many proteases offers slower cleavage of the linker in constrained compartments. In some embodiment, the cleavable linker may be a self-cleaving linker, e.g., a T2A peptide linker. In some embodiments, the linker may comprise a “ribosome skipping” sequence, e.g., a tPT2A linker.

Examples of molecules suitable for use in linkers described herein include a negatively charged sulfonate group; lipids, such as a poly (— CH2-) hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturated variants thereof, hydroxylated variants thereof, amidated or otherwise N-containing variants thereof; noncarbon linkers; carbohydrate linkers; phosphodiester linkers, or other molecule capable of covalently linking two or more components of an expression repressor. Non-covalent linkers are also included, such as hydrophobic lipid globules to which the polypeptide is linked, for example through a hydrophobic region of a polypeptide or a hydrophobic extension of a polypeptide, such as a series of residues rich in leucine, isoleucine, valine, or perhaps also alanine, phenylalanine, or even tyrosine, methionine, glycine, or other hydrophobic residues. Components of an expression repressor may be linked using charge-based chemistry, such that a positively charged component of an expression repressor is linked to a negative charge of another component.

Targeting Moieties

The present disclosure provides, e.g., expression repressors comprising a targeting moiety that specifically targets, e.g., binds, a genomic sequence element (e.g., a promoter, a TSS, or an anchor sequence) in, proximal to, and/or operably linked to a target gene. Targeting moieties may specifically bind a DNA sequence, e.g., a DNA sequence associated with a target gene, e.g., MYC. Any molecule or compound that specifically binds a DNA sequence may be used as a targeting moiety.

In some embodiments, a targeting moiety targets, e.g., binds, a component of a genomic complex (e.g., ASMC). In some embodiments, a targeting moiety targets, e.g., binds, an expression control sequence (e.g., a promoter or enhancer) operably linked to a target gene. In some embodiments, a targeting moiety targets, e g., binds, a target gene or a part of a target gene. The target of a targeting moiety' may be referred to as its targeted component. A targeted component may be any genomic sequence element operably linked to a target gene, or the target gene itself, including but not limited to a promoter, enhancer, anchor sequence, exon, intron, UTR encoding sequence, a splice site, or a transcription start site. In some embodiments, a targeting moiety binds specifically to one or more target anchor sequences (e g., within a cell) and not to non-targeted anchor sequences (e g., within the same cell).

In some embodiments, a targeting moiety may be or comprise a CRISPR/Cas domain, a TAL effector domain, a Zn finger domain, peptide nucleic acid (PNA) or a nucleic acid molecule. In some embodiments, an expression repressor comprises one effector moiety. In some embodiments, an expression repressor comprises a plurality of targeting moieties, wherein each targeting moiety does not detectably bind, e.g., does not bind, to another targeting moiety. In some embodiments, an expression repression system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors comprises a targeting moiety, wherein each targeting moiety does not detectably bind, e.g., does not bind, to another targeting moiety. In some embodiments, an expression repression system comprises a first expression repressor comprising a first targeting moiety and a second expression repressor comprising a second targeting moiety, wherein the first targeting moiety does not detectably bind, e.g., does not bind, to the second targeting moiety. In some embodiments, an expression repression system comprises a first expression repressor comprising a first targeting moiety and a second expression repressor comprising a second targeting moiety, wherein the first targeting moiety does not detectably bind, e.g., does not bind, to another first targeting moiety, and the second targeting moiety does not detectably bind, e.g., does not bind, to another second targeting moiety. In some embodiments, a targeting moiety for use in the compositions and methods described herein is functional (e.g., binds to a DNA sequence) in a monomeric, e.g., non-dimeric, state.

In some embodiments, binding of a targeting moiety to a targeted component decreases binding affinity of the targeted component for another transcription factor, genomic complex component, or genomic sequence element. In some embodiments, a targeting moiety binds to its target sequence with a K_D of less than or equal to 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.002, or 0.001 nM (and optionally, a KD of at least 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.002, or 0.001 nM). In some embodiments, a targeting moiety binds to its target sequence with a KD of 0.001 nM to 500 nM, e.g., 0.1 nM to 5 nM, e.g., about 0.5 nM. In some embodiments, a targeting moiety binds to a non-target sequence with aKc of at least 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, or 100,000 nM (and optionally, does not appreciably bind to a non-target sequence). In some embodiments, a targeting moiety does not bind to a non-target sequence.

In some embodiments, a targeting moiety comprises a nucleic acid sequence complementary to a targeted component, e.g., a regulatory element (e.g., promoter or enhancer) of a target gene, e.g., MYC. In some embodiments, a targeting moiety comprises a nucleic acid sequence that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% complementary to a targeted component.

In some embodiments, a targeting moiety may be or comprise a CRISPR/Cas domain, a TAL effector domain, a Zn finger domain, or a nucleic acid molecule.

In some embodiments, the targeting moiety of an expression repressor comprises no more than 100, 90, 80, 70, 60, 50, 40, 30, or 20 nucleotides (and optionally at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides). In some embodiments, an expression repressor or the effector moiety of a fusion molecule, comprises no more than 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 amino acids (and optionally at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or 1900 amino acids). In some embodiments, an expression repressor or the effector moiety of a fusion molecule, comprises 100- 2000, 100-1900, 100-1800, 100-1700, 100-1600, 100-1500, 100-1400, 100-1300, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-2000, 200- 1900, 200-1800, 200-1700, 200-1600, 200-1500, 200-1400, 200-1300, 200-1200, 200-1100, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-2000, 300-1900, 300-1800, 300- 1700, 300-1600, 300-1500, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-900, 300-800, 300- 700, 300-600, 300-500, 300-400, 400-2000, 400-1900, 400-1800, 400-1700, 400-1600, 400-1500, 400- 1400, 400-1300, 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500- 2000, 500-1900, 500-1800, 500-1700, 500-1600, 500-1500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-2000, 600-1900, 600-1800, 600-1700, 600-1600, 600-1500, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800, 600-700, 700-2000, 700-1900, 700-1800, 700-1700, 700-1600, 700-1500, 700-1400, 700-1300, 700-1200, 700-1100, 700- 1000, 700-900, 700-800, 800-2000, 800-1900, 800-1800, 800-1700, 800-1600, 800-1500, 800-1400, 800- 1300, 800-1200, 800-1100, 800-1000, 800-900, 900-2000, 900-1900, 900-1800, 900-1700, 900-1600, 900-1500, 900-1400, 900-1300, 900-1200, 900-1100, 900-1000, 1000-2000, 1000-1900, 1000-1800, 1000-1700, 1000-1600, 1000-1500, 1000-1400, 1000-1300, 1000-1200, 1000-1100, 1100-2000, 1100- 1900, 1100-1800, 1100-1700, 1100-1600, 1100-1500, 1100-1400, 1100-1300, 1100-1200, 1200-2000, 1200-1900, 1200-1800, 1200-1700, 1200-1600, 1200-1500, 1200-1400, 1200-1300, 1300-2000, 1300- 1900, 1300-1800, 1300-1700, 1300-1600, 1300-1500, 1300-1400, 1400-2000, 1400-1900, 1400-1800, 1400-1700, 1400-1600, 1400-1500, 1500-2000, 1500-1900, 1500-1800, 1500-1700, 1500-1600, 1600- 2000, 1600-1900, 1600-1800, 1600-1700, 1700-2000, 1700-1900, 1700-1800, 1800-2000, 1800-1900, or 1900-2000 amino acids.

An expression repressor or a system comprising an expression repressor as disclosed herein, may comprise nucleic acid, e.g., one or more nucleic acids. The term “nucleic acid” refers to any compound that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, “nucleic acid” refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is or comprises more than 50% ribonucleotides and is referred to herein as a ribonucleic acid (RNA). In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more “peptide nucleic acids”, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively, or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 - methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5 -bromouridine, C5 -fluorouridine, C5 -iodouridine, C5 -propynyl-uridine, C5 -propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof), hi some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2 ’-fluororibose, ribose, 2 ’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. As used herein, “recombinant” when used to describe a nucleic acid refers to any nucleic acid that does not naturally occur. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, nucleic acids may have a length from about 2 to about 5000 nts, about 10 to about 100 nts, about 50 to about 150 nts, about 100 to about 200 nts, about 150 to about 250 nts, about 200 to about 300 nts, about 250 to about 350 nts, about 300 to about 500 nts, about 10 to about 1000 nts, about 50 to about 1000 nts, about 100 to about 1000 nts, about 1000 to about 2000 nts, about 2000 to about 3000 nts, about 3000 to about 4000 nts, about 4000 to about 5000 nts, or any range therebetween. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

In some embodiments, the targeting moiety comprises or is a nucleic acid sequence, a protein, protein fusion, or a membrane translocating polypeptide. In some embodiments, the targeting moiety is selected from an exogenous conjunction nucleating molecule, a nucleic acid encoding the conjunction nucleating molecule, or a fusion of a sequence targeting polypeptide and a conjunction nucleating molecule. The conjunction nucleating molecule may be, e g., CTCF, cohesin, USF1, YY1, TATA-box binding protein associated factor 3 (TAF3), ZNF143 binding motif. In some embodiments, a targeting moiety comprises or is a polymer or polymeric moiety , e.g., a polymer of nucleotides (such as an oligonucleotide), a peptide nucleic acid, a peptide-nucleic acid mixmer, a peptide or polypeptide, a polyamide, a carbohydrate, etc.

In some embodiments, a targeting moiety comprises or is nucleic acid. In some embodiments, an effector moiety comprises or is nucleic acid. In some embodiments, a nucleic acid that may be included in a moiety may be or comprise DNA, RNA, and/or an artificial or synthetic nucleic acid or nucleic acid analog or mimic. For example, in some embodiments, a nucleic acid may be or include one or more of genomic DNA (gDNA), complementary DNA (cDNA), a peptide nucleic acid (PNA), a peptide-nucleic acid mixmer, a peptide- oligonucleotide conjugate, a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a polyamide, a triplex- forming oligonucleotide, an antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA or other RNAi molecule (e.g., that targets a non-coding RNA as described herein and/or that targets an expression product of a particular gene associated with a targeted genomic complex as described herein), etc. A nucleic acid sequence suitable for use in a modulating agent may include modified oligonucleotides (e.g., chemical modifications, such as modifications that alter backbone linkages, sugar molecules, and/or nucleic acid bases) and/or artificial nucleic acids. In some embodiments, a nucleic acid sequence includes, but is not limited to, genomic DNA, cDNA, peptide nucleic acids (PNA) or peptide oligonucleotide conjugates, locked nucleic acids (LNA), bridged nucleic acids (BNA), polyamides, triplex forming oligonucleotides, modified DNA, antisense DNA oligonucleotides, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNA or DNA molecules. In some embodiments, a nucleic acid may include one or more residues that is not a naturally -occurring DNA or RNA residue, may include one or more linkages that is/are not phosphodiester bonds (e.g., that may be, for example, phosphorothioate bonds, etc.), and/or may include one or more modifications such as, for example, a 2’0 modification such as 2’-0meP. A variety of nucleic acid structures useful in preparing synthetic nucleic acids is known in the art (see, for example, WO2017/0628621 and W02014/012081) those skilled in the art will appreciate that these may be utilized in accordance with the present disclosure.

Some examples of nucleic acids include, but are not limited to, a nucleic acid that hybridizes to an target gene, e.g., MYC, (e.g., gRNA or antisense ssDNA as described herein elsewhere), a nucleic acid that hybridizes to an exogenous nucleic acid such as a viral DNA or RNA, nucleic acid that hybridizes to an RNA, a nucleic acid that interferes with gene transcription, a nucleic acid that interferes with RNA translation, a nucleic acid that stabilizes RNA or destabilizes RNA such as through targeting for degradation, a nucleic acid that interferes with a DNA or RNA binding factor through interference of its expression or its function, a nucleic acid that is linked to a intracellular protein or protein complex and modulates its function, etc.

In some embodiments, an expression repressor comprises one or more nucleoside analogs. In some embodiments, a nucleic acid sequence may include in addition or as an alternative to one or more natural nucleosides, e.g., purines or pyrimidines, e.g., adenine, cytosine, guanine, thymine and uracil, one or more nucleoside analogs. In some embodiments, a nucleic acid sequence includes one or more nucleoside analogs. A nucleoside analog may include, but is not limited to, a nucleoside analog, such as 5 -fluorouracil; 5 -bromouracil, 5 -chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 4- m ethylbenzimidazole, 5 -(carboxyhydroxylmethyl) uracil, 5 -carboxymethylaminomethyl -2 -thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, dihydrouridine, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1 -metlrylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2 -methyladenine, 2- methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2 -thiouracil, beta-D-mannosylqueosine, 5’- mctlioxycarbox mcthyluraci I. 5 -methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5 -oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosinc. 5 -methy 1-2 -thiouracil, 2-thiouracil, 4- thiouracil, 5 -methyluracil, uracil-5-oxyacetic acid methylester, uracil-5 -oxyace tic acid (v), 5-methyl-2- thiouracil, 3-(3-amino-3-N-2 -carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine, 3 -nitropyrrole, inosine, thiouridine, queuosine, wyosine, diaminopurine, isoguanine, isocytosine, diaminopyrimidine, 2,4- difluorotoluene, isoquinoline, pyrrolo[2,3-P]pyridine, and any others that can base pair with a purine or a pyrimidine side chain.

CRISPR/Cas Domains

In some embodiments, a targeting moiety is or comprises a CRISPR/Cas domain. A CRISPR/Cas protein can comprise a CRISPR/Cas effector and optionally one or more other domains. A CRISPR/Cas domain typically has structural and/or functional similarity to a protein involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, e g., a Cas protein. The CRISPR/Cas domain optionally comprises a guide RNA, e.g., single guide RNA (sgRNA). In some embodiments, the gRNA comprised by the CRISPR/Cas domain is noncovalently bound by the CRISPR/Cas domain.

CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e. g., Cas9 or Cpfl) to cleave foreign DNA. For example, in a typical CRISPR/Cas system, an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences. Three classes (I-III) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). The crRNA contains a “guide RNA”, typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence. crRNA also contains a region that binds to the tracrRNA to fonn a partially double-stranded structure which is cleaved by Rnase III, resulting in a crRNA/tracrRNA hybrid. A crRNA/tracrRNA hybrid then directs Cas9 endonuclease to recognize and cleave a target DNA sequence. A target DNA sequence must generally be adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease; however, PAM sequences appear throughout a given genome. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5’-NGG (Streptococcus pyogenes), 5’-NNAGAA (Streptococcus thermophilus CRISPR1), 5’-NGGNG (Streptococcus thermophilus CRISPR3), and 5’- NNNGATT (Neisseria meningiditis). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5’-NGG, and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5’ from) the PAM site. Another class II CRISPR system includes the type V endonuclease Cpfl, which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.). Cpfl -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpfl system requires only Cpfl nuclease and a crRNA to cleave a target DNA sequence. Cpfl endonucleases, are associated with T-rich PAM sites, e. g., 5’-TTN. Cpfl can also recognize a 5’-CTA PAM motif. Cpfl cleaves atarget DNA by introducing an offset or staggered double-strand break with a 4- or 5 -nucleotide 5' overhang, for example, cleaving atarget DNA with a 5 -nucleotide offset or staggered cut located 18 nucleotides downstream from (3 ’ from) from a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5 -nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt- end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759 - 771.

A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method. Specific examples of Cas proteins include class II systems including Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Cpfl, C2C1, or C2C3. In some embodiments, a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species. In some embodiments a particular Cas protein, e.g., a particular Cas9 protein, is selected to recognize a particular protospacer-adjacent motif (PAM) sequence. In some embodiments, a DNA-targeting moiety includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9. In certain embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In certain embodiments, a Cas protein may be from a gram-positive bacteria or a gram-negative bacteria. In certain embodiments, a Cas protein may be from a Streptococcus (e.g., a S. pyogenes, or a S. thermophilus), a Francisella (e.g., an F. novicida), a Staphylococcus (e.g., an S. aureus), an Acidaminococcus (e.g., an Acidaminococcus sp. BV3L6), a Neisseria (e.g., an N. meningitidis), a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter.

In some embodiments, a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and/or function. In some embodiments, the PAM is or comprises, from 5’ to 3’, NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT, where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G. hr some embodiments, a Cas protein is a protein listed in Table 1. In some embodiments, a Cas protein comprises one or more mutations altering its PAM. In some embodiments, a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises DI 135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R, K548V, and N 2R mutations or analogous substitutions to the amino acids corresponding to said positions.

Table 1

In some embodiments, the Cas protein is modified to deactivate the nuclease, e.g., nuclease- deficient Cas. In some embodiments, the Cas protein is a Cas9 protein. Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA, a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 generates only a single-strand break; a catalyticalh inactive Cas9 (“dCas9”) does not cut target DNA. In some embodiments, dCas binding to a DNA sequence may interfere with transcription at that site by steric hindrance. In some embodiments, a DNA-targeting moiety is or comprises a catalytically inactive Cas, e.g., dCas. Many catalytically inactive Cas proteins are known in the art. In some embodiments, dCas9 comprises mutations in each endonuclease domain of tire Cas protein, e.g., D10A and H840A mutations.

In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a DI 1A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a H969A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises aN995A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises DI 1A, H969A, and N995A mutations or analogous substitutions to the amino acids corresponding to said positions.

In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a H557A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises D10A and H557A mutations or analogous substitutions to the amino acids corresponding to said positions.

In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D839A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a H840A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises aN863A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises D10A, D839A, H840A, and N863A mutations or analogous substitutions to the amino acids corresponding to said positions.

In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a E993A mutation or an analogous substitution to the amino acid corresponding to said position.

In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D917A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a E1006A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D1255A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises D917A, E1006A, and D1255A mutations or analogous substitutions to the amino acids corresponding to said positions.

In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D587A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a H588A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a N611A mutation or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises D16A, D587A, H588A, and N611A mutations or analogous substitutions to the amino acids corresponding to said positions.

In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) targeting moiety and one or more effector moiety, wherein the one or more targeting moiety is or comprises a CRISPR/Cas domain comprising a Cas protein, e.g., catalytically inactive Cas9 protein, e.g., dCas9, or a functional variant or fragment thereof, hr some embodiments, dCas9 comprises an amino acid sequence of SEQ ID NO: 17:

DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVA YHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL

ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGV EDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK

QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSR ERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVAAIV

PQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER

GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNIKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKAT AKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE

VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY

NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGD (SEQ ID NO: 17)

In some embodiments, the dCas9 is encoded by a nucleic acid sequence of SEQ ID NO: 50:

GACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGGGCCGT

GATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGG

CACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGG

CCACCCGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTA

CCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTG

GAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACA

TCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCT

GGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATC AAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACA AGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCC

AGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGA

ACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCT GAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTG CAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACC AGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATC

CTGCGGGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACG

ACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAA

GTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGC

GCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCG

AGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAA

CGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAG

GACTTCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGAT

CCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGAAAT

CCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCA

GAGCTTCATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCC

AAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACG

TGACCGAGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGA

CCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAG

AAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCC

TGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGA

GAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATG

ATCGAGGAGCGGCTGAAAACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGA

AGCGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGA

CAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAATCCGACGGCTTCGCCAACCGGAAC

TTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGG

TGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCAT

CAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGG

CACAAGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCC

AGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCAGCC

AGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTA

CTACCTGCAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGAGC

GACTACGACGTGGCCGCCATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACA

AGGTGCTGACCCGGAGCGACAAGGCCCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAGG

TGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCG

GAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGC

TTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGG

ACAGCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGA

TCACCCTGAAATCCAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGG GAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCC TGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGA CGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTC TTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCG GAAGCGGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCG GGACTTCGCCACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAAACC GAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAACAGCGACAAGC TGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGT GGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAATCC GTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCG ACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAA GTACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCGGCGAGCTG CAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCC ACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGC AGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGAT CCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCC ATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGC CGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCAGCACCAAGGAGGTG CTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCTGA GCCAGCTGGGCGGCGAC (SEQ ID NO: 50)

In some embodiments, a targeting moiety may comprise a Cas domain comprising or linked (e.g., covalently) to a gRNA. A gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas-protein binding and a user-defined ~20 nucleotide targeting sequence for a genomic target. In practice, guide RNA sequences are generally designed to have a length of between 17 - 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to the targeted nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. Gene editing has also been achieved using a chimeric “single guide RNA” (“sgRNA”), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective for use with Cas proteins; see, for example, Hendel et al. (2015) Nature Biotechnol., 985 - 991. The exemplary guide RNA sequences arc disclosed in Table 2 and Table 3. In some embodiments, a gRNA comprises a nucleic acid sequence that is complementary to a DNA sequence associated with a target gene. In some embodiments, the DNA sequence is, comprises, or overlaps an expression control element that is operably linked to the target gene. In some embodiments, a gRNA comprises a nucleic acid sequence that is at least 90, 95, 99, or 100% complementary’ to a DNA sequence associated with a target gene. In some embodiments, a gRNA for use with a DNA-targeting moiety that comprises a Cas molecule is an sgRNA.

In some embodiments, a gRNA for use with a CRISPR/Cas domain specifically binds a target sequence associated with CTCF. In some embodiments, a gRNA for use with a CRISPR/Cas domain specifically binds a target sequence associated with the promoter. In some embodiments, the gRNA binds a target sequence listed in Table 2 or Table 3. In some embodiments, an expression repressor described herein binds to a target sequence listed in Table 2 or Table 3.

Table 2: Exemplary gRNA sequences

Table 3: Exemplary gRNA sequences

In some embodiments, an expression repression system comprises a first expression repressor comprising a first DNA-targeting moiety and a second expression repressor comprising a second DNA- targeting moiety, wherein the first DNA-targeting moiety comprises or is a first CRISPR/Cas domain and the second DNA-targeting moiety comprises or is a second CRISPR/Cas domain. In some embodiments, the first CRISPR/Cas domain comprises a first CRISPR/Cas protein and first guide RNA, and the second CRISPR/Cas domain comprises a second CRISPR/Cas protein and a second guide RNA. In some embodiments, the first CRISPR/Cas protein does not appreciably bind (e.g., does not bind) the second guide RNA, e.g., binds with a KD of at least 10, 20, 50, 100, 1000, or 10,000 nM, and the second CRISPR/Cas protein does not appreciably bind (e.g., does not bind) the first guide RNA, e.g., binds with a KD of at least 10, 20, 50, 100, 1000, or 10,000 nM.

TAL Effector domains

In some embodiments, a DNA-targeting moiety is or comprises a TAL effector domain. A TAL effector domain, e.g., a TAL effector domain that specifically binds a DNA sequence, comprises a plurality of TAL effector repeats or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effector repeats (e.g., N- and/or C-terminal of the plurality of TAL effector domains) wherein each TAL effector repeat recognizes a nucleotide. A TAL effector protein can comprise a TAL effector domain and optionally one or more other domains. Many TAL effector domains are known to those of skill in the art and are commercially available, e.g., from Thermo Fisher Scientific.

TALEs are natural effector proteins secreted by numerous species of bacterial pathogens including the plant pathogen Xanthomonas which modulates gene expression in host plants and facilitates bacterial colonization and survival. The specific binding of TAL effectors is based on a central repeat domain of tandemly arranged nearly identical repeats of typically 33 or 34 amino acids (the repeatvariable di-residues, RVD domain).

Members of the TAL effectors family differ mainly in the number and order of their repeats. The number of repeats ranges from 1.5 to 33.5 repeats and the C-terminal repeat is usually shorter in length (e.g., about 20 amino acids) and is generally referred to as a “half-repeat”. Each repeat of the TAL effector features a one-repeat-to-one-base-pair correlation with different repeat types exhibiting different base-pair specificity (one repeat recognizes one base-pair on the target gene sequence). Generally, the smaller the number of repeats, the weaker the protein-DNA interactions. A number of 6.5 repeats has been shown to be sufficient to activate transcription of a reporter gene (Scholze et al., 2010).

Repeat to repeat variations occur predominantly at amino acid positions 12 and 13, which have therefore been termed “hypervariable” and which are responsible for the specificity of the interaction with the target DNA promoter sequence, as shown in Table 4 listing exemplary repeat variable di-residues (RVD) and their correspondence to nucleic acid base targets.

Table 4: RVDs and Nucleic Acid Base Specificity

Accordingly, it is possible to modify the repeats of a TAL effector to target specific DNA sequences. Further studies have shown that the RVD NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5 ' base targeted by the first repeat, but the exact mechanism of this recognition is not known. More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXalO and AvrBs3.

Accordingly , the TAL effector repeat of the TAL effector domain of the present disclosure may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain strain 756C and Xanthomonas oryzae pv. Oryzicolastiam BLS256 (Bogdanove et al. 2011). As used herein, the TAL effector domain in accordance with the present disclosure comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and/or C-terminal side of the RVD domain) also from the naturally occurring TAL effector. It may comprise more or fewer repeats than the RVD of the naturally occurring TAL effector domain. The TAL effector domain of the present disclosure is designed to target a given DNA sequence based on the above code and others known in the art. The number of TAL effector repeats (e.g., monomers or modules) and their specific sequence are selected based on the desired DNA target sequence. For example, TAL effector repeats, may be removed or added in order to suit a specific target sequence. In an embodiment, the TAL effector domain of the present disclosure comprises between 6.5 and 33.5 TAL effector repeats. In an embodiment, TAL effector domain of the present disclosure comprises between 8 and 33.5 TAL effector repeats, e.g., between 10 and 25 TAL effector repeats, e.g., between 10 and 14 TAL effector repeats.

In some embodiments, tire TAL effector domain comprises TAL effector repeats that correspond to a perfect match to the DNA target sequence. In some embodiments, a mismatch between a repeat and a target base-pair on the DNA target sequence is permitted as along as it allows for the function of the expression repression system, e.g., the expression repressor comprising the TAL effector domain. In general, TALE binding is inversely correlated with the number of mismatches. In some embodiments, the TAL effector domain of an expression repressor of the present disclosure comprises no more than 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, 2 mismatches, or 1 mismatch, and optionally no mismatch, with the target DNA sequence. Without wishing to be bound by theory, in general the smaller the number of TAL effector repeats in the TAL effector domain, the smaller the number of mismatches will be tolerated and still allow for the function of the expression repression system, e.g., the expression repressor comprising the TAL effector domain. The binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, TAL effector domains having 25 TAL effector repeats or more may be able to tolerate up to 7 mismatches.

In addition to the TAL effector repeats, the TAL effector domain of the present disclosure may comprise additional sequences derived from a naturally occurring TAL effector. The length of the C- terminal and/or N-terminal sequence(s) included on each side of the TAL effector repeat portion of the TAL effector domain can vary and be selected by one skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al., have characterized a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL-effector based proteins and have identified key elements, which contribute to optimal binding to the target sequence and thus activation of transcription. Generally, it was found that transcriptional activity is inversely correlated with the length of N-terminus. Regarding the C-terminus, an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Accordingly, in some embodiments, the first 68 amino acids on the C-terminal side of the TAL effector repeats of the naturally occurring TAL effector is included in the TAL effector domain of an expression repressor of the present disclosure. Accordingly, in an embodiment, a TAL effector domain of the present disclosure comprises 1) one or more TAL effector repeats derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids from the naturally occurring TAL effector on the N-terminal side of the TAL effector repeats; and/or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids from the naturally occurring TAL effector on the C-terminal side of the TAL effector repeats.

In some embodiments, a modulating agent comprises a targeting moiety comprising an engineered DNA binding domain (DBD), e.g., a TAL effector comprising a TAL effector repeat that binds to a target sequence, e.g., a promoter or transcription start site (TSS)) sequence operably linked to a target gene (e.g., MYC), e.g., a sequence proximal to the transcription regulatory element, e.g., an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene (e.g., MYC), e.g., a sequence proximal to the anchor sequence. In some embodiments, the TAL effector domain can be engineered to carry epigenetic effector moieties to target sites.

Zn finger domains

In some embodiments, a DNA-targeting moiety is or comprises a Zn finger domain. A Zn finger domain comprises a Zn finger, e.g., a naturally occurring Zn finger or engineered Zn finger, or fragment thereof. Many Zn fingers are known to those of skill in the art and are commercially available, e.g., from Sigma-Aldrich. Generally, a Zn finger domain comprises a plurality of Zn fingers, wherein each Zn finger recognizes three nucleotides. A Zn finger protein can comprise a Zn finger domain and optionally one or more other domains.

In some embodiments, a Zn finger molecule comprises a non-naturally occurring Zn finger protein that is engineered to bind to a target DNA sequence of choice. See, for example, Beerli, et al. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by reference in their entireties.

An engineered Zn finger may have a novel binding specificity, compared to a naturally -occurring Zn finger. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; and WO 01/88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger proteins has been described, for example, in International Patent Publication No. WO 02/077227.

In addition, as disclosed in these and other references, zinc fingers and/or multi-fingered zinc finger domains may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. Tire proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227.

Zn fingers and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; International Patent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496.

In certain embodiments, the DNA-targeting moiety comprises aZn finger domain comprising an engineered zinc finger that binds (in a sequence-specific manner) to a target DNA sequence. In some embodiments, the Zn finger domain comprises one Zn finger or fragment thereof. In some embodiments, the Zn finger domain comprises a plurality of Zn fingers (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more Zn fingers (and optionally no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 Zn fingers). In some embodiments, the Zn finger domain comprises at least three Zn fingers. In some embodiments, the Zn finger domain comprises four, five or six Zn fingers. In some embodiments, the Zn finger domain comprises 8, 9, 10, 11 or 12 Zn fingers. In some embodiments, a Zn finger domain comprising three Zn fingers recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger domain comprising four Zn fingers recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger domain comprising six Zn fingers recognizes a target DNA sequence comprising 18 to 21 nucleotides.

In some embodiments, a targeting domain comprises a two-handed Zn finger protein. Two handed zinc finger proteins are those proteins in which two clusters of zinc fingers are separated by intervening amino acids so that the two zinc finger domains bind to two discontinuous target DNA sequences. An example of a two-handed type of zinc finger binding protein is SIP 1, where a cluster of four zinc fingers is located at the amino terminus of the protein and a cluster of three Zn fingers is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these domains is able to bind to a unique target sequence and the spacing between the two target sequences can comprise many nucleotides.

In some embodiments, an expression repressor comprises a targeting moiety comprising an engineered DNA binding domain (DBD), e.g., a Zn finger domain comprising a Zn finger (ZFN) that binds to a target sequence, e.g., a promoter or transcription start site (TSS)) sequence operably linked to a target gene (e.g., MYC), e.g., a sequence proximal to the transcription regulatory element, e.g., an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene (e.g., MYC), e.g., a sequence proximal to the anchor sequence. In some embodiments, the ZFN can be engineered to carry epigenetic effector molecules to target sites. In some embodiments, the targeting moiety comprises a Zn Finger domain that comprises 2, 3, 4, 5, 6, 7, or 8 zinc fingers. The amino acid sequences of exemplary targeting moieties disclosed herein are listed in Table 5. The nucleotide sequences encoding exemplary targeting moieties disclosed herein are listed in Table 6. In some embodiments, an expression repressor or system described herein comprises a targeting moiety having a sequence set forth in Table 5, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, a nucleic acid described herein comprises a sequence set forth in

Table 6, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. Table 5: Amino acid sequences of exemplary targeting moieties

Table 6: Nucleotide sequences of exemplary targeting moieties

In some embodiments, an expression repression comprises a targeting moiety comprising an engineered DNA binding domain (DBD), e.g., a Zn finger domain comprising a Zn finger (ZFN) that binds to a target sequence, e.g., a promoter or transcription start site (TSS)) sequence operably linked to a target gene (e.g., MYC), e.g., a sequence proximal to the transcription regulatory element, e.g., an anchor sequence of an anchor sequence mediated conjunction (ASMC) comprising a target gene (e.g., MYC), e.g., a sequence proximal to the anchor sequence in mouse genome. In some embodiments, the ZFN can be engineered to carry epigenetic effector molecules to target sites. In some embodiments, the targeting moiety comprises a Zn Finger domain that comprises 2, 3, 4, 5, 6, 7, or 8 zinc fingers. The amino acid sequences of exemplary targeting moieties disclosed herein are listed in Table 7. The nucleotide sequences encoding exemplary targeting moieties disclosed herein are listed in Table 8. In some embodiments, an expression repressor or system described herein comprises a targeting moiety having a sequence set forth in Table 7, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, a nucleic acid described herein comprises a sequence set forth in Table 8, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity thereto.

Table 7: Amino acid sequences of exemplary mouse-specific targeting moieties

Table 8: Nucleotide sequences of exemplary mouse-specific targeting moieties

Nucleic acid molecule

In some embodiments, a targeting moiety is or comprises a DNA-binding domain from a nuclease. For example, the recognition sequences of homing endonucleases and meganucleases such as I- Scel, I-Ceul, PI-PspI, Pl-Sce, 1-SceIV, I-CsmI, I-PanI, I-SceII, I-Ppol, I-SceIII, I-Crel, I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort, et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon, et al. (1989) Gene 82: 115-118; Perler, et al. (1994) Nucleic Acids Res. 22: 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble, et al. (1996); J. Mol. Biol. 263:163-180; Argast, et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. In addition, the DNA-binding specificity of homing endonucleases and meganucleases can be engineered to bind nonnatural target sites. See, for example, Chevalier, et al. (2002) Molec. Cell 10:895-905; Epinat, et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth, et al. (2006) Nature 441:656-659; Paques, et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No. 2007/0117128.

In some embodiments, a DNA-targeting moiety comprises or is nucleic acid. In some embodiments, a nucleic acid that may be included in a DNA-targeting moiety, may be or comprise DNA, RNA, and/or an artificial or synthetic nucleic acid or nucleic acid analog or mimic. For example, in some embodiments, a nucleic acid may be or include one or more of genomic DNA (gDNA), complementary DNA (cDNA), a peptide nucleic acid (PNA), a peptide- oligonucleotide conjugate, a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a polyamide, a triplex- forming oligonucleotide, an antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA or other RNAi molecule (e.g., that targets a non-coding RNA as described herein and/or that targets an expression product of a particular gene associated with a targeted genomic complex as described herein), etc. In some embodiments, a nucleic acid may include one or more residues that is not a naturally-occurring DNA or RNA residue, may include one or more linkages that is/are not phosphodiester bonds (e.g., that may be, for example, phosphorothioate bonds, etc.), and/or may include one or more modifications such as, for example, a 2’0 modification such as 2’-OmeP. A variety of nucleic acid structures useful in preparing synthetic nucleic acids is known in the art (see, for example, WO2017/0628621 and W02014/012081) those skilled in the art will appreciate that these may be utilized in accordance with the present disclosure.

A nucleic acid suitable for use in an expression repressor, e.g., in the DNA-targeting moiety, may include, but is not limited to, DNA, RNA, modified oligonucleotides (e.g., chemical modifications, such as modifications that alter backbone linkages, sugar molecules, and/or nucleic acid bases), and artificial nucleic acids. In some embodiments, a nucleic acid includes, but is not limited to, genomic DNA, cDNA, peptide nucleic acids (PNA) or peptide oligonucleotide conjugates, locked nucleic acids (LNA), bridged nucleic acids (BNA), polyamides, triplex forming oligonucleotides, modified DNA, antisense DNA oligonucleotides, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or other RNA or DNA molecules.

In some embodiments, a DNA-targeting moiety comprises a nucleic acid with a length from about 15-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120- 200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 215-190, 20-190, 30-190, 40-190, 50-190, 60-190, 70-190, 80-190, 90-190, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160- 190, 170-190, 180-190, 15-180, 20-180, 30-180, 40-180, 50-180, 60-180, 70-180, 80-180, 90-180, 100- 180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 15-170, 20-170, 30-170, 40-170, 50-170, 60-170, 70-170, 80-170, 90-170, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160- 170, 15-160, 20-160, 30-160, 40-160, 50-160, 60-160, 70-160, 80-160, 90-160, 100-160, 110-160, 120- 160, 130-160, 140-160, 150-160, 215-150, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90- 150, 100-150, 110-150, 120-150, 130-150, 140-150, 15-140, 20-140, 30-140, 40-140, 50-140, 60-140, 70- 140, 80-140, 90-140, 100-140, 110-140, 120-140, 130-140, 15-130, 20-130, 30-130, 40-130, 50-130, 60- 130, 70-130, 80-130, 90-130, 100-130, 110-130, 120-130, 215-120, 20-120, 30-120, 40-120, 50-120, 60- 120, 70-120, 80-120, 90-120, 100-120, 110-120, 15-110, 20-110, 30-110, 40-110, 50-110, 60-110, 70- 110, 80-110, 90-110, 100-110, 15-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 15-90, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 15-80, 20-80, 30-80, 40-80, 50-80, 60-80, 70-80, 15-70, 20-70, 30-70, 40-70, 50-70, 60-70, 15-60, 20-60, 30-60, 40-60, 50-60, 15-50, 20-50, 30-50, 40-50, 15-40, 20-40, 30-40, 15-30, 20-30, or 15-20 nucleotides, or any range therebetween.

Effector moieties

In some embodiments, expression repressors of the present disclosure comprise one or more effector moieties. In some embodiments, an effector moiety, when used as part of an expression repressor or an expression repression system described herein, decreases expression of a target gene in a cell.

In some embodiments, the effector moiety has functionality unrelated to the binding of the targeting moiety. For example, effector moieties may target, e.g., bind, a genomic sequence element or genomic complex component proximal to the genomic sequence element targeted by the targeting moiety or recruit a transcription factor. As a further example, an effector moiety may comprise an enzymatic activity, e.g., a genetic modification functionality.

In some embodiments, an effector moiety comprises an epigenetic modifying moiety. In some embodiments, an effector moiety comprises a DNA modifying functionality, e.g., a DNA methyltransferase. In some embodiments, an effector moiety is or comprises a protein chosen from MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional variant or fragment of any thereof.

In some embodiments, an effector moiety comprises a transcription repressor. In some embodiments the transcription repressor blocks recruitment of a factor that stimulates or promotes transcription, e.g., of the target gene. In some embodiments, the transcription repressor recruits a factor that inhibits transcription, e.g., of the target gene. In some embodiments, an effector moiety, e.g., transcription repressor, is or comprises a protein chosen from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment of any thereof.

In some embodiments an effector moiety promotes epigenetic modification, e.g., directly or indirectly. For example, an effector moiety can indirectly promote epigenetic modification by recruiting an endogenous protein that epigenetically modifies the chromatin. An effector moiety can directly promote epigenetic modification by catalyzing epigenetic modification, wherein the effector moiety comprises enzymatic activity and directly places an epigenetic mark on the chromatin.

In some embodiments, an effector moiety comprises a histone modifying functionality, e.g., a histone methyltransferase, histone demethylase, or histone deacetylase activity. In some embodiments, a effector moiety is or comprises a protein chosen from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, or a functional variant or fragment of any thereof. In some embodiments, a effector moiety is or comprises a protein chosen from HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a fiinctional variant or fragment of any thereof

In some embodiments, an effector moiety comprises a protein having a functionality described herein. In some embodiments, an effector moiety is or comprises a protein selected from: KRAB (e.g., as according to NP 056209.2 or the protein encoded by NM 015394.5); a SET domain (e.g., the SET domain of: SETDB1 (e.g., as according to NP_001353347.1 or the protein encoded by NM 001366418.1); EZH2 (e.g., as according to NP-004447.2 or the protein encoded by NM 004456.5); G9A (e.g., as according to NP 001350618.1 or the protein encoded by NM OO 1363689.1); or SUV39H1 (e.g., as according to NP_003164.1 orthe protein encoded by NM 003173.4)); histone demethylase LSD1 (e.g., as according to NP 055828.2 or the protein encoded by NM_015013.4); FOG1 (e.g., the N- terminal residues of FOG1) (e.g., as according to NP_722520.2 or the protein encoded by

NM 153813.3); or KAP1 (e.g., as according to NP 005753.1 or the protein encoded by NM 005762.3); a functional fragment or variant of any thereof, or a polypeptide with a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any of the above-referenced sequences.

In some embodiments, a effector moiety is or comprises a protein selected from: DNMT3A (e.g., human DNMT3A) (e.g., as according to NP_072046.2 orthe protein encoded by NM 022552.4); DNMT3B (e.g., as according to NP_008823.1 orthe protein encoded by NM 006892.4); DNMT3L (e.g., as according to NP 787063.1 or the protein encoded by NM 175867.3); DNMT3A/3L complex, bacterial MQ1 (e.g., as according to CAA35058. 1 or P15840.3); a functional fragment of any thereof, or a polypeptide with a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any of the above-referenced sequences.

In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) targeting moiety and one or more effector moiety, wherein the one or more effector moiety is or comprises Krueppel-associated box (KRAB) e.g., as according to NP 056209.2 or the protein encoded by NM_015394.5 or a functional variant or fragment thereof. In some embodiments, KRAB is a synthetic KRAB construct In some embodiments, KRAB comprises an amino acid sequence of SEQ ID NO: 18:

DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVIL RLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO: 18)

In some embodiments, the KRAB effector moiety is encoded by a nucleotide sequence of SEQ ID NO: 51. In some embodiments, a nucleotide sequence described herein comprises a sequence of SEQ ID NO: 51 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

GACGCCAAGAGCCTGACCGCCTGGAGCCGGACCCTGGTGACCTTCAAGGACGTGTTCGTGG ACTTCACCCGGGAGGAGTGGAAGCTGCTGGACACCGCCCAGCAGATCCTGTACCGGAACGT GATGCTGGAGAACTACAAGAACCTGGTGAGCCTGGGCTACCAGCTGACCAAGCCCGACGTG ATCCTGCGGCTGGAGAAGGGCGAGGAGCCCTGGCTGGTGGAGCGGGAGATCCACCAGGAGA CCCACCCCGACAGCGAGACCGCCTTCGAGATCAAGAGCAGCGTG (SEQ ID NO: 51)

In some embodiments, KRAB for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to the KRAB sequence of SEQ ID NO: 18. In some embodiments, an KRAB variant comprises one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 18.

In some embodiments, the polypeptide or the expression repressor is a fusion protein comprising a effector moiety that is or comprises KRAB and a DNA-targeting moiety. In some embodiments, the targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR/Cas domain, e.g., comprising a CRISPR/Cas protein, e.g., a dCas9 protein. In some embodiments, the polypeptide or the expression repressor comprises an additional moiety described herein. In some embodiments, the polypeptide or the expression repressor decreases expression of a target gene, e.g., MYC. In some embodiments, the polypeptide or the expression repressor may be used in methods of modulating, e.g., decreasing, gene expression, methods of treating a condition, or methods of epigenetically modify ing a target gene, e.g., MYC or transcription control element described herein, e.g., in place of an expression repression system. In some embodiments, an expression repression system comprises two or more (e.g., two, three, or four) expression repressors, wherein the first expression repressor comprises an effector moiety comprising the KRAB sequence of SEQ ID NO: 18, or a functional variant or fragment thereof. In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) targeting moiety and one or more effector moiety, wherein the one or more effector moiety is or comprises MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof. In some embodiments, MQ1 is Mollicutes spiroplasma MQ1. In some embodiments, MQ1 is Spiroplasma monobiae MQ1. In some embodiments, MQ1 is MQ1 from strain ATCC 33825 and/or corresponding to Uniprot ID P 15840. In some embodiments, MQ1 comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, MQ1 comprises an amino acid sequence of SEQ ID NO: 87. In some embodiments, an effector domain described herein comprises SEQ ID NO: 19 or 87, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

SKVENKTKKLRVFEAFAGIGAQRKALEKVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEY KSVSREEMIDYLENKTLSWNSKNPVSNGYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRTL KNIDLLTYSFPCQDLSQQGIQKGMKRGSGTRSGLLWEIERALDSTEKNDLPKYLLMENVGALLH KKNEEELNQWKQKLESLGYQNSIEVLNAADFGSSQARRRVFMISTLNEFVELPKGDKKPKSIKK VLNKIVSEKDILNNLLKYNLTEFKKTKSNINKASLIGYSKFNSEGYVYDPEFTGPTLTASGANSRI KIKDGSNIRKMNSDETFLYIGFDSQDGKRVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGG (SEQ ID NO: 19)

MSKVENKTKKLRVFEAFAGIGAQRKALEKVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHT KLEYKSVSREEMIDYLENKTLSWNSKNPVSNGYWKRKKDDELKIIYNAIKLSEKEGNIFD IRDLYKRTLKNIDLLTYSFPCQDLSQQGIQKGMKRGSGTRSGLLWEIERALDSTEKNDLP KYLLMENVGALLHKKNEEELNQWKQKLESLGYQNSIEVLNAADFGSSQARRRVFMISTLN EFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNLTEFKKTKSNINKASLIGYSKFNS EGYVYDPEFTGPTLTASGANSRIKIKDGSNIRKMNSDETFLYIGFDSQDGKRVNEIEFLT ENQKIFVCGNSISVEVLEAIIDKIGG (SEQ ID NO: 87)

In some embodiments, MQ 1 is encoded by a nucleotide sequence of SEQ ID NO: 52 or 132. hr some embodiments, a nucleic acid described herein comprises a sequence of SEQ ID NO: 52, 132 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

AGCAAGGTGGAGAACAAGACCAAGAAGCTGCGGGTGTTCGAGGCCTTCGCCGGCATCGGCG CCCAGCGGAAGGCCCTGGAGAAGGTGCGGAAGGACGAGTACGAGATCGTGGGCCTGGCCG AGTGGTACGTGCCCGCCATCGTGATGTACCAGGCCATCCACAACAACTTCCACACCAAGCTG

GAGTACAAGAGCGTGAGCCGGGAGGAGATGATCGACTACCTGGAGAACAAGACCCTGAGCT

GGAACAGCAAGAACCCCGTGAGCAACGGCTACTGGAAGCGGAAGAAGGACGACGAGCTGA

AGATCATCTACAACGCCATCAAGCTGAGCGAGAAGGAGGGCAACATCTTCGACATCCGGGA

CCTGTACAAGCGGACCCTGAAGAACATCGACCTGCTGACCTACAGCTTCCCCTGCCAGGACC

TGAGCCAGCAGGGCATCCAGAAGGGCATGAAGCGGGGCAGCGGCACCCGGAGCGGCCTGCT

GTGGGAGATCGAGCGGGCCCTGGACAGCACCGAGAAGAACGACCTGCCCAAGTACCTGCTG

ATGGAGAACGTGGGCGCCCTGCTGCACAAGAAGAACGAGGAGGAGCTGAACCAGTGGAAG

CAGAAGCTGGAGAGCCTGGGCTACCAGAACAGCATCGAGGTGCTGAACGCCGCCGACTTCG

GCAGCAGCCAGGCCCGGCGGCGGGTGTTCATGATCAGCACCCTGAACGAGTTCGTGGAGCT

GCCCAAGGGCGACAAGAAGCCCAAGAGCATCAAGAAGGTGCTGAACAAGATCGTGAGCGA

GAAGGACATCCTGAACAACCTGCTGAAGTACAACCTGACCGAGTTCAAGAAAACCAAGAGC

AACATCAACAAGGCCAGCCTGATCGGCTACAGCAAGTTCAACAGCGAGGGCTACGTGTACG

ACCCCGAGTTCACCGGCCCCACCCTGACCGCCAGCGGCGCCAACAGCCGGATCAAGATCAA

GGACGGCAGCAACATCCGGAAGATGAACAGCGACGAGACCTTCCTGTACATCGGCTTCGAC

AGCCAGGACGGCAAGCGGGTGAACGAGATCGAGTTCCTGACCGAGAACCAGAAGATCTTCG

TGTGCGGCAACAGCATCAGCGTGGAGGTGCTGGAGGCCATCATCGACAAGATCGGCGGC (SEQ ID NO: 52)

AGCAAGGUGGAGAACAAGACCAAGAAGCUGCGGGUGUUCGAGGCCUUCGCCGGCAUCGGC

GCCCAGCGGAAGGCCCUGGAGAAGGUGCGGAAGGACGAGUACGAGAUCGUGGGCCUGGCC

GAGUGGUACGUGCCCGCCAUCGUGAUGUACCAGGCCAUCCACAACAACUUCCACACCAAG

CUGGAGUACAAGAGCGUGAGCCGGGAGGAGAUGAUCGACUACCUGGAGAACAAGACCCU

GAGCUGGAACAGCAAGAACCCCGUGAGCAACGGCUACUGGAAGCGGAAGAAGGACGACG

AGCUGAAGAUCAUCUACAACGCCAUCAAGCUGAGCGAGAAGGAGGGCAACAUCUUCGACA

UCCGGGACCUGUACAAGCGGACCCUGAAGAACAUCGACCUGCUGACCUACAGCUUCCCCU

GCCAGGACCUGAGCCAGCAGGGCAUCCAGAAGGGCAUGAAGCGGGGCAGCGGCACCCGGA

GCGGCCUGCUGUGGGAGAUCGAGCGGGCCCUGGACAGCACCGAGAAGAACGACCUGCCCA

AGUACCUGCUGAUGGAGAACGUGGGCGCCCUGCUGCACAAGAAGAACGAGGAGGAGCUG

AACCAGUGGAAGCAGAAGCUGGAGAGCCUGGGCUACCAGAACAGCAUCGAGGUGCUGAA

CGCCGCCGACUUCGGCAGCAGCCAGGCCCGGCGGCGGGUGUUCAUGAUCAGCACCCUGAA

CGAGUUCGUGGAGCUGCCCAAGGGCGACAAGAAGCCCAAGAGCAUCAAGAAGGUGCUGA

ACAAGAUCGUGAGCGAGAAGGACAUCCUGAACAACCUGCUGAAGUACAACCUGACCGAGU UCAAGAAaACCAAGAGCAACAUCAACAAGGCCAGCCUGAUCGGCUACAGCAAGUUCAACA GCGAGGGCUACGUGUACGACCCCGAGUUCACCGGCCCCACCCUGACCGCCAGCGGCGCCA ACAGCCGGAUCAAGAUCAAGGACGGCAGCAACAUCCGGAAGAUGAACAGCGACGAGACCU UCCUGUACAUCGGCUUCGACAGCCAGGACGGCAAGCGGGUGAACGAGAUCGAGUUCCUGA CCGAGAACCAGAAGAUCUUCGUGUGCGGCAACAGCAUCAGCGUGGAGGUGCUGGAGGCCA UCAUCGACAAGAUCGGCGGC (SEQ ID NO: 132)

In some embodiments, MQ 1 for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to a wildtype MQ 1 (e.g., SEQ ID NO: 19). In some embodiments, an MQ1 variant comprises one or more amino acid substitutions, deletions, or insertions relative to a wildtype MQ1, e.g., the MQ1 of SEQ ID NO: 19. In some embodiments, an MQ1 variant comprises a K297P substitution. In some embodiments, an MQ1 variant comprises a N299C substitution. In some embodiments, an MQ1 variant comprises a E301Y substitution. In some embodiments, an MQ1 variant comprises a Q147L substitution (e.g., and has reduced DNA methyltransferase activity relative to wildtype MQ1). In some embodiments, an MQ1 variant comprises K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA binding affinity relative to wildtype MQ1). In some embodiments, an MQ1 variant comprises Q147L, K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA methyltransferase activity and DNA binding affinity relative to wildtype MQ1).

In some embodiments, the polypeptide or the expression repressor is a fusion protein comprising an effector moiety that is or comprises MQ1 and a targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR/Cas domain, a dCas9 domain. In some embodiments, the polypeptide or the expression repressor comprises an additional moiety described herein. In some embodiments, the polypeptide or the expression repressor decreases expression of a target gene, e.g., MYC. In some embodiments, the polvpcptidc or the expression repressor may be used in methods of modulating, e.g., decreasing, gene expression, methods of treating a condition, or methods of epigenetically modifying a target gene, e.g., MYC or transcription control element described herein, e.g., in place of an expression repression system. In some embodiments, an expression repression system comprises two or more (e.g., two, three, or four) expression repressors, wherein the first expression repressor comprises an effector moiety comprising MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof.

In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) targeting moiety and one or more effector moiety, wherein the one or more effector moiety is or comprises DNMT1, e.g., human DNMT1, or a functional variant or fragment thereof. In some embodiments, DNMT1 is human DNMT1, e.g., corresponding to Gene ID 1786, e.g., corresponding to UniProt ID P26358.2. hr some embodiments, DNMT1 comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, an effector domain described herein comprises a sequence according to SEQ ID NO: 20 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto:

VDLRTLDVFSGCGGLSEGFHQAGISDTLWAIEMWDPAAQAFRLNNPGSTVFTEDCNILLKLVMA GETTNSRGQRLPQKGDVEMLCGGPPCQGFSGMNRFNSRTYSKFKNSLVVSFLSYCDYYRPRFFL LENVRNFVSFKRSMVLKLTLRCLVRMGYQCTFGVLQAGQYGVAQTRRRAIILAAAPGEKLPLFP EPLHVFAPRACQLSVVVDDKKFVSNITRLSSGPFRTITVRDTMSDLPEVRNGASALEISYNGEPQS WFQRQLRGAQYQPILRDHICKDMSALVAARMRHIPLAPGSDWRDLPNIEVRLSDGTMARKLRY THHDRKNGRSSSGALRGVCSCVEAGKACDPAARQFNTLIPWCLPHTGNRHNHWAGLYGRLEW

DGFFSTTVTNPEPMGKQGRVLHPEQHRVVSVRECARSQGFPDTYRLFGNILDKHRQVGNAVPPP LAKAIGLEIKLCMLAKARESASAKIKEEEAAKD (SEQ ID NO: 20)

In some embodiments, DNMT1 is encoded by a nucleotide sequence of SEQ ID NO: 53. In some embodiments, a nucleic acid described herein comprises a sequence of SEQ ID NO: 53 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto

GTGGATCTGAGGACACTCGACGTGTTTAGCGGATGCGGCGGACTCTCCGAAGGCTTCCACCA

AGCCGGAATTTCCGACACACTCTGGGCCATTGAGATGTGGGACCCCGCCGCTCAAGCCTTCA GACTGAATAATCCCGGCTCCACCGTGTTCACCGAGGACTGCAACATTCTGCTGAAGCTGGTG ATGGCTGGCGAAACCACCAACTCTAGAGGCCAGAGGCTGCCCCAGAAGGGAGATGTGGAAA TGCTCTGTGGAGGCCCTCCTTGCCAAGGCTTCTCCGGCATGAACAGGTTCAACTCTAGAACA TACAGCAAGTTCAAGAACTCTCTGGTCGTGAGCTTTCTGAGCTACTGCGACTACTATAGACC TAGGTTCTTTCTGCTGGAGAACGTGAGAAATTTCGTGTCCTTCAAGAGGAGCATGGTGCTGA

AGCTGACACTGAGGTGTCTGGTGAGGATGGGCTACCAGTGCACATTCGGAGTGCTGCAAGCT

GGCCAGTACGGCGTGGCCCAGACCAGAAGGAGGGCCATCATTCTGGCTGCTGCCCCCGGCG AGAAACTCCCTCTGTTCCCCGAGCCCCTCCACGTGTTCGCCCCTAGAGCTTGCCAGCTGAGC GTGGTGGTCGACGATAAGAAGTTCGTGAGCAACATCACAAGGCTGTCCAGCGGACCCTTCA GAACCATTACCGTGAGGGATACCATGTCCGACCTCCCCGAGGTGAGGAATGGCGCCAGCGC TCTGGAGATTFCCTACAACGGCGAACCTCAGAGCTGGTTCCAAAGGCAGCTGAGAGGCGCTC AGTATCAGCCCATTCTGAGGGACCACATCTGCAAAGATATGAGCGCTCTGGTGGCCGCTAGA

ATGAGACATATTCCTCTGGCCCCCGGCAGCGACTGGAGAGATCTGCCCAATATTGAGGTGAG ACTCAGCGACGGAACAATGGCTAGAAAACTGAGGTACACCCATCATGATAGAAAGAACGGA AGGAGCAGCAGCGGCGCTCTGAGAGGAGTGTGTAGCTGCGTGGAAGCTGGCAAGGCTTGCG ATCCCGCCGCTAGGCAGTTCAATACCCTCATCCCTTGGTGTCTGCCTCACACCGGCAACAGA CACAATCATTGGGCTGGACTGTATGGAAGGCTCGAATGGGACGGCTTTTTCAGCACCACCGT GACCAATCCCGAACCTATGGGCAAGCAAGGAAGGGTGCTCCACCCCGAGCAGCATAGAGTC GTGTCCGTGAGAGAATGCGCTAGAAGCCAAGGCTTCCCCGACACCTATAGACTGTTCGGCAA CATTCTGGATAAGCACAGACAAGTGGGAAATGCTGTCCCTCCTCCTCTGGCCAAGGCTATCG GACTGGAGATCAAGCTGTGTATGCTCGCCAAAGCTAGGGAGAGCGCTTCCGCCAAGATTAA GGAGGAGGAGGCCGCCAAGGAC (SEQ ID NO: 53)

In some embodiments, DNMT1 for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to a DNMT sequence of SEQ ID NO: 20. In some embodiments, the effector domain comprises one or more amino acid substitutions, deletions, or insertions relative to wild type DNMT1. In some embodiments, the polypeptide is a fusion protein comprising a repressor domain that is or comprises DNMT1 and a targeting moiety. In some embodiments, the targeting moiety is or comprises a zinc finger domain, TAL domain, or CRISPR/Cas domain, e.g., a dCas9 domain. In some embodiments, an expression repression system comprises two or more (e.g., two, three, or four) expression repressors, wherein the first expression repressor comprises an effector moiety comprising DNMT1, or a functional variant or fragment thereof.

In another aspect, the disclosure is directed to an expression repressor or a polypeptide comprising one or more (e.g., one) targeting moiety and one or more effector moiety, wherein the one or more effector moiety is or comprises DNMT3a/3Lcomplex, or a functional variant or fragment thereof. In some embodiments, the DNMT3a/3L complex fusion construct. In some embodiments the DNMT3a/3L complex comprises DNMT3A (e.g., human DNMT3A) (e.g., as according to NP_072046.2 or the protein encoded by NM 022552.4). In some embodiments the DNMT3a/3L complex comprises DNMT3L (e.g., as according to NP 787063. 1 or the protein encoded by NM 175867.3). In some embodiments, DNMT3a/3L comprises an amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 114. In some embodiments, an effector domain described herein comprises SEQ ID NO: 21 or SEQ ID NO: 114, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

EWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVA MGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRI AKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTOVSNMSRLARQRL LGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMNPLEMFETVPVWR RQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGH TCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHG GSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYF STELTSSL (SEQ ID NO: 21)

NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRH QGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDA RPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRP LASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFG FPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLS LRGSHMNPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKD VEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDL DVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSSKLAAKW PTKLVKNCFLPLREYFKYFSTELTSSL (SEQ ID NO: 114)

In some embodiments, DNMT3a/3L is encoded by a nucleotide sequence of SEQ ID NO: 54. In some embodiments, a nucleic acid described herein comprises a sequence of SEQ ID NO: 54 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

AACCACGACCAGGAGTTCGACCCCCCCAAGGTGTACCCCCCCGTGCCCGCCGAGAAGCGGA AGCCCATCCGGGTGCTGAGCCTGTTCGACGGCATCGCCACCGGCCTGCTGGTGCTGAAGGAC CTGGGCATCCAGGTGGACCGGTACATCGCCAGCGAGGTGTGCGAGGACAGCATCACCGTGG GCATGGTGCGGCACCAGGGCAAGATCATGTACGTGGGCGACGTGCGGAGCGTGACCCAGAA GCACATCCAGGAGTGGGGCCCCTTCGACCTGGTGATCGGCGGCAGCCCCTGCAACGACCTG AGCATCGTGAACCCCGCCCGGAAGGGCCTGTACGAGGGCACCGGCCGGCTGTTCTTCGAGTT CTACCGGCTGCTGCACGACGCCCGGCCCAAGGAGGGCGACGACCGGCCCTTCTTCTGGCTGT TCGAGAACGTGGTGGCCATGGGCGTGAGCGACAAGCGGGACATCAGCCGGTTCCTGGAGAG CAACCCCGTGATGATCGACGCCAAGGAGGTGAGCGCCGCCCACCGGGCCCGGTACTTCTGG GGCAACCTGCCCGGCATGAACCGGCCCCTGGCCAGCACCGTGAACGACAAGCTGGAGCTGC AGGAGTGCCTGGAGCACGGCCGGATCGCCAAGTTCAGCAAGGTGCGGACCATCACCACCCG GAGCAACAGCATCAAGCAGGGCAAGGACCAGCACTTCCCCGTGTTCATGAACGAGAAGGAG GACATCCTGTGGTGCACCGAGATGGAGCGGGTGTTCGGCTTCCCCGTGCACTACACCGACGT GAGCAACATGAGCCGGCTGGCCCGGCAGCGGCTGCTGGGCCGGAGCTGGAGCGTGCCCGTG ATCCGGCACCTGTTCGCCCCCCTGAAGGAGTACTTCGCCTGCGTGAGCAGCGGCAACAGCAA CGCCAACAGCCGGGGCCCCAGCTTCAGCAGCGGCCTGGTGCCCCTGAGCCTGCGGGGCAGC CACATGAATCCTCTGGAGATGTTCGAGACAGTGCCCGTGTGGAGAAGGCAACCCGTGAGGG TGCTGAGCCTCTTCGAGGACATTAAGAAGGAGCTGACCTCTCTGGGCTTTCTGGAATCCGGC AGCGACCCCGGCCAGCTGAAACACGTGGTGGACGTGACCGACACAGTGAGGAAGGACGTGG AAGAGTGGGGCCCCTTTGACCTCGTGTATGGAGCCACACCTCCTCTCGGCCACACATGCGAT AGGCCTCCCAGCTGGTATCTCTTCCAGTTCCACAGACTGCTCCAGTACGCCAGACCTAAGCC CGGCAGCCCCAGACCCTTCTTCTGGATGTTCGTGGACAATCTGGTGCTGAACAAGGAGGATC TGGATGTGGCCAGCAGATTTCTGGAGATGGAACCCGTGACAATCCCCGACGTGCATGGCGG CTCTCTGCAGAACGCCGTGAGAGTGTGGTCCAACATCCCCGCCATTAGAAGCAGACACTGGG CTCTGGTGAGCGAGGAGGAACTGTCTCTGCTGGCCCAGAATAAGCAGTCCTCCAAGCTGGCC GCCAAGTGGCCCACCAAGCTGGTGAAGAACTGCTTTCTGCCTCTGAGGGAGTATTTCAAGTA TTTCAGCACCGAACTGACCAGCAGCCTG (SEQ ID NO: 54)

In some embodiments, DNMT3a/3L for use in a polypeptide or an expression repressor described herein is a variant, e.g., comprising one or more mutations, relative to the DNMT3a/3L of SEQ ID NO: 21 or SEQ ID NO: 114. In some embodiments, an DNMT3a/3L variant comprises one or more amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 21 or SEQ ID NO: 114. In some embodiments, the polypeptide or the expression repressor is a fusion protein comprising an effector moiety' that is or comprises DNMT3a/3L and a targeting moiety. In some embodiments, the targeting moiety' is or comprises a zinc finger domain, TAL domain, or CRISPR/Cas domain e.g., a dCas9 domain. In some embodiments, an expression repression system comprises two or more (e g., two, three, or four) expression repressors, wherein the first expression repressor comprises an effector moiety comprising DNMT3a/3L, or a functional variant or fragment thereof.

In some embodiments, an effector moiety is or comprises a polypeptide. In some embodiments, an effector moiety is or comprises a nucleic acid. In some embodiments, an effector moiety is a chemical, e.g., a chemical that modulates a cytosine I or an adenine(A) (e.g., Na bisulfite, ammonium bisulfite). In some embodiments, an effector moiety has enzymatic activity (e.g., methyl transferase, demethylase, nuclease (e.g., Cas9), or deaminase activity). An effector moiety may be or comprise one or more of a small molecule, a peptide, a nucleic acid, a nanoparticle, an aptamer, or a pharmaco-agent with poor PK/PD.

In some embodiments, an effector moiety, may comprise a peptide ligand, a full-length protein, a protein fragment, an antibody, an antibody fragment, and/or a targeting aptamer. In some embodiments, the protein may bind a receptor such as an extracellular receptor, neuropeptide, hormone peptide, peptide drug, toxic peptide, viral or microbial peptide, synthetic peptide, or agonist or antagonist peptide.

In some embodiments, an effector moiety may comprise antigens, antibodies, antibody fragments such as, e.g. single domain antibodies, ligands, or receptors such as, e.g., glucagon-like peptide-1 (GLP- 1), GLP-2 receptor 2, cholecystokinin B (CCKB), or somatostatin receptor, peptide therapeutics such as, e.g., those that bind to specific cell surface receptors such as G protein-coupled receptors (GPCRs) or ion channels, synthetic or analog peptides from naturally-bioactive peptides, anti-microbial peptides, poreforming peptides, tumor targeting or cytotoxic peptides, or degradation or self-destruction peptides such as an apoptosis-inducing peptide signal or photosensitizer peptide.

Peptide or protein moieties for use in effector moieties as described herein may also include small antigen-binding peptides, e.g., antigen binding antibody or antibody-like fragments, such as, e.g., single chain antibodies, nanobodies (see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21(7):1076-l 13). Such small antigen binding peptides may bind, e.g., a cytosolic antigen, a nuclear antigen, an intra-organellar antigen.

In some embodiments, an effector moiety comprises a dominant negative component (e.g., dominant negative moiety), e.g., a protein that recognizes and binds a sequence (e.g., an anchor sequence, e.g., a CTCF binding motif), but with an inactive (e.g., mutated) dimerization domain, e.g., a dimerization domain that is unable to form a functional anchor sequence-mediated conjunction), or binds to a component of a genomic complex (e.g., a transcription factor subunit, etc.) preventing formation of a functional transcription factor, etc. For example, the Zinc Finger domain of CTCF can be altered so that it binds a specific anchor sequence (by adding zinc fingers that recognize flanking nucleic acids), while the homo-dimerization domain is altered to prevent the interaction between engineered CTCF and endogenous forms of CTCF. In some embodiments, a dominant negative component comprises a synthetic nucleating polypeptide with a selected binding affinity for an anchor sequence within a target anchor sequence-mediated conjunction. In some embodiments, binding affinity may be at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher or lower than binding affinity of an endogenous nucleating polypeptide (e.g., CTCF) that associates with a target anchor sequence. A synthetic nucleating polypeptide may have between 30-90%, 30-85%, 30-80%, 30-70%, 50-80%, 50-90% amino acid sequence identity to a corresponding endogenous nucleating polypeptide. A nucleating polypeptide may modulate (e.g., disrupt), such as through competitive binding, e.g., competing with binding of an endogenous nucleating polypeptide to its anchor sequence.

In some embodiments, an effector moiety comprises an antibody or fragment thereof. In some embodiments, target gene (e.g., MY C) expression is altered via use of effector moieties that are or comprise one or more antibodies or fragments thereof. In some embodiments, gene expression is altered via use of effector moieties that are or comprise one or more antibodies (or fragments thereof) and dCas9.

In some embodiments, an antibody or fragment thereof for use in an effector moiety may be monoclonal. An antibody may be a fusion, a chimeric antibody, a non-humanized antibody, a partially or fully humanized antibody, etc. As will be understood by one of skill in the art, format of antibody(ies) used may be the same or different depending on a given target.

In some embodiments, an effector moiety, comprises a conjunction nucleating molecule, a nucleic acid encoding a conjunction nucleating molecule, or a combination thereof. A conjunction nucleating molecule may be, e.g., CTCF, cohesin, USF1, YY1, TATA-box binding protein associated factor 3 (TAF3), ZNF143 binding motif, or another polypeptide that promotes formation of an anchor sequence-mediated conjunction. A conjunction nucleating molecule may be an endogenous polypeptide or other protein, such as a transcription factor, e g., autoimmune regulator (AIRE), another factor, e.g., X- inactivation specific transcript (XIST), or an engineered polypeptide that is engineered to recognize a specific DNA sequence of interest, e.g., having a zinc finger, leucine zipper or bHLH domain for sequence recognition. A conjunction nucleating molecule may modulate DNA interactions within or around the anchor sequence -mediated conjunction (e.g., associated with or comprising the genomic sequence element targeted by the targeting moiety). For example, a conjunction nucleating molecule can recruit other factors to an anchor sequence that alters an anchor sequence -mediated conjunction formation or disruption.

A conjunction nucleating molecule may also have a dimerization domain for homo- or heterodimerization. One or more conjunction nucleating molecules, e.g., endogenous and engineered, may interact to form an anchor sequence-mediated conjunction. In some embodiments, a conjunction nucleating molecule is engineered to further include a stabilization domain, e.g., cohesion interaction domain, to stabilize an anchor sequence-mediated conjunction. In some embodiments, a conjunction nucleating molecule is engineered to bind a target sequence, e g., target sequence binding affinity is modulated. In some embodiments, a conjunction nucleating molecule is selected or engineered with a selected binding affinity for an anchor sequence within an anchor sequence -mediated conjunction.

Conjunction nucleating molecules and their corresponding anchor sequences may be identified through use of cells that harbor inactivating mutations in CTCF and Chromosome Conformation Capture or 3C-based methods, e.g., Hi-C or high-throughput sequencing, to examine topologically associated domains, e.g., topological interactions between distal DNA regions or loci, in the absence of CTCF. Long-range DNA interactions may also be identified. Additional analyses may include ChlA-PET analysis using a bait, such as Cohesin, YY1 or USF1, ZNF143 binding motif, and MS to identify complexes that are associated with a bait. In some embodiments, an effector moiety comprises a DNA-binding domain of a protein. In some embodiments, a DNA binding domain of an effector moiety enhances or alters targeting of a modulating agent but does not alone achieve complete targeting by a modulating agent (e.g., the targeting moiety is still needed to achieve targeting of the modulating agent). In some embodiments, a DNA binding domain enhances targeting of a modulating agent. In some embodiments, a DNA binding domain enhances efficacy of a modulating agent. DNA-binding proteins have distinct structural motifs, e.g., that play a key role in binding DNA, known to those of skill in the art. In some embodiments, a DNA-binding domain comprises a helix-tum-helix (HTH) motif, a common DNA recognition motif in repressor proteins. Such a motif comprises two helices, one of which recognizes DNA (aka recognition helix) with side chains providing binding specificity’. Such motifs are commonly used to regulate proteins that are involved in developmental processes. Sometimes more than one protein competes for the same sequence or recognizes the same DNA fragment. Different proteins may differ in their affinity for the same sequence, or DNA conformation, respectively through H-bonds, salt bridges and Van der Waals interactions.

In some embodiments, a DNA-binding domain comprises a helix-hairpin-helix (HhH) motif. DNA-binding proteins with a HhH structural motif may be involved in non-sequence-specific DNA binding that occurs via the formation of hydrogen bonds between protein backbone nitrogen and DNA phosphate groups.

In some embodiments, a DNA-binding domain comprises a helix-loop -helix (HLH) motif. DNA- binding proteins with an HLH structural motif are transcriptional regulatory proteins and are principally related to a wide array of developmental processes. An HLH structural motif is longer, in terms of residues, than HTH or HhH motifs. Many of these proteins interact to form homo- and hetero-dimers. A structural motif is composed of two long helix regions, with an N-terminal helix binding to DNA, while a complex region allows the protein to dimerize.

In some embodiments, a DNA-binding domain comprises a leucine zipper motif. In some transcription factors, a dimer binding site with DNA forms a leucine zipper. This motif includes two amphipathic helices, one from each subunit, interacting with each other resulting in a left-handed coiled- coil super secondary structure. A leucine zipper is an interdigitation of regularly spaced leucine residues in one helix with leucines from an adjacent helix. Mostly, helices involved in leucine zippers exhibit a heptad sequence (abcdefg) with residues a and d being hydrophobic and other residues being hydrophilic. Leucine zipper motifs can mediate either homo- or Aeteradimer formation.

In some embodiments, a DNA-binding domain comprises a Zn finger domain, where a Zn⁺⁺ ion is coordinated by 2 Cys and 2 His residues. Such a transcription factor includes a trimer with the stoichiometry PP ‘a. An apparent effect of Zn⁺⁺ coordination is stabilization of a small complex structure instead of hydrophobic core residues. Each Zn-finger interacts in a conformationally identical manner with successive triple base pair segments in the major groove of the double helix. Protein-DNA interaction is determined by two factors: (i) H-bonding interaction between a-helix and DNA segment, mostly between Arg residues and Guanine bases, (ii) H-bonding interaction with DNA phosphate backbone, mostly with Arg and His. An alternative Zn-finger motif chelates Zn⁺⁺ with 6 Cys.

In some embodiments, a DNA-b inding domain comprises a TATA box binding protein (TBP). TBP was first identified as a component of the class II initiation factor TFIID. These binding proteins participate in transcription by all three nuclear RNA polymerases acting as subunit in each of them. Structure of TBP shows two a/p structural domains of 89-90 amino acids. The C-terminal or core region of TBP binds with high affinity to a TATA consensus sequence (TATAa/tAa/t) recognizing minor groove determinants and promoting DNA bending. TBP resemble a molecular saddle. The binding side is lined with central 8 strands of a 10-stranded anti-parallel p-sheet. The upper surface contains four a-helices and binds to various components of transcription machinery.

In some embodiments, a DNA-binding domain is or comprises a transcription factor. Transcription factors (TFs) may be modular proteins containing a DNA-binding domain that is responsible for specific recognition of base sequences and one or more effector domains that can activate or repress transcription. TFs interact with chromatin and recruit protein complexes that serve as coactivators or corepressors.

In some embodiments, an effector moiety comprises one or more RNAs (e.g., gRNA) and dCas9. In some embodiments, one or more RNAs is/are targeted to a genomic sequence element via dCas9 and target-specific guide RNA. As will be understood by one of skill in the art, RNAs used for targeting may be the same or different depending on a given target. An effector moiety may comprise an aptamer, such as an oligonucleotide aptamer or a peptide aptamer. Aptamer moieties are oligonucleotide or peptide aptamers.

An effector moiety may comprise an oligonucleotide aptamer. Oligonucleotide aptamers are single -stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind to pre-selected targets including proteins and peptides with high affinity and specificity.

Oligonucleotide aptamers are nucleic acid species that may be engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers provide discriminate molecular recognition and can be produced by chemical synthesis. In addition, aptamers possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. Both DNA and RNA aptamers show robust binding affinities for various targets. For example, DNA and RNA aptamers have been selected for t lysozyme, thrombin, human immunodeficiency virus trans-acting responsive element (HIV TAR), hemin, interferon y, vascular endothelial growth factor (VEGF), prostate specific antigen (PSA), dopamine, and the non-classical oncogene, heat shock factor 1 (HSF1).

Diagnostic techniques for aptamer-based plasma protein profiling includes aptamer plasma proteomics. This technology will enable future multi -biomarker protein measurements that can aid diagnostic distinction of disease versus healthy states.

An effector moiety may comprise a peptide aptamer moiety. Peptide aptamers have one (or more) short variable peptide domains, including peptides having low molecular weight, 12-14 kDa. Peptide aptamers may be designed to specifically bind to and interfere with protein-protein interactions inside cells.

Peptide aptamers are artificial proteins selected or engineered to bind specific target molecules. These proteins include of one or more peptide complexes of variable sequence. They are typically isolated from combinatorial libraries and often subsequently improved by directed mutation or rounds of variable region mutagenesis and selection. In vivo, peptide aptamers can bind cellular protein targets and exert biological effects, including interference with the normal protein interactions of their targeted molecules with other proteins. In particular, a variable peptide aptamer complex attached to a transcription factor binding domain is screened against a target protein attached to a transcription factor activating domain. In vivo binding of a peptide aptamer to its target via this selection strategy is detected as expression of a downstream yeast marker gene. Such experiments identify particular proteins bound by aptamers, and protein interactions that aptamers disrupt, to cause a given phenotype. In addition, peptide aptamers derivatized with appropriate functional moieties can cause specific post-translational modification of their target proteins or change subcellular localization of the targets. Peptide aptamers can also recognize targets in vitro. They have found use in lieu of antibodies in biosensors and used to detect active isofonns of proteins from populations containing both inactive and active protein forms. Derivatives known as tadpoles, in which peptide aptamer “heads” are covalently linked to unique sequence double-stranded DNA “tails”, allow quantification of scarce target molecules in mixtures by PCR (using, for example, the quantitative real-time polymerase chain reaction) of their DNA tails.

Peptide aptamer selection can be made using different systems, but the most used is currently a yeast two-hybrid system. Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopannings. Among peptides obtained from biopannings, mimotopes can be considered as a kind of peptide aptamers. Peptides panned from combinatorial peptide libraries have been stored in a special database with named MimoDB.

An exemplary effector moiety may include, but is not limited to: ubiquitin, bicyclic peptides as ubiquitin ligase inhibitors, transcription factors, DNA and protein modification enzymes such as topoisomerases, topoisomerase inhibitors such as topotecan, DNA methyltransferases such as the DNMT family (e.g., DNMT3A, DNMT3B, DNMT3a/3L, MQ1), protein methyltransferases (e.g., viral lysine methyltransferase (vSET), protein-lysine N-methyltransferase (SMYD2), deaminases (e.g., APOBEC, UG1), histone methyltransferases such as enhancer of zeste homolog 2 (EZH2), PRMT1, histone-lysine- N-methyltransferase (Setdbl), histone methyltransferase (SET2), euchromatic histone-lysine N- methyltransferase 2 (G9a), histone-lysine N-methyltransferase (SUV39H1), and G9a), histone deacetylase (e.g., HDAC1, HDAC2, HDAC3), enzymes with a role in DNA demethylation (e.g., the TET family enzymes catalyze oxidation of 5 -methylcytosine to 5 -hydroxymethylcytosine and higher oxidative derivatives), protein demethylases such as KDM1A and lysine-specific histone demethylase 1 (LSD1), helicases such as DHX9, deacetylases (e.g., sirtuin 1, 2, 3, 4, 5, 6, or 7), kinases, phosphatases, DNA- intercalating agents such as ethidium bromide, SYBR green, and proflavine, efflux pump inhibitors such as peptidomimetics like phenylalanine arginyl p-naphthylamide or quinoline derivatives, nuclear receptor activators and inhibitors, proteasome inhibitors, competitive inhibitors for enzymes such as those involved in lysosomal storage diseases, protein synthesis inhibitors, nucleases (e.g., Cpfl, Cas9, zinc finger nuclease), specific domains from proteins, such as a KRAB domain, and fusions of one or more thereof (e.g., dCas9-DNMT, dCas9-MQl, dCas9-KRAB).

In some embodiments, a candidate domain may be determined to be suitable for use as an effector moiety by methods known to those of skill in the art. For example, a candidate effector moiety may be tested by assaying whether, when the candidate effector moiety is present in the nucleus of a cell and appropriately localized (e.g., to a target gene or transcription control element operably linked to said target gene, e.g., via a targeting moiety), the candidate effector moiety decreases expression of the target gene in the cell, e.g., decreases the level of RNA transcript encoded by the target gene (e.g., as measured by RNASeq or Northern blot) or decreases the level of protein encoded by the target gene (e.g., as measured by ELISA).

In some embodiments, an expression repressor comprises a plurality of effector moiety, wherein each effector moiety does not detectably bind, e g., does not bind, to another effector moiety. In some embodiments, an expression repression system comprises a first expression repressor comprising a first effector moiety and a second expression repressor comprising a second effector moiety, wherein the first effector moiety does not detectably bind, e.g., does not bind, to the second effector moiety'. In some embodiments, an expression repression system comprises a plurality of expression repressors, wherein each member of the plurality of expression repressors comprises an effector moiety, wherein each effector moiety does not detectably bind, e.g., does not bind, to another effector moiety. In some embodiments, an expression repression system comprises a first expression repressor comprising a first effector moiety and a second expression repressor comprising a second effector moiety, wherein the first effector moiety does not detectably bind, e.g., does not bind, to the second effector moiety. In some embodiments, an expression repression system comprises a first expression repressor comprising a first effector moiety and a second expression repressor comprising a second effector moiety, wherein the first effector moiety does not detectably bind, e.g., does not bind, to another first effector moiety, and the second effector moiety does not detectably bind, e.g., does not bind, to another second effector moiety. In some embodiments, an effector moiety for use in the compositions and methods described herein is functional in a monomeric, e.g., non-dimeric, state.

In some embodiments, an effector moiety is or comprises an epigenetic modifying moiety, e.g., that modulates the two-dimensional structure of chromatin (i.e., that modulate structure of chromatin in a way that would alter its two-dimensional representation).

Epigenetic modify ing moieties useful in methods and compositions of the present disclosure include agents that affect epigenetic markers, e.g., DNA methylation, histone methylation, histone acetylation, histone sumoylation, histone phosphorylation, and RNA-associated silencing. Exemplary epigenetic enzymes that can be targeted to a genomic sequence element as described herein include DNA methylases (e.g., DNMT3a, DNMT3b, DNMT3a/3L, MQ1), DNA demethylation (e.g., the TET family), histone methyltransferases, histone deacetylase (e.g., HDAC1, HDAC2, HDAC3), sirtuin 1, 2, 3, 4, 5, 6, or 7, lysine-specific histone demethylase 1 (LSD1), histone-lysine-N -methyltransferase (Setdbl), euchromatic histone-lysine N-methyltransferase 2 (G9a), histone-lysine N-methyltransferase (SUV39H1), enhancer of zeste homolog 2 (EZH2), viral lysine methyltransferase (vSET), histone methyltransferase (SET2), and protein-lysine N-methyltransferase (SMYD2). Examples of such epigenetic modifying agents are described, e.g., in de Groote et al. Nuc. Acids Res. (2012): 1-18.

In some embodiments, an expression repressor, e.g., comprising an epigenetic modifying moiety, useful herein comprises or is a construct described in Koferle et al. Genome Medicine 7.59 (2015): 1-3 incorporated herein by reference. For example, in some embodiments, an expression repressor comprises or is a construct found in Table 1 of Koferle et al., e.g., histone deacetylase, histone methyltransferase, DNA demethylation, or H3K4 and/or H3K9 histone demethylase described in Table 1 (e.g., dCas9-p300, TALE-TET1, ZF-DNMT3A, or TALE-LSD1).

In some embodiments, an effector moiety comprises a component of a gene editing system e.g., a CRISPR/Cas domain, e.g., a Zn Finger domain, e.g., a TAL effector domain. In some embodiments, an epigenetic modifying moiety may comprise a polypeptide (e.g., peptide or protein moiety) linked to a gRNA and a targeted nuclease, e.g., a Cas9, e.g., a wild type Cas9, a nickase Cas9 (e.g., Cas9 D10A), a catalytically inactive Cas9 (dCas9), eSpCas9, Cpfl, C2C1, or C2C3, or a nucleic acid encoding such a nuclease.

As used herein, a “biologically active portion of an effector domain” is a portion that maintains function (e.g., completely, partially, minimally) of an effector domain (e.g., a “minimal” or “core” domain). In some embodiments, fusion of a dCas9 with all or a portion of one or more effector domains of an epigenetic modifying agent (such as a DNA methylase or enzyme with a role in DNA demethylation, e.g., DNMT3a, DNMT3b, DNMT3L, a DNMT inhibitor, combinations thereof, TET family enzymes, protein acetyl transferase or deacetylase, dCas9-DNMT3a/3L, dCas9- DNMT3a/3L/KRAB, dCas9/VP64) creates a chimeric protein that is linked to the polypeptide and useful in the methods described herein. An effector moiety comprising such a chimeric protein is referred to as either a genetic modifying moiety (because of its use of a gene editing system component, Cas9) or an epigenetic modifying moiety (because of its use of an effector domain of an epigenetic modifying agent).

In some embodiments, provided technologies are described as comprising a gRNA that specifically targets a target gene. In some embodiments, the target gene is an oncogene, a tumor suppressor, or a MYC mis-regulation disorder related gene. In some embodiments, the target gene is MYC.

In some embodiments, technologies provided herein include methods of delivering one or more genetic modifying moieties (e.g., CRISPR system components) described herein to a subject, e.g., to a nucleus of a cell or tissue of a subject, by linking such a moiety to a targeting moiety as part of a fusion molecule. In some embodiments, technologies provided herein include methods of delivering one or more genetic modifying moieties (e.g., CRISPR system components) described herein to a subject, e.g., to a nucleus of a cell or tissue of a subject, by encapsulating the one or more genetic modifying moieties (e.g., CRISPR system components) in a lipid nanoparticle.

Additional Moieties

An expression repressor may further comprise one or more additional moieties (e.g., in addition to one or more targeting moieties and one or more effector moieties). In some embodiments, an additional moiety' is selected from a tagging or monitoring moiety, a cleavable moiety (e g., a cleavable moiety positioned between a DNA-targeting moiety and an effector moiety or at the N- or C-terminal end of a polypeptide), a small molecule, a membrane translocating polypeptide, or a pharmaco-agent moiety. Exemplary Expression Repressors

The following exemplary expression repressors are presented for illustration purposes only and are not intended to be limiting.

In some embodiments, an expression repressor comprises a targeting moiety comprising dCas9, e.g., an S. aureus dCas9, and an effector moiety comprising MQ1, e.g., bacterial MQ1. In some embodiments, the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 68 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor). In some embodiments, the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 119. In some embodiments, a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 68, 119 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. dCas9-MQl nucleotide sequence:

GAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGC AGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAG ACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCG AGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGAG CATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAG AGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCA AGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGT GGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCATG GAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGG TGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCCG GAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGC AAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGG ACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGA GCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTG AGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCACC TGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGAC CGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCA CCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAAGCGGCCCGCCGC CACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGCCCGGGACAGCAAGGTGGAGAACAA GACCAAGAAGCTGCGGGTGTTCGAGGCCTTCGCCGGCATCGGCGCCCAGCGGAAGGCCCTG GAGAAGGTGCGGAAGGACGAGTACGAGATCGTGGGCCTGGCCGAGTGGTACGTGCCCGCCA TCGTGATGTACCAGGCCATCCACAACAACTTCCACACCAAGCTGGAGTACAAGAGCGTGAG

CCGGGAGGAGATGATCGACTACCTGGAGAACAAGACCCTGAGCTGGAACAGCAAGAACCCC

GTGAGCAACGGCTACTGGAAGCGGAAGAAGGACGACGAGCTGAAGATCATCTACAACGCCA

TCAAGCTGAGCGAGAAGGAGGGCAACATCTTCGACATCCGGGACCTGTACAAGCGGACCCT

GAAGAACATCGACCTGCTGACCTACAGCTTCCCCTGCCAGGACCTGAGCCAGCAGGGCATCC

AGAAGGGCATGAAGCGGGGCAGCGGCACCCGGAGCGGCCTGCTGTGGGAGATCGAGCGGG

CCCTGGACAGCACCGAGAAGAACGACCTGCCCAAGTACCTGCTGATGGAGAACGTGGGCGC

CCTGCTGCACAAGAAGAACGAGGAGGAGCTGAACCAGTGGAAGCAGAAGCTGGAGAGCCT

GGGCTACCAGAACAGCATCGAGGTGCTGAACGCCGCCGACTTCGGCAGCAGCCAGGCCCGG

CGGCGGGTGTTCATGATCAGCACCCTGAACGAGTTCGTGGAGCTGCCCAAGGGCGACAAGA

AGCCCAAGAGCATCAAGAAGGTGCTGAACAAGATCGTGAGCGAGAAGGACATCCTGAACA

ACCTGCTGAAGTACAACCTGACCGAGTTCAAGAAAACCAAGAGCAACATCAACAAGGCCAG

CCTGATCGGCTACAGCAAGTTCAACAGCGAGGGCTACGTGTACGACCCCGAGTTCACCGGCC

CCACCCTGACCGCCAGCGGCGCCAACAGCCGGATCAAGATCAAGGACGGCAGCAACATCCG

GAAGATGAACAGCGACGAGACCTTCCTGTACATCGGCTTCGACAGCCAGGACGGCAAGCGG

GTGAACGAGATCGAGTTCCTGACCGAGAACCAGAAGATCTTCGTGTGCGGCAACAGCATCA

GCGTGGAGGTGCTGGAGGCCATCATCGACAAGATCGGCGGCCCCAGCAGCGGCGGCAAGCG

GCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGA

CGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTG

GCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGG

AAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 68)

AAGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAG

AAGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCC

TGGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAG

CAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGC

GCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC

GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGAT

GGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGAC

AAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGA

AGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCT

GCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG

GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTA CAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTG

AGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGA

AGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAG

AGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACG

ACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAG

AACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGG

CCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCT

GAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGC

AAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCA

TCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGA

GGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTG

GGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACC

GGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGG

GGCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACT

TCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTT

CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTC

ACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCT

TCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGT

GACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAG

ATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGA

TCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGT

GCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCC

CACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCC

GGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGA

CTTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCC

TGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGA

GCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAG

GTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGA

TGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGC

GGATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGA

ACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA

CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCC

CAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCC

GGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGC GGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGA

GCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACC

CGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACG

AGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGA

CTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG

ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG

CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG

CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA

GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC

GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA

GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA

GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC

AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG

TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT

GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG

GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC

GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG

CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG

GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG

AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT

GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC

CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA

CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC

ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAAGCGGCCCGCCG

CCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGCCCGGGACAGCAAGGTGGAGAACA

AGACCAAGAAGCTGCGGGTGTTCGAGGCCTTCGCCGGCATCGGCGCCCAGCGGAAGGCCCT

GGAGAAGGTGCGGAAGGACGAGTACGAGATCGTGGGCCTGGCCGAGTGGTACGTGCCCGCC

ATCGTGATGTACCAGGCCATCCACAACAACTTCCACACCAAGCTGGAGTACAAGAGCGTGA

GCCGGGAGGAGATGATCGACTACCTGGAGAACAAGACCCTGAGCTGGAACAGCAAGAACCC

CGTGAGCAACGGCTACTGGAAGCGGAAGAAGGACGACGAGCTGAAGATCATCTACAACGCC

ATCAAGCTGAGCGAGAAGGAGGGCAACATCTTCGACATCCGGGACCTGTACAAGCGGACCC

TGAAGAACATCGACCTGCTGACCTACAGCTTCCCCTGCCAGGACCTGAGCCAGCAGGGCATC

CAGAAGGGCATGAAGCGGGGCAGCGGCACCCGGAGCGGCCTGCTGTGGGAGATCGAGCGG

GCCCTGGACAGCACCGAGAAGAACGACCTGCCCAAGTACCTGCTGATGGAGAACGTGGGCG CCCTGCTGCACAAGAAGAACGAGGAGGAGCTGAACCAGTGGAAGCAGAAGCTGGAGAGCC TGGGCTACCAGAACAGCATCGAGGTGCTGAACGCCGCCGACTTCGGCAGCAGCCAGGCCCG GCGGCGGGTGTTCATGATCAGCACCCTGAACGAGTTCGTGGAGCTGCCCAAGGGCGACAAG AAGCCCAAGAGCATCAAGAAGGTGCTGAACAAGATCGTGAGCGAGAAGGACATCCTGAAC AACCTGCTGAAGTACAACCTGACCGAGTTCAAGAAAACCAAGAGCAACATCAACAAGGCCA GCCTGATCGGCTACAGCAAGTTCAACAGCGAGGGCTACGTGTACGACCCCGAGTTCACCGG CCCCACCCTGACCGCCAGCGGCGCCAACAGCCGGATCAAGATCAAGGACGGCAGCAACATC CGGAAGATGAACAGCGACGAGACCTTCCTGTACATCGGCTTCGACAGCCAGGACGGCAAGC GGGTGAACGAGATCGAGTTCCTGACCGAGAACCAGAAGATCTTCGTGTGCGGCAACAGCAT CAGCGTGGAGGTGCTGGAGGCCATCATCGACAAGATCGGCGGCCCCAGCAGCGGCGGCAAG CGGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTAC GACGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTC TGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTA GGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 119)

In some embodiments, an expression repressor comprises the amino acid sequence of SEQ ID NOs: 35 or 151. In some embodiments, an expression repressor described herein comprises an amino acid sequence of SEQ ID NO: 35, 151, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. dCas9-MQl Protein sequence:

MAPKKKRKVGHGVPAADKKYSIGLAIGTNSVGWAVITOEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDKRP AATKKAGQAKKKKARDSKVENKTKKLRVFEAFAGIGAQRKALE KVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEYKSVSREEMIDYLENKTLSWNSKNPVSN GYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRTLKNIDLLTYSFPCQDLSQQGIQKGMKRG SGTRSGLLWEIERALDSTEKNDLPKYLLMENVGALLHKKNEEELNQWKQKLESLGYQNSIEVLN AADFGSSQARRRVFMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNLTEFKKTKS NINKASLIGYSKFNSEGYVYDPEFTGPTLTASGANSRIKIKDGSNIRKMNSDETFLYIGFDSQDGK RVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGGPSSGGKRPAATKKAGQAKKKKGSYPYDVPDY A (SEQ ID NO: 35)

MAPKKKRKVGHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDDQ RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDKRP AATKKAGQAKKKKARDSKVENKTKKLRVFEAFAGIGAQRKALE KVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEYKSVSREEMIDYLENKTLSWNSKNPVSN GYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRTLKNIDLLTYSFPCQDLSQQGIQKGMKRG SGTRSGLLWEIERALDSTEKNDLPKYLLMENVGALLHKKNEEELNQWKQKLESLGYQNSIEVLN AADFGSSQARRRVFMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNLTEFKKTKS NINKASLIGYSKFNSEGYVYDPEFTGPTLTASGANSRIKIKDGSNIRKMNSDETFLYIGFDSQDGK RVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGGPSSGGKRPAATKKAGQAKKKKGS (SEQ ID NO: 151)

In some embodiments, an expression repressor comprises a targeting moiety comprising dCas9, e.g., an S. pyogenes dCas9, and an effector moiety comprising KRAB, e.g., a KRAB domain. In some embodiments, the expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 67 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor). In some embodiments, a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 67 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. dCas9-KRAB nucleotide sequence:

AAGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAG AAGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCC TGGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAG CAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGC GCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGC GGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGAT GGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGAC AAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGA AGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCT GCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTA

CAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTG

AGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGA

AGAAGAACGGCCTGT CGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAG

AGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACG

ACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAG

AACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGG

CCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCT

GAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGC

AAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCA

TCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGA

GGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTG

GGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACC

GGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGG

GGCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACT

TCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTT

CGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTC

ACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCT

TCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGT

GACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAG

ATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGA

TCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGT

GCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCC

CACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCC

GGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGA

CTTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCC

TGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGA

GCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAG

GTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGA

TGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGC

GGATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGA

ACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTA

CGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCC

CAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCC GGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGC

GGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGA

GCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACC

CGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACG

AGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGA

CTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG

ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG

CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG

CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA

GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC

GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA

GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA

GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC

AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG

TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT

GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG

GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC

GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG

CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG

GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG

AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT

GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC

CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA

CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC

ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAAGCGGCCCGCCG

CCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGCCAGCGACGCCAAGAGCCTGACCG

CCTGGAGCCGGACCCTGGTGACCTTCAAGGACGTGTTCGTGGACTTCACCCGGGAGGAGTGG

AAGCTGCTGGACACCGCCCAGCAGATCCTGTACCGGAACGTGATGCTGGAGAACTACAAGA

ACCTGGTGAGCCTGGGCTACCAGCTGACCAAGCCCGACGTGATCCTGCGGCTGGAGAAGGG

CGAGGAGCCCTGGCTGGTGGAGCGGGAGATCCACCAGGAGACCCACCCCGACAGCGAGACC

GCCTTCGAGATCAAGAGCAGCGTGAGCGGCGGCAAGCGGCCCGCCGCCACCAAGAAGGCCG

GCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGAGCGGC

CGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCA

CCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAA (SEQ ID NO: 67)

In some embodiments, an expression repressor comprises the amino acid sequence of SEQ ID NOs: 34 or 150. In some embodiments, a nucleic acid described herein comprises an amino acid sequence of SEQ ID NO: 34, 150, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. dCas9-KRAB Protein sequence:

MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLED SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI

AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDKRPAATKKAGQAKKKKASDAKSLTAWSRTLVTFKDVFVDFTREEWK LLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIK SSVSGGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 34) MAPKKKRKVGHGVPAADKKYSIGLAIGTNSVGWAVITOEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS

MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDKRPAATKKAGQAKKKKASDAKSLTAWSRTLVTFKDVFVDFTREEWK LLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIK SSVSGGKRPAATKKAGQAKKKKGS (SEQ ID NO: 150)

In some embodiments, an expression repressor comprises a DNA-targeting moiety comprising dCas9, e.g., an S. aureus dCas9, and an effector moiety comprising DNMT1, e.g., human DNMT1. In some embodiments, the expression repressor is encoded by the nucleic acid sequence of SEQ ID NO: 69 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor). In some embodiments, a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 69 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. dCas9-DNMTl nucleotide sequence

AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA

AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT

GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC

AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG

CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG

GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG

GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA

AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA

GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG

CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG

CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC

AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA

GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA

GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA

GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA

CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA

ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC

CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG

AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA

AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT

CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG

GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG

GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG

GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG

GCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTT

CGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTC

GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA

CCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTT

CCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTG

ACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGA

TCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGAT

CATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTG

CTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCC ACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCG

GCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGAC

TTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCT

GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAG

CACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGG

TGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGAT

GGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCG

GATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAA

CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTAC

GTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCC

AGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCG

GGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCG

GCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAG

CGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCC

GGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGA

GAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGAC

TTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG

ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG

CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG

CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA

GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC

GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA

GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA

GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC

AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG

TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT

GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG

GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC

GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG

CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG

GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG

AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT

GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC

CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC

ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAGCGGCGGCAAGC

GGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGTCGGGCGGGGGTGGCT

CAGTGGATCTGAGGACACTCGACGTGTTTAGCGGATGCGGCGGACTCTCCGAAGGCTTCCAC

CAAGCCGGAATTTCCGACACACTCTGGGCCATTGAGATGTGGGACCCCGCCGCTCAAGCCTT

CAGACTGAATAATCCCGGCTCCACCGTGTTCACCGAGGACTGCAACATTCTGCTGAAGCTGG

TGATGGCTGGCGAAACCACCAACTCTAGAGGCCAGAGGCTGCCCCAGAAGGGAGATGTGGA

AATGCTCTGTGGAGGCCCTCCTTGCCAAGGCTTCTCCGGCATGAACAGGTTCAACTCTAGAA

CATACAGCAAGTTCAAGAACTCTCTGGTCGTGAGCTTTCTGAGCTACTGCGACTACTATAGA

CCTAGGTTCTTTCTGCTGGAGAACGTGAGAAATTTCGTGTCCTTCAAGAGGAGCATGGTGCT

GAAGCTGACACTGAGGTGTCTGGTGAGGATGGGCTACCAGTGCACATTCGGAGTGCTGCAA

GCTGGCCAGTACGGCGTGGCCCAGACCAGAAGGAGGGCCATCATTCTGGCTGCTGCCCCCG

GCGAGAAACTCCCTCTGTTCCCCGAGCCCCTCCACGTGTTCGCCCCTAGAGCTTGCCAGCTG

AGCGTGGTGGTCGACGATAAGAAGTTCGTGAGCAACATCACAAGGCTGTCCAGCGGACCCT

TCAGAACCATTACCGTGAGGGATACCATGTCCGACCTCCCCGAGGTGAGGAATGGCGCCAG

CGCTCTGGAGATTTCCTACAACGGCGAACCTCAGAGCTGGTTCCAAAGGCAGCTGAGAGGC

GCTCAGTATCAGCCCATTCTGAGGGACCACATCTGCAAAGATATGAGCGCTCTGGTGGCCGC

TAGAATGAGACATATTCCTCTGGCCCCCGGCAGCGACTGGAGAGATCTGCCCAATATTGAGG

TGAGACTCAGCGACGGAACAATGGCTAGAAAACTGAGGTACACCCATCATGATAGAAAGAA

CGGAAGGAGCAGCAGCGGCGCTCTGAGAGGAGTGTGTAGCTGCGTGGAAGCTGGCAAGGCT

TGCGATCCCGCCGCTAGGCAGTTCAATACCCTCATCCCTTGGTGTCTGCCTCACACCGGCAA

CAGACACAATCATTGGGCTGGACTGTATGGAAGGCTCGAATGGGACGGCTTTTTCAGCACCA

CCGTGACCAATCCCGAACCTATGGGCAAGCAAGGAAGGGTGCTCCACCCCGAGCAGCATAG

AGTCGTGTCCGTGAGAGAATGCGCTAGAAGCCAAGGCTTCCCCGACACCTATAGACTGTTCG

GCAACATTCTGGATAAGCACAGACAAGTGGGAAATGCTGTCCCTCCTCCTCTGGCCAAGGCT

ATCGGACTGGAGATCAAGCTGTGTATGCTCGCCAAAGCTAGGGAGAGCGCTTCCGCCAAGA

TTAAGGAGGAGGAGGCCGCCAAGGACGGAGGTGGCGGATCGGGAAAGCGGCCCGCCGCCA

CCAAGAAGGCCGGTCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTA

CGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCT

TCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAA (SEQ ID NO: 69) In some embodiments, an expression repressor comprises the amino acid sequence of SEQ ID NOs: 36, or 152. In some embodiments, an expression repressor described herein comprises an amino acid sequence of SEQ ID NO: 36, 152, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. dCas9-DNMTl Protein sequence:

MAPKKKRKVGHGVPAADKKYSIGLAIGTNSVGWAVITOEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEET1TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDSGGKRPAATKKAGQAKKKKSGGGGSVDLRTLDVFSGCGGLSEGFHQ

AGISDTLWAIEMWDPAAQAFRLNNPGSTVFTEDCNILLKLVMAGETTNSRGQRLPQKGDVEML CGGPPCQGFSGMNRFNSRTYSKFKNSLVVSFLSYCDYYRPRFFLLENVRNFVSFKRSMVLKLTLR CLVRMGYQCTFGVLQAGQYGVAQTRRRAIILAAAPGEKLPLFPEPLHVFAPRACQLSVVVDDKK FVSNITRLSSGPFRTITVRDTMSDLPEVRNGASALEISYNGEPQSWFQRQLRGAQYQPILRDHICK DMSALVAARMRHIPLAPGSDWRDLPNIEVRLSDGTMARKLRYTHHDRKNGRSSSGALRGVCSC VEAGKACDPAARQFNTLIPWCLPHTGNRHNHWAGLYGRLEWDGFFSTTVTNPEPMGKQGRVL HPEQHRVVSVRECARSQGFPDTYRLFGNILDKHRQVGNAVPPPLAKAIGLEIKLCMLAKARESAS AKIKEEEAAKDGGGGSGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 36)

MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITOEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDSGGKRPAATKKAGQAKKKKSGGGGSVDLRTLDVFSGCGGLSEGFHQ AGISDTLWAIEMWDPAAQAFRLNNPGSTVFTEDCNILLKLVMAGETTNSRGQRLPQKGDVEML CGGPPCQGFSGMNRFNSRTYSKFKNSLVVSFLSYCDYYRPRFFLLENVRNFVSFKRSMVLKLTLR CLVRMGYQCTFGVLQAGQYGVAQTRRRAIILAAAPGEKLPLFPEPLHVFAPRACQLSVVVDDKK

FVSNITRLSSGPFRTITVRDTMSDLPEVRNGASALEISYNGEPQSWFQRQLRGAQYQPILRDHICK DMSALVAARMRHIPLAPGSDWRDLPNIEVRLSDGTMARKLRYTHHDRKNGRSSSGALRGVCSC VEAGKACDPAARQFNTLIPWCLPHTGNRHNHWAGLYGRLEWDGFFSTTVTNPEPMGKQGRVL HPEQHRVVSVRECARSQGFPDTYRLFGNILDKHRQVGNAVPPPLAKAIGLEIKLCMLAKARESAS AKIKEEEAAKDGGGGSGKRPAATKKAGQAKKKKGS (SEQ ID NO: 152) In some embodiments, an expression repressor comprises a DNA-targeting moiety comprising dCas9, e.g., an S. aureus dCas9, and an effector moiety comprising DNMT13a/3L. In some embodiments, the expression repressor is encoded by the nucleic acid sequence of SEQ ID NO: 70 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor). In some embodiments, a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 70 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. dCas9-DNMT3a/3L nucleotide sequence AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG GCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTT

CGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTC

GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA

CCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTT

CCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTG

ACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGA

TCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGAT

CATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTG

CTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCC

ACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCG

GCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGAC

TTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCT

GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAG

CACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGG

TGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGAT

GGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCG

GATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAA

CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTAC

GTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCC

AGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCG

GGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCG

GCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAG

CGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCC

GGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGA

GAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGAC

TTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG

ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG

CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG

CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA

GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC

GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA

GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA

GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC

AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT

GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG

GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC

GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG

CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG

GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG

AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT

GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC

CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA

CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC

ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAGCGCCGGCGGCG

GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCCCCAAGAAGAAGCGGAAGG

TGGCCGCCGCCGGCAGCAACCACGACCAGGAGTTCGACCCCCCCAAGGTGTACCCCCCCGT

GCCCGCCGAGAAGCGGAAGCCCATCCGGGTGCTGAGCCTGTTCGACGGCATCGCCACCGGC

CTGCTGGTGCTGAAGGACCTGGGCATCCAGGTGGACCGGTACATCGCCAGCGAGGTGTGCG

AGGACAGCATCACCGTGGGCATGGTGCGGCACCAGGGCAAGATCATGTACGTGGGCGACGT

GCGGAGCGTGACCCAGAAGCACATCCAGGAGTGGGGCCCCTTCGACCTGGTGATCGGCGGC

AGCCCCTGCAACGACCTGAGCATCGTGAACCCCGCCCGGAAGGGCCTGTACGAGGGCACCG

GCCGGCTGTTCTTCGAGTTCTACCGGCTGCTGCACGACGCCCGGCCCAAGGAGGGCGACGAC

CGGCCCTTCTTCTGGCTGTTCGAGAACGTGGTGGCCATGGGCGTGAGCGACAAGCGGGACAT

CAGCCGGTTCCTGGAGAGCAACCCCGTGATGATCGACGCCAAGGAGGTGAGCGCCGCCCAC

CGGGCCCGGTACTTCTGGGGCAACCTGCCCGGCATGAACCGGCCCCTGGCCAGCACCGTGA

ACGACAAGCTGGAGCTGCAGGAGTGCCTGGAGCACGGCCGGATCGCCAAGTTCAGCAAGGT

GCGGACCATCACCACCCGGAGCAACAGCATCAAGCAGGGCAAGGACCAGCACTTCCCCGTG

TTCATGAACGAGAAGGAGGACATCCTGTGGTGCACCGAGATGGAGCGGGTGTTCGGCTTCC

CCGTGCACTACACCGACGTGAGCAACATGAGCCGGCTGGCCCGGCAGCGGCTGCTGGGCCG

GAGCTGGAGCGTGCCCGTGATCCGGCACCTGTTCGCCCCCCTGAAGGAGTACTTCGCCTGCG

TGAGCAGCGGCAACAGCAACGCCAACAGCCGGGGCCCCAGCTTCAGCAGCGGCCTGGTGCC

CCTGAGCCTGCGGGGCAGCCACATGAATCCTCTGGAGATGTTCGAGACAGTGCCCGTGTGGA

GAAGGCAACCCGTGAGGGTGCTGAGCCTCTTCGAGGACATTAAGAAGGAGCTGACCTCTCT

GGGCTTTCTGGAATCCGGCAGCGACCCCGGCCAGCTGAAACACGTGGTGGACGTGACCGAC

ACAGTGAGGAAGGACGTGGAAGAGTGGGGCCCCTTTGACCTCGTGTATGGAGCCACACCTC

CTCTCGGCCACACATGCGATAGGCCTCCCAGCTGGTATCTCTTCCAGTTCCACAGACTGCTCC

AGTACGCCAGACCTAAGCCCGGCAGCCCCAGACCCTTCTTCTGGATGTTCGTGGACAATCTG GTGCTGAACAAGGAGGATCTGGATGTGGCCAGCAGATTTCTGGAGATGGAACCCGTGACAA TCCCCGACGTGCATGGCGGCTCTCTGCAGAACGCCGTGAGAGTGTGGTCCAACATCCCCGCC ATTAGAAGCAGACACTGGGCTCTGGTGAGCGAGGAGGAACTGTCTCTGCTGGCCCAGAATA AGCAGTCCTCCAAGCTGGCCGCCAAGTGGCCCACCAAGCTGGTGAAGAACTGCTTTCTGCCT CTGAGGGAGTATTTCAAGTATTTCAGCACCGAACTGACCAGCAGCCTGAGCGGCGGCAAGC GGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACG ACGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCT GGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAG GAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO: 70)

In some embodiments, an expression repressor comprises the amino acid sequence of SEQ ID NO: 37 or 153. In some embodiments, an expression repressor described herein comprises an amino acid sequence of SEQ ID NO: 37 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. dCas9-DNMT3a/3L protein sequence MAPKKKRKVGHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLED SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS

LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSNHDQEFDPPKVYPPVP AEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQ KHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFEN VVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLE HGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLAR QRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMNPLEMFETVP VWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPP LGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPD

VHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYF KYFSTELTSSLSGGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 37)

MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL

TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSNHDQEFDPPKVYPPVP AEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQ KHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFEN VVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLE HGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLAR QRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMNPLEMFETVP VWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPP LGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPD VHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYF

KYFSTELTSSLSGGKRPAATKKAGQAKKKKGS (SEQ ID NO: 153)

In some embodiments, an expression repressor comprises a targeting moiety comprising a Zn Finger domain, and an effector moiety comprising KRAB, e.g., a KRAB domain. In some embodiments, the expression repressors are encoded by a nucleic acid sequence of any of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 189, 194, 195, and 196 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor). The nucleic acid sequences of these exemplary expression repressors are disclosed in Table 9. In some embodiments, a nucleic acid described herein comprises a nucleic acid sequence of any of SEQ ID NOs: 55-60, 189, 194-196, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. In some embodiments, the nucleic acid sequence comprises a poly-A sequence, and in other embodiments, the nucleic acid lacks the poly-A sequence.

Table 9: Nucleotide sequences of exemplary ZF-KRAB effectors

In some embodiments, an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., having an amino acid sequence according to any of SEQ ID NO: 5-10 or 169-172), and an effector moiety comprising KRAB (e.g., an amino acid sequence SEQ ID NO: 18), e.g., a KRAB domain, hr some embodiments, an expression repressor described herein comprises an amino sequence of any of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 139-144, 177-180, or 183-186. The protein sequence of these exemplary expression repressors are disclosed in Table 10. In some embodiments, an expression repressor described herein comprises an amino acid sequence of any of SEQ ID NOs: 22-27, 139-144, 177-180, 183-186 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

Table 10: Amino Acid sequences of exemplary Zinc Finger-KRAB effectors

In some embodiments, an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., one encoded by a nucleotide sequence of any of SEQ ID NO: 44-49 or 115), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., one encoded by a nucleotide sequence of SEQ ID NO: 52). In some embodiments, the expression repressors are encoded by the nucleic sequence of SEQ ID NOs: 61, 62, 63, 64, 65, 66, 116, 117, 118, or 130. The nucleic acid sequence of these exemplary expression repressors are disclosed in Table 11. In some embodiments, a nucleic acid described herein comprises a nucleic acid sequence of any of SEQ ID NO: 61-66, 116-118, 130 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. In some embodiments, the nucleic acid sequence comprises a poly-A sequence, and in other embodiments, the nucleic acid lacks the poly-A sequence. For example, in some embodiments, a nucleic acid described herein comprises a sequence according to any of SEQ ID NO: 61-66, 116-118, or 130 (or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto), but lacking the 3’ poly-A sequence, or comprising a 3’ poly-A sequence of a shorter length.

Table 11: Nucleotide sequences of exemplary ZF-MQ1 effectors

In some embodiments, an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., comprising an amino acid sequence of any of SEQ ID NO: 11-14), and an effector moiety' comprising MQ1, e.g., abacterial MQ1 (e.g., SEQ ID NO: 19). In some embodiments, the expression repressor comprises an amino sequence of any of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 129, and 145-149. The protein sequence of these exemplary expression repressors are disclosed in Table 12. In some embodiments, an expression repressor described herein comprises an amino acid sequence of any of SEQ ID NOs: 28-33, 129 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

Table 12 Amino acid sequences of exemplary ZF-MQ1 effectors

In some embodiments an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., having an amino acid sequence of any of SEQ ID NO: 11-14), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., SEQ ID NO: 87). In some embodiments, an expression repressor comprises a targeting moiety comprising a Zn

Finger domain (e.g., one encoded by a nucleotide sequence of any of SEQ ID NO: 166-168), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., one encoded by a nucleotide sequence of SEQ ID NO: 52). In some embodiments, the expression repressors are encoded by the nucleic sequence of SEQ ID NOs: 157, 158, or 159. The nucleic acid sequence of these exemplary expression repressors are disclosed in Table 13. In some embodiments, a nucleic acid described herein comprises a nucleic acid sequence of any of SEQ ID NO: 166-168 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. In some embodiments, the nucleic acid sequence comprises a poly -A sequence, and in other embodiments, the nucleic acid lacks the poly-A sequence. For example, in some embodiments, a nucleic acid described herein comprises a sequence according to any of SEQ ID NO: 166-168 (or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto), but lacking the 3’ poly-A sequence, or comprising a 3’ poly -A sequence of a shorter length.

Table 13: Nucleotide sequences of exemplary mouse-specific ZF-MQ1 effectors

In some embodiments, an expression repressor comprises a targeting moiety comprising a Zn Finger domain (e.g., comprising an amino acid sequence of any of SEQ ID NO:154-156), and an effector moiety comprising MQ1, e.g., abacterial MQ1 (e.g., SEQ ID NO: 19). In some embodiments, the expression repressor comprises an amino sequence of any of SEQ ID NOs: 160-165. The protein sequences of these exemplary expression repressors are disclosed in Table 14. In some embodiments, an expression repressor described herein comprises an amino acid sequence of any of SEQ ID NOs: 160-165 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

Table 14: Amino acid sequences of exemplary ZF-MQ1 effectors

In some embodiments, the present disclosure provides an expression repressor system comprising a first targeting moiety comprising a first ZF, a first effector moiety comprising a DNA methyltransferase, e.g., MQ1 or a functional fragment thereof, a second targeting moiety comprising a second ZF, and a second effector moiety comprising KRAB, e.g., a KRAB domain. In some embodiments, the expression repressor system is encoded by a first nucleic acid encoding the first targeting moiety and first effector moiety, wherein expression is driven by a first promoter or IRES, and a second nucleic acid encoding the second targeting moiety and second effector moiety, wherein expression is driven by a second promoter or IRES. In some embodiments, mono-cistronic sequences are used. In some embodiments, the nucleic acid encoding the expression repressor system is a multi-cistronic sequence. In some embodiments, the multi-cistronic sequence is a bi-cistronic sequence. In some embodiments, the multi-cistronic sequence comprises a sequence encoding the first expression repressor and a sequence encoding the second expression repressor. In some embodiments, the multi-cistronic sequence encodes a self-cleavable peptide sequence, e.g., a 2A peptide sequence, e.g., a T2A peptide sequence, a P2A sequence. In some embodiments, the multi-cistronic sequence encodes a T2A peptide sequence and a P2A peptide sequence. In some embodiments, the multi-cistronic sequence encodes a tandem 2A sequence, e g., a tPT2A sequence. In some embodiments, the multi-cistronic construct encodes, from 5’ to 3’, (i) a first nuclear localization signal, e.g., a SV40 NLS, (ii) a first targeting moiety, e.g., a DNA binding domain, e.g., a zinc finger binding domain, e.g., ZF-9, (iii) a first effector moiety, e.g., a DNA methyltransferase, e.g., MQ1, (iv) a second nuclear localization signal, e.g., a nucleoplasmin NLS, (v) a linker, e.g., atPT2A linker, (vi) a third nuclear localization signal, e.g., a SV40NLS, (vii) a second targeting moiety, e.g., a DNA binding domain, e g., a zinc finger binding domain, e.g., ZF-3, (viii) a second effector moiety, e.g., a transcription repressor moiety, e g., KRAB, and (ix) a fourth nuclear localization signal, e.g., a nucleoplasmin NLS. In some embodiments, the bi-cistronic construct further comprises a polyA tail. In some embodiments, upon transcription of the bi-cistronic gene construct, a single mRNA transcript encoding the first expression repressor, and the second expression repressor are produced, which upon translation gets cleaved, e.g., after the glycine residue within the 2A peptide, to yield the first expression repressor and the second expression repressor as two separate proteins. In some embodiments, the first and the second expression repressor are separated by “ribosome-skipping”. In some embodiments the first expression repressor and/ or the second expression repressor retains a fragment of the 2A peptide after ribosome skipping. In some embodiments, the expression level of the first and second expression repressor are equal. In some embodiments, the expression level of the first and the second expression repressor are different. In some embodiments, the protein level of the first expression repressor is within 1%, 2%, 5%, or 10% of (greater than or less than) the protein level of the second expression repressor.

In some embodiments, a system encoded by a bi-cistronic nucleic acid decreases expression of a target gene (e.g., MYC) at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, in a cell, than an otherwise similar system wherein the first and second expression repressor are encoded by mono-cistronic nucleic acids.

In some embodiments, the bi-cistronic sequence encodes an amino acid of SEQ ID NO: 91, 92, 121, 122, 181, 182, 187, 188, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. In some embodiments, an expression repressor system comprises a targeting moiety comprising a Zn Finger domain (e.g., comprising an amino acid sequence of any of SEQ ID NO:7 or 13), and an effector moiety comprising MQ1, e.g., a bacterial MQ1 (e.g., SEQ ID NO: 19) or KRAB, e.g., a KRAB domain (e.g., SEQ ID NO: 18). In some embodiments, the expression repressor comprises an amino sequence of any of SEQ ID NOs: 91, 92, 121, 122, 181, 182, 187, 188. The protein sequence of these exemplary expression repressor systems are disclosed in Table 15. In some embodiments, an expression repressor system described herein comprises an amino acid sequence of any of SEQ ID NOs: 91-92, 121-122, 181, 182, 187, 188, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

In some embodiments, the bi-cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 93 or 112 (e g., a nucleic acid (e.g., cDNA) encoding the expression repressor) or SEQ ID NO: 94 or 113 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor). In some embodiments, the bi- cistronic sequence comprises nucleic acid sequence of SEQ ID NO: 196 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor) or SEQ ID NO: 197 (e.g., a nucleic acid (e.g., cDNA) encoding the expression repressor), hr some embodiments, a nucleic acid described herein comprises a nucleic acid sequence of SEQ ID NO: 93, 94, 112, 113, 196, or 197, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto. The nucleic acid sequence encoding these exemplary expression repressor systems are disclosed in Table 15. In some embodiments, the nucleic acid sequence comprises a poly-A sequence, and in other embodiments, the nucleic acid lacks the poly -A sequence. Table 15: Amino acid sequences of, and Nucleic acid sequences encoding, exemplary expression repressor systems

In some embodiments, an expression repressor comprises a nuclear localization sequence (NLS). In some embodiments, the expression repressor comprises an NLS, e.g., an SV40 NLS at the N-terminus. In some embodiments, the expression repressor comprises an NLS, e.g., a nucleoplasmin NLS at the C- terminus. In some embodiments, the expression repressor comprises a first NLS at the N-terminus and a second NLS at the C-terminus. In some embodiments the first and the second NLS have the same sequence. In some embodiments, the first and the second NLS have different sequences. In some embodiments, the expression repression repressor comprises an SV40 NLS, e.g., the expression repressor comprises a sequence according to PKKKRK (SEQ ID NO: 135). In some embodiments, the N-terminal sequence comprises an NLS and a spacer, e.g., having a sequence according to: MAPKKKRKVGIHGVPAAGSSGS (SEQ ID NO: 88). In some embodiments, the expression repressor comprises a C-terminal sequence comprising one or more of, e.g., any two or all three of: a spacer, a nucleoplasmin nuclear localization sequence and an HA-tag: e.g., SGGKRPAATKKAGQAKKKGSYPYDVPDYA (SEQ ID NO: 89). In some embodiments, the expression repressor comprises an epitope tag, e.g., an HA tag: YPYDVPDYA (SEQ ID NO: 90). For example, the expression repressor may comprise two copies of the epitope tag.

While an epitope tag is useful in many research contexts, it is sometimes desirable to omit an epitope tag in a therapeutic context. Accordingly, in some embodiments, the expression repressor lacks an epitope tag. In some embodiments, an expression repressor described herein comprises a sequence provided herein (or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto), but lacking the HA tag of SEQ ID NO: 90. In some embodiments, a nucleic acid described herein comprises a sequence provided herein (or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto), but lacking a region encoding the HA tag of SEQ ID NO: 90. In some embodiments, the expression repressor comprises a nucleoplasmin NLS, e.g., the expression repressor comprises a sequence of KRPAATKKAGQAKKK (SEQ ID NO: 136). In some embodiments, the expression repressor does not comprise an NLS. In some embodiments, the expression repressor does not comprise an epitope tag. In some embodiments the expression repressor does not comprise an HA tag. In some embodiments, the expression repressor does not comprise an HA tag sequence according to SEQ ID NO: 90.

In some embodiments, the present disclosure provides an expression repressor system comprises a self-cleaving peptide. Self-cleavmg peptides, first discovered in picomaviruses, are peptides of between 19 to 22 amino acids in length and are usually found between two proteins in some members of the picomavirus family. Using self-cleaving proteins, picomaviruses are capable of producing equimolar levels of multiple genes from the same mRNA. Such self-cleaving proteins are known to be found in other species of viruses and a person skilled in the art, based on the information provided herein, will be readily able to determine a suitable substitution for the self-cleaving protein disclosed herein, if required. In some embodiments, an expression repressor system comprises a self-cleaving peptide, e.g., a 2A selfcleaving peptide. In some embodiments, the 2A peptide comprises a single cleavage site, e.g., a 2A peptide, e.g., a P2A, a T2A, a E2A, or a F2A peptide. In some embodiments the self-cleaving peptide, e.g., a 2A peptide, comprises two cleavage sites, e.g., pPT2A, or P2A-T2A-E2A. In some embodiments, an expression repressor system comprises a self-cleaving peptide comprising a plurality of cleavage sites, e.g., a T2A self-cleaving peptide and a P2A self-cleaving peptide. In some embodiments, the 2A peptide gets cleaved after translation. In some embodiments, the self-cleaving peptide produces two or more fragments after cleaving. In some embodiments, the 2A peptide fragments comprise the sequences of SEQ ID NO: 126-127. In some embodiments, the 2A self-cleaving peptide comprises a sequence of SEQ ID NO: 120, 124, 125 or derivative thereof. In some embodiments, SEQ ID NO: 95 comprises a sequence of a self-cleaving peptide.

PSSGGKRPAATKKAGQAKKKKGSYPYDVPDYAGSYPYDVPDYAATNFSLLKQAGDVEENPGPT SAGKLGSGEGRGSLLTCGDVEENPGPLEGSSGSGSPKKKRKVGIHGVPAAGSSGS (SEQ ID NO: 95)

EGRGSLLTCGDVEENPGP (SEQ ID NO: 120) ATNFSLLKQAGDVEENPGPTSAGKLGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 124) ATNFSLLKQAGDVEENPGP (SEQ ID NO: 125)

ATNFSLLKQAGDVEENPG (SEQ ID NO: 126)

PTSAGKLGSGEGRGSLLTCGDVEENPG (SEQ ID NO: 127) P

It is of course understood that although a 2A sequence, e.g., tPT2A sequence (e.g., according to SEQ ID NO: 124), may be referred to in the scientific literature and herein as a self-cleaving peptide, this is according to a non-limiting theory. According to another non-limiting theory, in some embodiments, a 2A sequence acts via ribosome-skipping. For instance, an mRNA encoding a 2A sequence may induce ribosome skipping, wherein the ribosome fails to form a peptide bond while translating the 2A region, resulting in a release of the first part of the translation product. The ribosome then produces the second part of the translation product. Overall, it is well established that a 2A sequence placed between a first sequence and a second sequence will lead to the production of a first protein comprising the first sequence and a separate, second protein comprising the second sequence. This disclosure is not bound by any particular theory as to the molecular mechanism by which this is achieved.

Functional Characteristics

An expression repressor or a system of the present disclosure can be used to decrease expression of a target gene, e.g., MYC, in a cell. In general, an expression repressor or a system as described herein binds (e.g., via a targeting moiety) a genomic sequence element proximal to and/or operably linked to a target gene, e.g., MYC. In some embodiments, binding of the expression repressor or the system to the genomic sequence element modulates (e.g., decreases) expression of the target gene, e.g., MYC. For example, binding of an expression repressor or a system comprising an effector moiety that inhibits recruitment of components of the transcription machinery to the genomic sequence element may modulate (e.g., decrease) expression of the target gene, e.g., MYC. As a further example, binding of an expression repressor or a system comprising an effector moiety with an enzymatic activity (e.g., an epigenetic modifying moiety) may modulate (e.g., decrease) expression of the target gene, e.g., MYC) through the localized enzymatic activity of the effector moiety. As a further example, both binding of an expression repressor or a system to a genomic sequence element and the localized enzymatic activity of an expression repressor or a system may contribute to the resulting modulation (e.g., decrease) in expression of the target gene, e.g., MYC.

In some embodiments, decreasing expression comprises decreasing the level of RNA, e.g., mRNA, encoded by the target gene e.g., MYC. In some embodiments, decreasing expression comprises decreasing the level of a protein encoded by the target gene e.g., MYC. hi some embodiments, decreasing expression comprises both decreasing the level of mRNA and protein encoded by the target gene e.g., MYC. In some embodiments, the expression of a target gene in a cell contacted by or comprising the expression repressor or the expression repression system disclosed herein is at least 1.05x (i.e., 1.05 times), l. lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7.x, 8x, 9x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or lOOx lower than the level of expression of the target gene in a cell not contacted by or comprising the expression repressor or the expression repression system disclosed herein. Expression of a target gene e.g., MYC may be assayed by methods known to those of skill in the art, including RT-PCR, ELISA, Western blot, and the methods of Examples 2-9. Expression level of a target gene, e.g., MYC in a subject, e.g., a patient, e.g., a patient having a MYC mis-regulation disorder, e.g., a patient having a hepatic disease, a patient having a neoplasia and/or viral or alcohol related hepatic disease, e.g., a patient having a hepatocarcinoma, e.g., a patient having a hepatocarcinoma subtype SI or hepatocarcinoma subtvpe S2, may be assessed by evaluating blood (e.g., whole blood) levels of the target gene, e.g., MYC, e.g., by the method of either Oglesbee et al. Clin Chem. 2013 Oct;59(10):1461-9. Doi:

10.1373/clinchem.2013.207472 or Deutsch et al. J Neurol Neurosurg Psychiatry. 2014 Sep;85(9):994- 1002. Doi: 10.1136/jnnp-2013-306788, the contents of which are hereby incorporated by reference in their entirety.

An expression repressor or a system of the present disclosure can be used to decrease expression of a target gene e.g., MY C in a cell for a time period. In some embodiments, the expression of a target gene e.g., MYC in a cell contacted by or comprising the expression repressor or the system is appreciably decreased for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, 14, or 15 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely). Optionally, the expression of a target gene, e.g., MYC in a cell contacted by or comprising the expression repressor or the system is appreciably decreased for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years. In some embodiments, the expression of a target gene e.g., MYC in a cell contacted by or comprising the expression repressor or the system is appreciably decreased for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions. An expression repressor or a system of the present disclosure can be used to methylate CpG nucleotides in a target promoter, e.g., MYC promoter. In some embodiments, the transcriptional changes in MY C expression correlates to percentage of CpG methylation. In some embodiments, the methylation persists for at least 1 days, at least 2 days, at least 5 days, at least 7 days, at least 10 days, at least 15 days, or at least 20 days post-treatment with an expression repressor or a system disclosed herein.

An expression repressor or a system of the present disclosure can be used to decrease the viability of a cell comprising the target locus, e.g., MYC locus. In some embodiments, expression repressor or a system of the present disclosure can be used to decrease the viability of a plurality of cells comprising the target locus, e.g., MYC locus. In some embodiments, the number of viable cells contacted by or comprising the expression repressor, or the system is appreciably decreased by 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to number of viable cells in a control population of cells that is not contacted by or does not comprise the expression repressor or the system.

In some embodiments, an expression repressor or a system of the present disclosure can be used to decrease the viability of a plurality of cells comprising cancer cells and non-cancer cells. In some embodiments, an expression repressor or a system of the present disclosure can be used to decrease the viability of the plurality of cancer cells more than it decreases the viability of the plurality of non-cancer cells. In some embodiments, an expression repressor or a system of the present disclosure can be used to decrease the viability of the plurality of cancer cells 1.05x (i.e., 1.05 times), l.lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx more than it decreases the viability of the plurality of non-cancer cells.

In some embodiments, an expression repressor or a system of the present disclosure can be used to decrease the viability of a plurality of cells comprising infected cells and uninfected cells. In some embodiments, an expression repressor or a system of the present disclosure can be used to decrease the viability of the plurality of infected cells more than it decreases the viability of the plurality of uninfected cells. In some embodiments, an expression repressor or a system of the present disclosure can be used to decrease the viability of the plurality of infected cells 1.05x (i.e., 1.05 times), l.lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 50x, or lOOx more than it decreases the viability of the plurality of uninfected cells.

An expression repression system may comprise a plurality of expression repressors, where each expression repressor comprises an effector moiety with a different functionality than the effector moiety of another expression repressor. For example, an expression repression system may comprise two expression repressors, where the first expression repressor comprises a first effector moiety comprising an epigenetic modifying moiety e.g., DNA methyltransferase, e.g., MQ1 and the second expression repressor comprises a second effector moiety comprising a transcription repressor, e.g., KRAB. In some embodiments, the second expression repressor does not comprise a second effector moiety. In some embodiments, an expression repression system comprises expression repressors comprising a combination of effector moieties whose functionalities are complementary to one another with regard to inhibiting expression of a target gene, e.g., MYC, where the functionalities together enable inhibition of expression and, optionally, do not inhibit or negligibly inhibit expression when present individually. In some embodiments, an expression repression system comprises a plurality of expression repressors, wherein each expression repressor comprises an effector moiety that complements the effector moieties of each other expression repressor, e.g., each effector moiety decreases expression of a target gene, e.g., MYC.

In some embodiments, an expression repression system comprises expression repressors comprising a combination of effector moieties whose functionalities synergize with one another with regards to inhibiting expression of a target gene, e.g., MYC. Without wishing to be bound by theory, epigenetic modifications to a genomic locus may be cumulative, in that multiple repressive epigenetic markers (e.g., multiple different types of epigenetic markers and/or more extensive marking of a given type) individually together reduce expression more effectively than individual modifications alone (e.g., producing a greater decrease in expression and/or a longer-lasting decrease in expression). In some embodiments, an expression repression system comprises a plurality of expression repressors, wherein each expression repressor comprises an effector moiety that synergizes with the effector moieties of each other expression repressor, e.g., each effector moiety decreases expression of a target gene, e.g., MYC.

In some embodiments, an expression repressor or a system modulates (e.g., decreases) expression of a target gene, e.g., MY C by altering one or more epigenetic markers associated with the target gene, e.g., MYC or an expression control sequence operably linked thereto. In some embodiments, altering comprises decreasing the level of an epigenetic marker associated with the target gene, e.g., MYC or an expression control sequence operably linked thereto. Epigenetic markers include, but are not limited to, DNA methylation, histone methylation, and histone deacetylation.

In some embodiments, altering the level of an epigenetic marker decreases the level of the epigenetic marker associated with the target gene, e.g., MYC or an expression control sequence operably linked thereto by at least 1.05x (i.e., 1.05 times), l. lx, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or lOOx lower than the level of the epigenetic marker associated with the target gene, e.g., MYC or an expression control sequence operably linked thereto in a cell not contacted by or comprising the expression repressor or the system. The level of an epigenetic marker may be assayed by methods known to those of skill in the art, including whole genome bisulfite sequencing, reduced representation bisulfite sequencing, bisulfite amplicon sequencing, methylation arrays, pyrosequencing, ChlP-seq, or ChlP-qPCR. hi some embodiments, the changes (e.g., increase or decrease) in epigenetic marker e.g., DNA methylation may be assayed using bisulfite genomic sequencing at precise genomic coordinates according to hgl9 reference genome, e.g., in between chr8: 129188693- 129189048 according to hgl9 reference genome. In some embodiments, the changes (e.g., increase or decrease) in epigenetic marker e.g., DNA methylation may be assayed using bisulfite genomic sequencing at a genomic location according to SEQ ID NO: 123. CAGAGAAGGAGGAAGTTAATTCACATTCTTAATTTTTTCTAAGGGCAAAAAAAAAAAAAAAATGCAC CAGCTCATTTTCCATCTCTGCTTGGGTCATCAGTGTGCATTGTGAGCCTGTACAAAGGCCTTAGACGGG GAATGCTGCCGAGAGCATCACCTTTTATGTCTTCTTTTATATGAAATGTGCCACTTCCCCACTAACCCT GGCTCTGGGCTCTGCCTCTGCTCTCCTGATGGTGTGTTTATGGTGGATTCAGCATTCTGGGCCACACAA GGAAGCTGCAGGGGGTGTCCAAGTTCACATGTCCCCGCATTCCAGGCGAATGTTTCTGACATTGAGCA ATGATATGGCTCT (SEQ ID NO: 123)

An expression repressor or the system of the present disclosure can be used to alter the level of an epigenetic marker associated with the target gene, e.g., MYC or an expression control sequence operably linked thereto in a cell for a time period. In some embodiments, the level of the epigenetic marker associated with the target gene or an expression control sequence operably linked thereto in a cell contacted by or comprising the expression repressor or the system is appreciably decreased for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely). Optionally, the level of an epigenetic marker associated with the target gene, e.g., MYC or an expression control sequence operably linked thereto in a cell contacted by or comprising the expression repressor or the system is appreciably decreased for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years.

Combinations of Repressors

In some embodiments, an expression repression system comprises a first expression repressor comprising a first effector moiety and a second expression repressor comprising a second effector moiety wherein the first effector moiety and second effector moiety are different from one another. In some embodiments, the first effector moiety is or comprises a first epigenetic modifying moiety (e.g., that increases or decreases a first epigenetic marker) or functional fragment thereof and the second effector moiety is or comprises a second epigenetic modifying moiety (e.g., that increases or decreases a second epigenetic marker) or functional fragment thereof. In some embodiments, the first effector moiety is or comprises a DNA methyltransferase or functional fragment thereof and the second effector moiety is or comprises a KRAB or functional fragment thereof. In some embodiments, the first effector moiety is or comprises a histone deacetylase or functional fragment thereof and the second effector moiety is or comprises a KRAB or functional fragment thereof. In some embodiments, the first effector moiety is or comprises a histone methyltransferase or functional fragment thereof and the second effector moiety n is or comprises a KRAB or functional fragment thereof. In some embodiments, the first effector moiety is or comprises a histone demethylase or functional fragment thereof and the second effector moiety is or comprises a KRAB or functional fragment thereof. In some embodiments, the first effector moiety is or comprises MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C. KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2 or a functional fragment of any thereof, and the second effector moiety is or comprises KRAB (e g., a KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional fragment of any thereof.

In some embodiments, the first effector moiety is or comprises KRAB (e.g., a KRAB domain), MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional fragment of any thereof, and the second effector moiety is or comprises MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, NO66, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2 or a functional fragment of any thereof.

In some embodiments, the first effector moiety is or comprises bacterial MQ 1 or a functional variant or fragment thereof, and the second effector moiety is or comprises KRAB or a functional variant or fragment thereof.

In some embodiments, the first effector moiety is or comprises DNMT3A or a functional variant or fragment thereof, and the second effector moiety is or comprises KRAB or a functional variant or fragment thereof.

In some embodiments, the first effector moiety is or comprises DNMT3B or a functional variant or fragment thereof, and the second effector moiety is or comprises KRAB or a functional variant or fragment thereof.

In some embodiments, the first effector moiety is or comprises DNMT3L or a functional variant or fragment thereof, and the second effector moiety is or comprises KRAB or a functional variant or fragment thereof.

In some embodiments, the first effector moiety is or comprises DNMT3a/3L complex or a functional variant or fragment thereof, and the second effector moiety is or comprises KRAB or a functional variant or fragment thereof. Target Sites

Expression repressors or expression repressor systems disclosed herein are useful for modulating, e.g., decreasing, expression of atarget gene, e.g., MYC in cell, e.g., in a subject or patient. A target gene, e.g., MYC may be any gene known to those of skill in the art. In some embodiments, a target gene, e.g., MYC is associated with a disease or condition in a subject, e.g., a mammal, e.g., a human, bovine, horse, sheep, chicken, rat, mouse, cat, or dog. A target gene may include coding sequences, e.g., exons, and/or non-coding sequences, e.g., introns, 3’UTR, or 5’UTR. In some embodiments, a target gene is operably linked to a transcription control element.

A targeting moiety suitable for use in an expression repressor or an expression repressor of system described herein may bind, e.g., specifically bind, to any site within atarget gene, e.g., MYC, transcription control element operably linked to a target gene, e.g., MYC to an anchor sequence (e.g., an anchor sequence proximal to a target gene or associated with an anchor sequence-mediated conjunction operably linked to atarget gene, e.g., MYC (e.g., an anchor sequence-mediated conjunction is operably linked to a target gene if disruption of the conjunction alters expression of the target gene, e g., MYC)), or to a regulatory element located in a super enhancer region (e.g., a regulatory element located in a super enhancer region of MYC).

In some embodiments, an expression repressor described herein binds at a site or at a location that is proximal to the site. For example, a targeting moiety may bind to a first site that is proximal to a repressor (the second site), and the effector moiety associated with said targeting moiety may epigenetically modify the first site such that the enhancer’s effect on expression of a target gene is modified, substantially the same as if the second site (the enhancer sequence) had been bound and/or modified. In some embodiments, a site proximal to atarget gene (e.g., an exon, intron, or splice site within the target gene), proximal to a transcription control element operably linked to the target gene, e.g., MYC, or proximal to an anchor sequence is less than 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 25 base pairs from the target gene, e.g., MYC (e.g., an exon, intron, or splice site within the target gene, e.g., MYC), transcription control element, or anchor sequence (and optionally at least 20, 25, 50, 100, 200, or 300 base pairs from the target gene, e g., MY C (e.g., an exon, intron, or splice site within the target gene), transcription control element, or anchor sequence).

In some embodiments, a targeting moiety binds to atarget gene, e.g., MYC. In some embodiments, a DNA-targeting moiety binds to a site within an exon of a target gene, e.g., MYC. In some embodiments, a targeting moiety binds to a site within an intron of atarget gene, e.g., MYC. In some embodiments, a targeting moiety binds to a site at the boundary of an exon and an intron, e.g., a splice site, of a target gene, e.g., MYC. In some embodiments, a targeting moiety binds to a site within the 5 ’UTR of a target gene, e.g., MY C. In some embodiments, a targeting moiety binds to a site within the 3 ’UTR of a target gene, e.g., MYC. Target genes include, but are not limited to the gene encoding MYC.

In some embodiments, a targeting moiety binds to a transcription control element operably linked to a target gene (e.g., MY C), e.g., a promoter or enhancer. In some embodiments, a targeting moiety binds to a portion of or a site within a promoter operably linked to a target gene, e.g., MYC. In some embodiments, a targeting moiety binds to the transcription start site of a target gene, e.g., MYC. In some embodiments, a targeting moiety binds to a portion of or a site within an enhancer operably linked to a target gene, e.g., MYC. In some embodiments, a genomic complex (e.g., ASMC) co-localizes two or more genomic sequence elements, wherein the two or more genomic sequence elements include a promoter. A promoter is, typically, a sequence element that initiates transcription of an associated gene. Promoters are typically near the 5’ end of a gene, not far from its transcription start site. As those of ordinary skill are aware, transcription of protein-coding genes in eukaryotic cells is typically initiated by binding of general transcription factors (e.g., TFIID, TFIIE, TFIIH, FUSE, CT-element etc.) and Mediator to core promoter sequences as a preinitiation complex that directs RNA polymerase II to the transcription start site, and in many instances remains bound to the core promoter sequences even after RNA polymerase escapes and elongation of the primary transcript is initiated. In some embodiments, a promoter includes a sequence element such as TATA, Inr, DPE, or BRE, but those skilled in the art are well aware that such sequences are not necessarily required to define a promoter. Those skilled in the art are familiar with a variety of positive (e.g., enhancers) or negative (e.g., repressors or silencers) transcription control elements that are associated with genes. In some embodiments, a transcription control element is a transcription factor binding site. Typically, when a cognate regulatory protein is bound to such a transcription control element, transcription from the associated gene(s) is altered (e.g., increased or decreased). In some embodiments, a targeting moiety binds to a genomic sequence located within a genomic coordinate GRCh37: chr8: 129162465-129212140.

In some embodiments, a targeting moiety binds to a target sequence comprised by or partially comprised by a genomic sequence element. In some embodiments, the genomic sequence element is or comprises an expression control sequence. In some embodiments, the genomic sequence element is or comprises the target gene, e.g., MY C or a part of the target gene, e.g., MYC. hi some embodiments, a targeting moiety binds to a target sequence that is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases long (and optionally no more than 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 bases long). In some embodiments, a targeting moiety binds to a target sequence that is 10-30, 15-30, 15-25, 18-24, 19-23, 20-23, 21-23, or 22- 23 bases long. In some embodiments, the target sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bases long. In some embodiments, the genomic sequence element is or comprises an anchor sequence.

Each ASMC comprises one or more anchor sequences, e.g., a plurality. In some embodiments, anchor sequences can be manipulated or altered to modulate (e.g., disrupt) a naturally occurring genomic complex (e.g., ASMC) or to form a new genomic complex (e.g., ASMC) (e.g., to form a non-naturally occurring genomic complex (e.g., ASMC) with an exogenous or altered anchor sequence). In some embodiments, an anchor sequence-mediated conjunction can be disrupted to alter, e g., inhibit, e.g., decrease expression of a target gene. Such disruptions may modulate gene expression by, e.g., changing topological structure of DNA, e.g., by modulating the ability of a target gene to interact with a transcription control element (e.g., enhancing and silencing/repressive sequences).

In some embodiments, a targeting moiety binds to an anchor sequence, e.g., an anchor sequence proximal to a target gene, e.g., MYC or associated with an anchor sequence-mediated conjunction (ASMC) operably linked to a target gene, e.g., MYC (e.g., an anchor sequence-mediated conjunction is operably linked to a target gene, e g., MYC if disruption of the conjunction alters expression of the target gene, e.g., MYC). In general, an anchor sequence is a genomic sequence element to which a genomic complex component, e.g., nucleating polypeptide binds specifically. In some embodiments, binding of a genomic complex component to an anchor sequence nucleates complex formation, e.g., ASMC formation. In some embodiments, a targeting moiety binds to a target gene, e.g., MYC locus. A locus is generally defined to encompass transcribed region, promoter, and anchor sites of an ASMC comprising a target gene, e.g., MYC. In some embodiments, a targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86 or 199-206. In some embodiments, the first targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86 and the second targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86, wherein the first and the second targeting moiety binds to the same sequence. In some embodiments, the first targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86 and the second targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 75-86 wherein the first and the second targeting moiety binds to different sequences. In some embodiments, the first targeting moiety binds to a sequence comprising any of SEQ ID NOs: 83, 203, or 206 and the second targeting moiety binds to a sequence comprising SEQ ID NO:77. hi some embodiments, the first targeting moiety binds to a sequence comprising SEQ ID NO: 77 and the second targeting moiety binds to a sequence comprising any of SEQ ID NOs: 83, 203, or 206. In some embodiments, the first targeting moiety binds to a sequence comprising any of SEQ ID NOs: 83, 203, or 206 and the second targeting moiety binds to a sequence comprising any of SEQ ID NOs: 199, 204, or 205. In some embodiments, the first targeting moiety binds to a sequence comprising any of SEQ ID NOs: 199, 204, or 205 and the second targeting moiety binds to a sequence comprising any of SEQ ID NOs:83, 203, or 206. In some embodiments, the first targeting moiety binds to a sequence comprising any of SEQ ID NOs: 83, 203, or 206 and the second targeting moiety binds to a sequence comprising SEQ ID NO: 201. In some embodiments, a nucleic acid encoding the first and second expression repressors comprises a first region that encodes the first expression repressor, wherein the first region is upstream of a second region that encodes the second expression repressor. In some embodiments, a nucleic acid encoding the first and second expression repressors comprises a first region that encodes the first expression repressor, wherein the first region is downstream of a second region that encodes the second expression repressor. In some embodiments, the first targeting moiety binds to a sequence comprising any one of SEQ ID NOs: 75-86 or 199-206, and the second targeting moiety (e.g., a CRISPR/Cas domain comprising a gRNA) binds to a sequence comprising any one of SEQ ID NOS: 1-4. In some embodiments, a targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96-110. In some embodiments, the first targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96-110 and the second targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96-110, wherein the first and the second targeting moiety binds to the same sequence. In some embodiments, the first targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96- 110 and the second targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 96-110 wherein the first and the second targeting moiety binds to different sequences. In some embodiments, the first targeting moiety binds to a sequence comprising any one of SEQ ID NOs: 96-110, and the second targeting moiety (e.g., a CRISPR/Cas domain comprising a gRNA) binds to a sequence comprising any one of SEQ ID NOS: 1-4. In some embodiments, the first targeting moiety binds to a sequence comprising any one of the SEQ ID Nos. disclosed in tables 2, 3, or 16, and the second targeting moiety (e.g., a CRISPR/Cas domain comprising a gRNA) binds to a sequence comprising any one of the SEQ ID Nos. disclosed in tables 2, 3, or 16.

Exemplary target sequences are disclosed in Table 16.

Table 16: Exemplary target sequences

In some embodiments, an expression repressor binds a genomic locus having a sequence set forth herein, e.g., any one of SEQ ID NOS: 1-4, 75-86, 96-110, or 199-206. It is understood that, in many cases, the genomic locus being bound comprises double stranded DNA, and this locus can be described by giving the sequence of its sense strand or its antisense strand. Thus, a gRNA having a given spacer sequence may cause expression repressor to bind to a particular genomic locus, wherein one strand of the genomic locus has a sequence similar or identical to the spacer sequence, and the other strand of the genomic locus has the complementary sequence. Typically, gRNA binding to the genomic locus will involve some unwinding of the genomic locus and pairing of the gRNA spacer with the strand to which it the spacer complementary.

In some embodiments, a targeting moiety binds to an anchor sequence, e g., an anchor sequence proximal to a target gene, e.g., MYC or associated with an anchor sequence-mediated conjunction (ASMC) operably linked to a target gene, e.g., MYC (e.g., an anchor sequence-mediated conjunction is operably linked to a target gene, e.g., MY C if disruption of the conjunction alters expression of the target gene, e.g., MYC) in mouse genome. In general, an anchor sequence is a genomic sequence element to which a genomic complex component, e.g., nucleating polypeptide binds specifically. In some embodiments, binding of a genomic complex component to an anchor sequence nucleates complex formation, e.g., ASMC formation. In some embodiments, a targeting moiety binds to a target gene, e.g., MYC locus. A locus is generally defined to encompass transcribed region, promoter, and anchor sites of an ASMC comprising a target gene, e.g., MYC. In some embodiments, a targeting moiety binds to a sequence comprising any one of SEQ ID NOS: 190-192. In some embodiments, the targeting moiety binds to a sequence comprising any one of the SEQ ID Nos. disclosed in Table 17. Exemplary target sequences in mouse genome are disclosed in Table 17.

Table 17: Exemplary target sequences in mouse genome

In some embodiments, an expression repressor binds a genomic locus having a sequence set forth herein, e.g., any one of SEQ ID NOS: 190-192. It is understood that, in many cases, the genomic locus being bound comprises double stranded DNA, and this locus can be described by giving the sequence of its sense strand or its antisense strand.

In one embodiment, the anchor sequence-mediated conjunction comprises a loop, such as an intra-chromosomal loop. In certain embodiments, the anchor sequence-mediated conjunction has a plurality of loops. One or more loops may include a first anchor sequence, a nucleic acid sequence, a transcriptional control sequence, and a second anchor sequence, hi another embodiment, at least one loop includes, in order, a first anchor sequence, a transcriptional control sequence, and a second anchor sequence, or a first anchor sequence, a nucleic acid sequence, and a second anchor sequence. In yet another embodiment, either one or both of the nucleic acid sequences and the transcriptional control sequence is located within or outside the loop. In still another embodiment, one or more of the loops comprises a transcriptional control sequence.

In some embodiments, the anchor sequence-mediated conjunction includes a TATA box, a CAAT box, a GC box, or a CAP site. In some embodiments, the anchor sequence-mediated conjunction comprises a plurality of loops, and where the anchor sequence-mediated conjunction comprises at least one of an anchor sequence, a nucleic acid sequence, and a transcriptional control sequence in one or more of the loops.

In some embodiments, chromatin structure is modified by substituting, adding, or deleting one or more nucleotides within an anchor sequence. In some embodiments, chromatin structure is modified by substituting, adding, or deleting one or more nucleotides within an anchor sequence of an anchor sequence-mediated conjunction. In some embodiments, transcription is inhibited by inclusion of an activating loop or exclusion of a repressive loop. In one such embodiment, the anchor sequence -mediated conjunction excludes a transcriptional control sequence that decreases transcription of the nucleic acid sequence. In some embodiments, transcription is repressed by inclusion of a repressive loop or exclusion of an activating loop, hi one such embodiment, the anchor sequence-mediated conjunction includes a transcriptional control sequence that decreases transcription of the nucleic acid sequence. The anchor sequences may be non-contiguous with one another. In embodiments with noncontiguous anchor sequences, the first anchor sequence may be separated from the second anchor sequence by about 500bp to about 500Mb, about 750bp to about 200Mb, about Ikb to about 100Mb, about 25kb to about 50Mb, about 50kb to about 1Mb, about lOOkb to about 750kb, about 150kb to about 15 500kb, or about 175kb to about 500kb. In some embodiments, the first anchor sequence is separated from the second anchor sequence by about 500bp, 600bp, 700bp, 800bp, 900bp, Ikb, 5kb, lOkb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 55kb, 60kb, 65kb, 70kb, 75kb, 80kb, 85kb, 90kb, 95kb, lOOkb, 125kb, 150kb, 175kb, 200kb, 225kb, 250kb, 275kb, 300kb, 350kb, 400kb, 500kb, 600kb, 700kb, 800kb, 900kb, 1Mb, 2Mb, 3Mb, 4Mb, 5Mb, 6Mb, 7Mb, 8Mb, 9Mb, 10Mb, 15Mb, 20Mb, 25Mb, 50Mb, 75Mb, 20 100Mb, 200Mb, 300Mb, 400Mb, 500Mb, or any size therebetween.

In some more embodiments, the targeting moiety introduces at least one of the following: at least one exogenous anchor sequence; an alteration in at least one conjunction nucleating molecule binding site, such as by altering binding affinity for the conjunction nucleating molecule; a change in an orientation of at least one common nucleotide sequence, such as a CTCF binding motif, YY 1 binding motif, ZNF143 binding motif, or other binding motif mentioned herein; and a substitution, addition or deletion in at least one anchor sequence, such as a CTCF binding motif, YY1 binding motif, ZNF143 binding motif, or other binding motif mentioned herein.

In some embodiments, an anchor sequence comprises a nucleating polypeptide binding motif, e.g., a CTCF-binding motif: N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/G)(C/T/A)(G/A/C) (SEQ ID NO: 71), where N is any nucleotide.

A CTCF-binding motif may also be in an opposite orientation, e.g., (G/A/C)(C/T/A)(C/G)(G/A)(C/A/T)GG(G/T)(G/A)GA(C/T/A)(C/G)(A/T/G)CC(G/A/T)N(T/C/G)N (SEQ ID NO: 72). Where N is any nucleotide

In some embodiments, an anchor sequence comprises SEQ ID NO: 71 or SEQ ID NO: 72 or a sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to either SEQ ID NO: 71 or SEQ ID NO: 72.

In some embodiments, an anchor sequence comprises a nucleating polypeptide binding motif, e.g., a YY1 -binding motif: CCGCCATNTT, where N is any nucleotide.

A YYl-binding motif may also be in an opposite orientation, e.g., AANATGGCGG, where N is any nucleotide.

In some embodiments, an anchor sequence-mediated conjunction comprises at least a first anchor sequence and a second anchor sequence. For example, in some embodiments, a first anchor sequence and a second anchor sequence may each comprise a nucleating polypeptide binding motif, e.g., each comprises a CTCF binding motif.

In some embodiments, a first anchor sequence and second anchor sequence comprise different sequences, e.g., a first anchor sequence comprises a CTCF binding motif, and a second anchor sequence comprises an anchor sequence other than a CTCF binding motif. In some embodiments, each anchor sequence comprises a nucleating polypeptide binding motif and one or more flanking nucleotides on one or both sides of a nucleating polypeptide binding motif.

Two CTCF-binding motifs (e.g., contiguous or non-contiguous CTCF binding motifs) that can form an ASMC may be present in a genome in any orientation, e.g., in the same orientation (tandem) either 5 ’-3’ (left tandem, e.g., the two CTCF-binding motifs that comprise SEQ ID NO:71) or 3 ’-5’ (right tandem, e.g., the two CTCF-binding motifs comprise SEQ ID NO:72), or convergent orientation, where one CTCF-binding motif comprises SEQ ID NO:71 and another other comprises SEQ ID NO:72.

In some embodiments, an anchor sequence comprises a CTCF binding motif associated with a target gene (e.g., MYC), wherein the target gene is associated with a disease, disorder and/or condition, e.g., MYC mis-regulating disorder, e.g., hepatic disorder, (e g., hepatocarcinoma) or lung cancer.

In some embodiments, methods of the present disclosure comprise modulating, e.g., disrupting, a genomic complex (e.g., ASMC), e.g., by modifying chromatin structure, by substituting, adding, or deleting one or more nucleotides within an anchor sequence, e.g., a nucleating polypeptide binding motif. One or more nucleotides may be specifically targeted, e.g., a targeted alteration, for substitution, addition or deletion within an anchor sequence, e.g., a nucleating polypeptide binding motif.

In some embodiments, a genomic complex (e.g., ASMC) may be altered by changing an orientation of at least one nucleating polypeptide binding motif. In some embodiments, an anchor sequence comprises a nucleating polypeptide binding motif, e.g., CTCF binding motif, and a targeting moiety introduces an alteration in at least one nucleating polypeptide binding motif, e.g., altering binding affinity for a nucleating polypeptide.

In some embodiments, before administration of an expression repressor or system described herein, the target gene, e.g., MYC has a defined state of expression, e.g., in a diseased state. For example, the target gene, e.g., MYC may have a high level of expression in a disease cell. By disrupting the anchor sequence-mediated conjunction, expression of the target gene, e.g., MYC may be decreased.

A targeting moiety suitable for use in an expression repressor of an expression repression system described herein may bind, e.g., specifically bind, to a site comprising at least 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,

45, 46, 47, 48, 49, or 50 nucleotides or base pairs (and optionally no more 50, 49, 48, 47, 46, 45, 44, 43,

42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides or base pairs). In some embodiments, a DNA-targeting moiety binds to a site comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides or base pairs.

Expression repression systems of the present disclosure may comprise two or more expression repressors. In some embodiments, the expression repressors of an expression repressor system each comprise a different targeting moiety.

In some embodiments, an expression repression system comprises a first expression repressor comprising a targeting moiety that binds a target gene, e.g., an exon, intron, or exon intron boundary (e.g., splice site), and second expression repressor comprising a targeting moiety that binds the target gene, e.g., an exon, intron, or exon intron boundary (e.g., splice site). In some embodiments, an expression repression system comprises a first expression repressor comprising a targeting moiety that binds a target gene, e.g., an exon, intron, or exon intron boundary (e.g., splice site), and second expression repressor comprising a targeting moiety that binds to a transcription control element (e.g., promoter or enhancer) operably linked to the target gene, e g., MYC. In some embodiments, an expression repression system comprises a first expression repressor comprising a targeting moiety that binds to a transcription control element (e.g., promoter or enhancer) operably linked to a target gene, and a second expression repressor comprising a targeting moiety that binds to a transcription control element (e.g., promoter or enhancer) operably linked to the target gene. In some embodiments, an expression repression system comprises a first expression repressor comprising a targeting moiety that binds to an anchor sequence proximal to a target gene, e.g., MYC or associated with an anchor sequence-mediated conjunction operably linked to a target gene, e.g., MYC, and a second expression repressor comprising a targeting moiety that binds to a transcription control element (e.g., promoter or enhancer) operably linked to the target gene, e.g., MYC. In some embodiments, an expression repression system comprises a first expression repressor comprising a targeting moiety that binds to an anchor sequence proximal to a target gene, e.g., MY C or associated with an anchor sequence-mediated conjunction operably linked to a target gene, e.g., MYC, and a second expression repressor comprising a targeting moiety that binds to the target gene (e.g., MYC), e.g., an exon, intron, or exon intron boundary (e.g., splice site). In some embodiments, an expression repression system comprises a first expression repressor comprising a targeting moiety that binds to an anchor sequence proximal to a target gene, e.g., MYC or associated with an anchor sequence-mediated conjunction operably linked to a target gene, e.g., MYC, and a second expression repressor comprising a targeting moiety that binds to an anchor sequence proximal to the target gene, e.g., MY C or associated with an anchor sequence-mediated conjunction operably linked to the target gene, e.g., MYC.

In some embodiments, an expression repression system comprises a first expression repressor comprising a targeting moiety that binds to a first site, e g., in a promoter operably linked to a target gene, e.g., MYC, and a second expression repressor comprising a targeting moiety that binds to a second site, e.g., in the promoter operably linked to a target gene, e.g., MYC. The first site and second site may be different and non-overlapping sites, e.g., the first site and second site do not share any sequence in common. The first site and second site may be different but overlapping sites, e.g., the first site and second site comprise different sequences but share some sequence in common.

In some embodiments, the target gene is MYC. In some embodiments, MYC is located on human chromosome 8. In some embodiments, the expression repressor or the expression repressor system as described herein binds to the transcription start site (TSS) of MYC.

Other Compositions

Nucleic acids and Vectors

The present disclosure is further directed, in part, to nucleic acids encoding expression repressors or expression repression systems described herein. In some embodiments, an expression repressor may be provided via a composition comprising a nucleic acid encoding the expression repressor, wherein the nucleic acid is associated with sufficient other sequences to achieve expression of the expression repressor, in a system of interest (e.g., in a particular cell, tissue, organism, etc.). In some embodiments, an expression repression system may be provided via a composition comprising a nucleic acid encoding the expression repression system, e.g., expression repressor(s) of the expression repression system, wherein the nucleic acid is associated with sufficient other sequences to achieve expression of the expression repression system, e.g., expression repressor(s) of the expression repression system, in a system of interest (e g., in a particular cell, tissue, organism, etc.).

In some particular embodiments, the present disclosure provides compositions of nucleic acids that encode an expression repressor or polypeptide portion thereof. In some such embodiments, provided nucleic acids may be or include DNA, RNA, or any other nucleic acid moiety or entity as described herein, and may be prepared by any technology described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro or in vivo transcription, etc.). In some embodiments, provided nucleic acids that encode an expression repressor or polypeptide portion thereof may be operationally associated with one or more replication, integration, and/or expression signals appropriate and/or sufficient to achieve integration, replication, and/or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.).

In some embodiments, a composition for delivering an expression repressor described herein is or comprises a vector, e.g., a viral vector, comprising one or more nucleic acids encoding an expression repressor or one or more components of an expression repressor as described herein. In some particular embodiments, the present disclosure provides compositions of nucleic acids that encode an expression repression system, one or more expression repressors, or polypeptide portions thereof. In some such embodiments, provided nucleic acids may be or include DNA, RNA, or any other nucleic acid moiety or entity as described herein, and may be prepared by any technology described herein or otherwise available in the art (e.g., synthesis, cloning, amplification, in vitro or in vivo transcription, etc.). In some embodiments, provided nucleic acids that encode an expression repression system, one or more expression repressors, or polypeptide portions thereof may be operationally associated with one or more replication, integration, and/or expression signals appropriate and/or sufficient to achieve integration, replication, and/or expression of the provided nucleic acid in a system of interest (e.g., in a particular cell, tissue, organism, etc.).

In some embodiments, a composition for delivering an expression repression system described herein ss or comprises a vector, e.g., a viral vector, comprising one or more nucleic acids encoding one or more components of an expression repression system, e.g., expression repressor(s) of the expression repression system as described herein.

In some embodiments, a composition for delivering an expression repressor described herein is or comprises RNA, e.g., mRNA, comprising one or more nucleic acids encoding an expression repressor or one or more components of an expression repressor, as described herein.

In some embodiments, a composition for delivering an expression repression system described herein is or comprises RNA, e.g., mRNA, comprising one or more nucleic acids encoding one or more components of an expression repression system, e.g., expression repressor(s) of the expression repression system as described herein.

Nucleic acids as described herein or nucleic acids encoding a protein described herein, may be incorporated into a vector. Vectors, including those derived from retroviruses such as lentivirus. are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Examples of vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. An expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art, and described in a variety of virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.

Expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding tire gene of interest to a promoter and incorporating tire construct into an expression vector. Vectors can be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for expression of the desired nucleic acid sequence.

Additional promoter elements, e.g., enhancing sequences, may regulate frequency of transcriptional initiation. Typically, these sequences are located in a region 30-110 bp upstream of a transcription start site, although a number of promoters have recently been shown to contain functional elements downstream of transcription start sites as well.

In some embodiments, an expression repressor or system described herein acts at an enhancing sequence. In some embodiments, the enhancing sequence is an enhancer, a stretch enhancer, a shadow enhancer, a locus control region (LCR), or a super enhancer. In some embodiments, the super enhancer comprises a cluster of enhancers and other regulatory elements. In some embodiments, these sequences are located in a region .2- 2 Mb upstream or downstream of a transcription start site. In some embodiments, the region is a non-coding region. In some embodiments, the region contains at least one SNP associated with higher risk of developing cancer. In some embodiments, the region is associated with long-range regulation of a target gene, e.g., MYC. In some embodiments, the regions are cell-type specific. In some embodiments, a super-enhancer modifies (e.g., increases or decreases) target gene expression, e.g., MYC expression, by recruiting the target gene promoter, e.g., MYC promoter. In some embodiments, the super enhancer interacts with a target gene promoter, e.g., MYC promoter, through an enhancer docking site. In some embodiments, the enhancer docking site is an anchor sequence. In some embodiments, the enhancer docking site is located at least 100 bp, 200 bp, 500 bp, 1000 bp, 1500 bp, 2000 bp, or 3000 bp away from the target gene promoter, e.g., MYC promoter. In some embodiments, a super enhancer region is at least 100 bp, at least 200 bp, at least 300 bp, at least 500 bp, at least 1 kb, at least 2 kb, at least 3 kb, at least 5 kb, at least 10 kb, at least 15 kb, at least 20 kb, or at least 25 kb long.

Spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In a thymidine kinase (tk) promoter, spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. In some embodiments of a suitable promoter is Elongation Growth Factor-la (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia vims promoter, an Epstein-Barr vims immediate early promoter, a Rous sarcoma vims promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.

The present disclosure should not be interpreted to be limited to use of any particular promoter or category of promoters (e.g., constitutive promoters). For example, in some embodiments, inducible promoters are contemplated as part of the present disclosure. In some embodiments, use of an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence to which it is operatively linked, when such expression is desired. In some embodiments, use of an inducible promoter provides a molecular switch capable of turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

In some embodiments, an expression vector to be introduced can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some aspects, a selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate transcriptional control sequences to enable expression in the host cells. Useful selectable markers may include, for example, antibiotic-resistance genes, such as neo, etc.

In some embodiments, reporter genes may be used for identifying potentially transfected cells and/or for evaluating the functionality of transcriptional control sequences. In general, a reporter gene is a gene that is not present in or expressed by a recipient source (of a reporter gene) and that encodes a polypeptide whose expression is manifested by some easily detectable property, e g., enzymatic activity or visualizable fluorescence. Expression of a reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, a construct with a minimal 5 ’ flanking region that shows highest level of expression of reporter gene is identified as a promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for ability to modulate promoter-driven transcription.

Cells

The present disclosure is further directed, in part, to cells comprising an expression repressor or an expression repression system described herein. Any cell, e.g., cell line, e.g., a cell line suitable for expression of a recombinant polypeptide, known to one of skill in the art is suitable to comprise an expression repressor or an expression repression system described herein. In some embodiments, a cell, e.g., cell line, may be used to express an expression repressor or an expression repression system, e.g., expression repressor(s), described herein. In some embodiments, a cell, e.g., cell line, may be used to express or amplify a nucleic acid, e.g., a vector, encoding an expression repressor or an expression repression system, e.g., expression repressor(s), described herein. In some embodiments, a cell comprises a nucleic acid encoding an expression repressor or an expression repression sy stem, e.g., expression repressor(s), described herein.

In some embodiments, a cell comprises a first nucleic acid encoding a first component of an expression repression system, e.g., a first expression repressor, and a second nucleic acid encoding a second component of the expression repression system, e.g., a second expression repressor. In some embodiments, wherein a cell comprises nucleic acid encoding an expression repression system comprising two or more expression repressors, the sequences encoding each expression repressor are disposed on separate nucleic acid molecules, e.g., on different vectors, e.g., a first vector encoding a first expression repressor and a second vector encoding a second expression repressor. In some embodiments, the sequences encoding each expression repressor are disposed on the same nucleic acid molecule, e.g., on the same vector. In some embodiments, some or all of the nucleic acid encoding the expression repression system is integrated into the genomic DNA of the cell. In some embodiments, the nucleic acid encoding a first expression repressor of an expression repression system is integrated into the genomic DNA of a cell, and the nucleic acid encoding a second expression repressor of an expression repression system is not integrated into the genomic DNA of a cell (e.g., is situated on a vector). In some embodiments, the nucleic acid(s) encoding a first and a second expression repressor of an expression repression system are integrated into the genomic DNA of a cell, e.g., at the same (e.g., adjacent or colocalized) or different sites in the genomic DNA.

Examples of cells that may comprise and/or express an expression repression system or expression repressor described herein include, but are not limited to, hepatocytes, neuronal cells, endothelial cells, myocytes, and lymphocytes.

Tire present disclosure is further directed, in part, to a cell made by a method or process described herein. In some embodiments, the disclosure provides a cell produced by: providing an expression repressor or an expression repression system described herein, providing the cell, and contacting the cell with the expression repressor (or a nucleic acid encoding the expression repressor, or a composition comprising said expression repressor or nucleic acid) or the expression repression system (or a nucleic acid encoding the expression repression system, or a composition comprising said expression repression system or nucleic acid). In some embodiments, contacting a cell with an expression repressor comprises contacting the cell with a nucleic acid encoding the expression repressor under conditions that allow the cell to produce the expression repressor. In some embodiments, contacting a cell with an expression repressor comprises contacting an organism that comprises the cell with the expression repressor or a nucleic acid encoding the expression repressor under conditions that allow the cell to produce the expression repressor.

Without wishing to be bound by theory, a cell contacted with an expression repressor or an expression repression system described herein may exhibit: a decrease in expression of a target gene (e.g., MYC) and/or a modification of epigenetic markers associated with the target gene, e.g., MYC, a transcription control element operably linked to the target gene, e.g., MYC, or an anchor sequence proximal to the target gene or associated with an anchor sequence-mediated conjunction operably linked to the target gene, e.g., MY C compared to a similar cell that has not been contacted by the expression repressor or the expression repression system. In some embodiments, a cell exhibiting said decrease in expression of a target gene, e.g., MY C and/or modification of epigenetic markers does not comprise the expression repressor or the expression repression system. The decrease in expression and/or modification of epigenetic markers may persist, e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely) after contact with the expression repressor or the expression repression system.

In some embodiments, a cell previously contacted by an the expression repressor or an expression repression system retains the decrease in expression and/or modification of epigenetic markers after the expression repressor or the expression repression system is no longer present in the cell, e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely) after the expression repressor or the expression repression system is no longer present in the cell.

Methods of Making Expression Repression Systems and/or Expression Repressors

In some embodiments, an expression repressor comprises or is a protein and may thus be produced by methods of making proteins. In some embodiments, an expression repression system, e.g., the expression repressor(s) of an expression repression system, comprise one or more proteins and may thus be produced by methods of making proteins. As will be appreciated by one of skill, methods of making proteins or polypeptides (which may be included in modulating agents as described herein) are routine in the art. See, in general, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

A protein or polypeptide of compositions of the present disclosure can be biochemically synthesized by employing standard solid phase techniques. Such methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods can be used when a peptide is relatively short (e.g., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.

Solid phase synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses, 2^nd Ed., Pierce Chemical Company, 1984; and Coin, I., et al., Nature Protocols, 2:3247-3256, 2007.

For longer peptides, recombinant methods may be used. Methods of making a recombinant therapeutic polypeptide are routine in the art. See, in general, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

Exemplary methods for producing a therapeutic pharmaceutical protein or polypeptide involve expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under control of appropriate promoters. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter, and other 5 ’ or 3 ’ flanking non-transcribed sequences, and 5’ or 3’ non-translated sequences such as necessary' ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, splice, and polyadenylation sites may be used to provide other genetic elements required for expression of a heterologous DNA sequence. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

In cases where large amounts of tire protein or polypeptide are desired, it can be generated using techniques such as described by Brian Bray, Nature Reviews Drug Discovery, 2:587-593, 2003; and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

Various mammalian cell culture systems can be employed to express and manufacture recombinant protein. Examples of mammalian expression systems include CHO cells, COS cells, HeLA and BHK cell lines. Processes of host cell culture for production of protein therapeutics are described in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologies Manufacturing (Advances in Biochemical Engineering/Biotechnology), Springer (2014). Compositions described herein may include a vector, such as a viral vector, e.g., a lentiviral vector, encoding a recombinant protein. In some embodiments, a vector, e.g., a viral vector, may comprise a nucleic acid encoding a recombinant protein. Compositions described herein may include a lipid nanoparticle encapsulating a vector, such as a viral vector, e.g., a lentiviral vector, encoding a recombinant protein. In some embodiments, a lipid nanoparticle encapsulating a vector, e.g., a viral vector, may comprise a nucleic acid encoding a recombinant protein.

Purification of protein therapeutics is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010). Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

Proteins comprise one or more amino acids. Amino acids include any compound and/or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)I-COOH. hr some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino -terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide. Pharmaceutical Compositions, Formulation, Delivery, and Administration

The present disclosure is further directed, in part, to pharmaceutical compositions comprising an expression repressor or an expression repression system, e.g., expression repressor(s), described herein, to pharmaceutical compositions comprising nucleic acids encoding the expression repressor or the expression repression system, e.g., expression repressor(s), described herein, and/or to and/or compositions that deliver an expression repressor or an expression repression system, e.g., expression repressor(s), described herein to a cell, tissue, organ, and/or subject.

As used herein, the term “pharmaceutical composition” refers to an active agent (e.g., an expression repressor or nucleic acids of the expression receptor, e.g., an expression repression system, e.g., expression repressor(s) of an expression repressor system, or nucleic acid encoding the same), formulated together with one or more pharmaceutically acceptable carriers (e.g., pharmaceutically acceptable carriers known to those of skill in the art). In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition comprising an expression repressor of the present disclosure comprises an expression repressor or nucleic acid(s) encoding the same. In some embodiments, a pharmaceutical composition comprising an expression repression system of the present disclosure comprises or each of the expression repressors of the expression repression system or nucleic acid(s) encoding the same (e.g., if an expression repression system comprises a first expression repressor and a second expression repressor, the pharmaceutical composition comprises the first and second expression repressor). In some embodiments, a pharmaceutical composition comprises less than all of the expression repressors of an expression repression system comprising a plurality of expression repressors. For example, an expression repression system may comprise a first expression repressor and a second expression repressor, and a first pharmaceutical composition may comprise the first expression repressor or nucleic acid encoding the same and a second pharmaceutical composition may comprise the second expression repressor or nucleic acid encoding the same. In some embodiments, a pharmaceutical composition may comprise coformulation of one or more expression repressors, or nucleic acid(s) encoding tire same.

In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; trans-dermally; or nasally, pulmonary, and/or to other mucosal surfaces.

As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. In some embodiments, for example, materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

As used herein, the term “pharmaceutically acceptable salt”, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i. e. , salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). hi some embodiments, pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate, and aryl sulfonate.

In various embodiments, the present disclosure provides pharmaceutical compositions described herein with a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipient includes an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

Pharmaceutical preparations may be made following conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and fdling for hard gelatin capsule forms. When a liquid carrier is used, a preparation can be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous solution or suspension. Such a liquid formulation may be administered directly per os.

In some embodiments, pharmaceutical compositions may be formulated for delivery to a cell and/or to a subject via any route of administration. Modes of administration to a subject may include injection, infusion, inhalation, intranasal, intraocular, topical delivery, inter-cannular delivery, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracap sular, intra-orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intra-cerebrospinal, and intra-stemal injection and infusion. In some embodiments, administration includes aerosol inhalation, e.g., with nebulization. In some embodiments, administration is systemic (e.g., oral, rectal, nasal, sublingual, buccal, or parenteral), enteral (e.g., system-wide effect, but delivered through the gastrointestinal tract), or local (e.g., local application on the skin, intravitreal injection). In some embodiments, one or more compositions is administered systemically. In some embodiments, administration is non -parenteral and a therapeutic is a parenteral therapeutic. In some embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, inter-dermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may be a single dose. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, six, eight, ten, 12, 15 or 20 or more administrations may be given to the subject during one treatment or over a period of time as a treatment regimen.

In some embodiments, administrations may be given as needed, e.g., for as long as symptoms associated with the disease, disorder or condition persist. In some embodiments, repeated administrations may be indicated for the remainder of the subject’s life. Treatment periods may vary and could be, e.g., one day, two days, three days, one week, two weeks, one month, two months, three months, six months, a year, or longer.

In some embodiments, the nucleic acid encoding the expression repressor (e.g., bicistronic ZF9- MQ1 ZF3-KRAB) is present in the pharmaceutical composition at a concentration of about 0.5-2 mg/mL, e.g., about 0.5 - 1.5 mg/mL, e.g., about 1 mg/mL.

In some embodiments, the pharmaceutical composition (e.g., the pharmaceutical composition comprising the nucleic acid encoding the expression repressor, e.g., bicistronic ZF9-MQ1 ZF3-KRAB) is at a volume of about 2.5 - 7.5 ml, e.g., about 3-7 ml, about 4-6 ml, or about 5 ml. In some embodiments, the pharmaceutical composition comprises about 5 mg of nucleic acid encoding the expression repressor in a volume of about 5 ml.

In some embodiments, the pharmaceutical composition is diluted in normal saline to a total volume of 250 mL. In certain embodiments, the pharmaceutical composition comprising 1 mg/mL of nucleic acid encoding the expression repressor (e.g., bicistronic ZF9-MQ1_ZF3-KRAB) at a volume of 5 mL diluted in normal saline to a total volume of 250 mL.

Unless specified otherwise, when concentrations or doses of an expression repressor are provided using mg (e.g., in mg/ml or mg/kg), the mass in mg refers to the mass of the nucleic acid and the LNP together. Dosing Regimens

In methods of the inventions, the nucleic acid encoding an expression repressor, the immune checkpoint inhibitor polypeptide, or both may be administered according to a defined dosing regimen. The dosing regimen may include a defined dose, a defined interval between doses, a defined period of dosing, or any combination thereof. The dosing regimen can vary based on, e.g., the condition being treated, the severity of the disease, the subject’s individual parameters, including age, physiological condition, size and weight, duration of treatment, the type of treatment to be performed (if any), the particular route of administration and similar factors. Thus, the dosing regimen of the agents described herein can depend on such various parameters. The dosing regimen of an administered composition may also vary depending upon other factors as the subject’s sex, general medical condition, and severity of the disorder to be treated.

The dose may be within a defined range. For example, it may be desirable to provide the subject with a dose of a modulating agent or combination of modulating agents disclosed herein that is in the range of from about 1 mg/kg to 6 mg/kg as a single intravenous infusion, although a lower or higher dose also may be administered as circumstances dictate. In some embodiments, dose may include, but are not limited to, 0.001 mg/kg - 6 mg/kg, 0.001 mg/kg - 5.5 mg/kg, 0.001 mg/kg - 5 mg/kg, 0.001 mg/kg - 4.5 mg/kg, 0.001 mg/kg - 4 mg/kg, 0.001 mg/kg - 3.5 mg/kg, 0.001 mg/kg - 3 mg/kg, 0.001 mg/kg - 2.5 mg/kg, 0.001 mg/kg - 2 mg/kg, 0.001 mg/kg - 1.5 mg/kg, 0.001 mg/kg - 1 mg/kg, 0.001 mg/kg - 0.5 mg/kg, 0.001 mg/kg - 0.2 mg/kg, 0.001 mg/kg - 0.1 mg/kg, 0.001 mg/kg - 0.07 mg/kg, 0.001 mg/kg - 0.03 mg/kg, 0.001 mg/kg - 0.01 mg/kg, 0.001 mg/kg - 0.009 mg/kg, 0.001 mg/kg - 0.005 mg/kg, 0.002 mg/kg - 6.0 mg/kg, 0.002 mg/kg - 5.0 mg/kg, 0.002 mg/kg - 4 mg/kg, 0.002 mg/kg - 3.0 mg/kg, 0.002 mg/kg - 2.0 mg/kg, 0.002 mg/kg - 1.0 mg/kg, 0.002 mg/kg - 0.05 mg/kg, 0.02 mg/kg - 6 mg/kg, 0.02 mg/kg - 5 mg/kg, 0.02 mg/kg - 4 mg/kg, 0.02 mg/kg - 3 mg/kg, 0.02 mg/kg - 2 mg/kg, 0.02 mg/kg - 1 mg/kg, 0.02 mg/kg - 0.05 mg/kg, 0.05 mg/kg - 6 mg/kg, 0.05 mg/kg - 5 mg/kg, 0.05 mg/kg - 4 mg/kg, 0.05 mg/kg - 3 mg/kg, 0.05 mg/kg - 2 mg/kg, 0.05 mg/kg - 1 mg/kg, 0.05 mg/kg - 0.5 mg/kg, 0.08 mg/kg - 6 mg/kg, 0.08 mg/kg - 5 mg/kg, 0.08 mg/kg - 4 mg/kg, 0.08 mg/kg - 3 mg/kg, 0.08 mg/kg - 2 mg/kg, 0.08 mg/kg - 1 mg/kg, 0.15 mg/kg - 6 mg/kg, 0.15 mg/kg - 5 mg/kg, 0.15 mg/kg - 4 mg/kg, 0.15 mg/kg - 3 mg/kg, 0.15 mg/kg - 2 mg/kg, 0.15 mg/kg - 1 mg/kg, 0.05 mg/kg - 0.5 mg/kg, 1.0 mg/kg- 6mg/kg, 1.0 mg/kg-5 mg/kg, 1.0 mg/kg-4 mg/kg, 1.0-3.0mg/kg, 1.5 mg/kg-3.0mg/kg, 1.0 mg/kg - 1.5 mg/kg, 1.5 mg/kg - 3 mg/kg, 3 mg/kg - 4 mg/kg, 4 mg/kg - 5 mg/kg, or 5 mg/kg - 6 mg/kg. In some embodiments, a modulating agent as disclosed herein may be administered at about 0.001 mg/kg to about 6 mg/kg, about 0.001 mg/kg to about 5.5 mg/kg, about 0.001 mg/kg to about 5 mg/kg, about 0.001 mg/kg to about 4.5 mg/kg, about 0.001 mg/kg to about 4 mg/kg, about 0.001 mg/kg to about 3.5 mg/kg, about 0.001 mg/kg to about 3 mg/kg, about 0.001 mg/kg to about 2.5 mg/kg, about 0.001 mg/kg to about 2 mg/kg, about 0.001 mg/kg to about 1 .5 mg/kg, about 0.001 mg/kg to about 1 mg/kg, about 0.001 mg/kg to about 0.5 mg/kg, about 0.001 mg/kg to about 0.2 mg/kg, about 0.001 mg/kg to about 0.1 mg/kg, about 0.001 mg/kg to about 0.07 mg/kg, about 0.001 mg/kg to about 0.03 mg/kg, about 0.001 mg/kg to about 0.01 mg/kg, about 0.001 mg/kg to about 0.009 mg/kg, about 0.001 mg/kg to about 0.005 mg/kg, about 0.002 mg/kg to about 6 mg/kg, about 0.002 mg/kg to about 5.5 mg/kg, about 0.002 mg/kg to about 5 mg/kg, about 0.002 mg/kg to about 4.5 mg/kg, about 0.002 mg/kg to about 4 mg/kg, about 0.002 mg/kg to about 3.5 mg/kg, about 0.002 mg/kg to about 3 mg/kg, about 0.002 mg/kg to about 2.5 mg/kg, about 0.002 mg/kg to about 2 mg/kg, about 0.002 mg/kg to about 1.5 mg/kg, about 0.002 mg/kg to about 1 mg/kg, about 0.002 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about 6 mg/kg, about 0.01 mg/kg to about 5.5 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 4.5 mg/kg, about 0.01 mg/kg to about 4 mg/kg, about 0.01 mg/kg to about 3.5 mg/kg, about 0.01 mg/kg to about 3 mg/kg, about 0.01 mg/kg to about 2.5 mg/kg, about 0.01 mg/kg to about 2 mg/kg, about 0.01 mg/kg to about 1 .5 mg/kg, about 0.01 mg/kg to about 1 mg/kg, about 0.01 mg/kg to about 0.5 mg/kg, about 0.1 mg/kg to about 6 mg/kg, about 0.1 mg/kg to about

5.5 mg/kg, about 0. 1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 4.5 mg/kg, about 0.1 mg/kg to about 4 mg/kg, about 0. 1 mg/kg to about 3.5 mg/kg, about 0. 1 mg/kg to about 3 mg/kg, about 0.1 mg/kg to about 2.5 mg/kg, about 0.1 mg/kg to about 2 mg/kg, about 0. 1 mg/kg to about 1.5 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0. 1 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 6 mg/kg, about 0.5 mg/kg to about 5.5 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 4.5 mg/kg, about 0.5 mg/kg to about 4 mg/kg, about 0.5 mg/kg to about 3.5 mg/kg, about 0.5 mg/kg to about 3 mg/kg, about 0.5 mg/kg to about 2.5 mg/kg, about 0.5 mg/kg to about 2 mg/kg, about 0.5 mg/kg to about

1.5 mg/kg, about 0.5 mg/kg to about 1 mg/kg, about 1 mg/kg to about 6 mg/kg, about 1 mg/kg to about

5.5 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 4.5 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3.5 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 2.5 mg/kg, about 1 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 6 mg/kg, about 1 .5 mg/kg to about 5.5 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about

4.5 mg/kg, about 1.5 mg/kg to about 4 mg/kg, about 1.5 mg/kg to about 3.5 mg/kg, about 1 .5 mg/kg to about 3 mg/kg, about 1.5 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about 6 mg/kg, about 2 mg/kg to about 5.5 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2 mg/kg to about 4.5 mg/kg, about 2 mg/kg to about 4 mg/kg, about 2 mg/kg to about 3.5 mg/kg, about 2 mg/kg to about 3 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2.5 mg/kg to about 6 mg/kg, about 2.5 mg/kg to about 5.5 mg/kg, about 2.5 mg/kg to about 5 mg/kg, about 2.5 mg/kg to about 4.5 mg/kg, about 2.5 mg/kg to about 4 mg/kg, about 2.5 mg/kg to about 3.5 mg/kg, about 2.5 mg/kg to about 3 mg/kg, about 3 mg/kg to about 6 mg/kg, about 3 mg/kg to about 5.5 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 4.5 mg/kg, about 3 mg/kg to about 4 mg/kg, about 3 mg/kg to about 3.5 mg/kg, about 3.5 mg/kg to about 6 mg/kg, about 3.5 mg/kg to about 5.5 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 3.5 mg/kg to about 4.5 mg/kg, about 3.5 mg/kg to about 4 mg/kg, about 4 mg/kg to about 6 mg/kg, about 4 mg/kg to about 5.5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4 mg/kg to about 4.5 mg/kg, about 4.5 mg/kg to about 6 mg/kg, about 4.5 mg/kg to about 5.5 mg/kg, or about 4.5 mg/kg to about 5 mg/kg.

The dose may be a defined value. For example, the dose may be about 0.002 mg/kg, about 0.01 mg/kg, about 0.02 mg/kg, about 0.05 mg/kg, about 0.075 mg/kg, about 0.08 mg/kg, about 0.1 mg/kg, about 0.125 mg/kg, about 0.15 mg/kg, about 0.175 mg/kg, about 0.2 mg/kg, about 0.25 mg/kg, about 0.3 mg/kg, about 0.35 mg/kg, about 0.4 mg/kg, about 0.45 mg/kg, about 0.5 mg/kg, about 0.08 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, or about 6 mg/kg.

The dose may be administered for a defined dosing period (e.g., the length of time taken to administer a single infusion). For example, the dosing period may be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, or about 6 hours. In certain embodiments, the dosing period may be between about 80-120 minutes (±10 minutes).

Multiple doses may be administered at defined intervals. For example, the interval between doses may be about 1 days, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 12 days, about 13 days, about 14 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, about 10 weeks, or about 12 weeks. The dose may be repeated as needed, for example, once every day (e.g., for 1-30 days), once every 3 days (e.g., for 1-30 days) once every 5 days (e.g., for 1-30 days), once per week (e.g., for 1-6 weeks or for 2-5 weeks). The dose may be administered multiple times, e.g., once, or twice a week, or once every 1 or 2 weeks.

In some embodiments, the subject is provided with a dose of a modulating agent or combination of modulating agents disclosed herein that is in the range of from about 0.002 mg/kg to 6 mg/kg as multiple intravenous infusions although a lower or higher dose also may be administered as circumstances dictate. In some embodiments, the subject is provided with a dose of a modulating agent or combination of modulating agents disclosed herein that is in the range of from about 0.001 mg/kg to 6 mg/kg as multiple intravenous infusions although a lower or higher dose also may be administered as circumstances dictate. In some embodiments, the subject is provided with a dose of a modulating agent or combination of modulating agents disclosed herein that is in the range of from about 1 mg/kg to 6 mg/kg as multiple intravenous infusions although a lower or higher dose also may be administered as circumstances dictate.

A modulating agent or a combination of modulating agents as disclosed herein may be administered as one dose every 3-5 days, repeated for a total of at least 3 doses. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 3 mg/kg every 5 days for 25 days. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 1.0-5.0 mg/kg every 3-5 days for 1-10 doses. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 1.0- 3.0 mg/kg every 5 days for 3 doses then every 3 days for 3 doses. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 1 .0-3.0 mg/kg every 5 days for 4 doses then every 3 days for 3 doses. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 0.001 mg/kg - 1 .5 mg/kg every 14 days. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 0.002 mg/kg - 1.5 mg/kg every 14 days. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 6 mg/kg every 5 days for 1- 10 doses. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 3 mg/kg every 5 days for 1-10 doses. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 1 .5 mg/kg every 5 days for 2 doses, 3 mg/kg every 5 days for 3 doses, 3 mg/kg every 3 days for 1 dose. Alternatively, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 6 mg/kg at every 5 days or at 1.5 mg/kg once a day for 5 days with 2 days off. The dosing schedule can optionally be repeated at other intervals and dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule.

In some embodiments, the dosing of modulating agents or a combination of modulating agents may include a dosage of between 1.0 mg/kg to 6.0 mg/kg, optionally given either weekly, twice per week, or every other week. In some embodiments, the dosing of modulating agents or a combination of modulating agents may include a dosage of between 0.001 mg/kg to 1.5 mg/kg, optionally given either weekly, twice per week, or every other week. In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 0.001 mg/kg. In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 0.002 mg/kg. In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 0.02 mg/kg. In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 0.05 mg/kg. hr some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 0.08 mg/kg. In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 0.125 mg/kg. In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered at 0. 15 mg/kg. In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered every two weeks e.g., by way of intravenous infusion, hi some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered as an intravenous infusion (e.g., over about 80-120 minutes (±10 minute)) every two weeks. In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered as an intravenous infusion (e.g., over about 80-120 minutes (±10 minute)) every two weeks. In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered as an intravenous infusion once every 2-weeks (e.g., administered on Day 1 and Day 15 of a 28-day/4-week cycle). In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered at between about 0.02 mg/kg and 0.15 mg/kg as an intravenous infusion (e.g., over about 80-120 minutes (±10 minute)) once every 2-weeks over a 28-day cycle (e.g., administered on Day 1 and Day 15 of each 28-day cycle). In some embodiments, a modulating agent or a combination of modulating agents as disclosed herein may be administered at between about 0.002 mg/kg and 0.15 mg/kg as an intravenous infusion (e.g., over about 80-120 minutes (±10 minute)) once every 2-weeks over a 28-day cycle (e.g., administered on Day 1 and Day 15 of each 28-day cycle).

In some embodiments, when a subject experiences a grade 2 infusion-associate reaction, the intravenous infusion can be interrupted for treatment and/or infusion time may be extended. In certain embodiments, a subject experiencing a grade 2 infusion-associated reaction is administered a treatment of one or more of antihistamine(s), famotidine or the equivalent, corticosteroids, and bronchodilatorv therapy.

In some embodiments, when a subject experiences a grade 3 or 4 infusion-associate reaction during intravenous infusion, the intravenous infusion is stopped. In certain embodiments, a subject experiencing a grade 3 or 4 infusion-associated reaction may be administered one or more of: intravenous saline, methylprednisolone (e.g., 100 mg, e.g., intravenously) or steroid equivalent, diphenhydramine (e.g., 50 mg, e.g., intravenously), bronchodilatory therapy, and epinephrine.

In some embodiments, when a subject experiences a grade 1 (e.g., mild transient reaction; infusion interruption not indicated; intervention not indicated) infusion reaction or hypersensitivity, the method of administration of the expression repressor disclosed herein comprises decreasing infusion rate by 50% or stop infusion until recovery of symptoms, hi some embodiments, if the method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 1 (e.g., mild transient reaction; infusion interruption not indicated; intervention not indicated) infusion reaction or hypersensitivity, the method of administration comprises resuming at 50% of the rate prior to the reaction after the subjects symptoms have resolved and may be increased to full rate once 50% rate is tolerated.

In certain embodiments, when a subject experiences a grade 2 (e.g., therapy or infusion interruption indicated but responds promptly to symptomatic treatment) infusion reaction or hypersensitivity, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when the method of administration (e.g., IV infusion) is stopped due to a subject experiencing a grade 2 (e.g., therapy or infusion interruption indicated but responds promptly to symptomatic treatment) infusion reaction or hypersensitivity, the method of administration of the expression repressor disclosed herein may be restarted at 50% of the previous rate under continuous observation. In some embodiments, when the subject experiences a recurring grade 2 (e.g., therapy or infusion interruption indicated but responds promptly to symptomatic treatment) infusion reaction or hypersensitivity at a reinitiated infusion rate after premedication, the method of administration of the expression repressor disclosed herein is discontinued (e g., permanently).

In certain embodiments, when a subject experiences a grade 3 (e.g., prolonged; recurrence of symptoms following initial improvement) or grade 4 infusion reaction or hypersensitivity, the method of administration of the expression repressor disclosed herein is discontinued (e.g., permanently). In certain embodiments, when a subject experiences a grade 3 (e.g., prolonged; recurrence of symptoms following initial improvement) or grade 4 infusion reaction or hypersensitivity, after the method of administration of the expression repressor disclosed herein is discontinued (e.g., permanently), the subject is administered supplemental oxygen, fluids, and resuscitative measures as needed.

In some embodiments, when a subject experiences a grade 2 (e.g., painless edema) infusion site extravasation, the method of administration of the expression repressor disclosed herein comprises stopping infusion (e.g., immediately). In some embodiments, if the method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 2 (e.g., painless edema) infusion site extravasation, the method of administration comprises resuming at full rate after a vascular access away from the initial affected site is established.

In certain embodiments, when a subject experiences a grade 3 (e.g., erythema with associated symptoms) infusion site extravasation, the method of administration of the expression repressor disclosed herein comprises stopping infusion (e.g., immediately). In certain embodiments, if the method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 3 (e.g., erythema with associated symptoms) infusion site extravasation, the subject receives one or both of: 1) an infectious disease consultation, and 2) a plastic surge ry consultation, hi certain embodiments, if tire method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 3 (e.g., erythema with associated symptoms) infusion site extravasation, the method of administration may be resumed upon resolution to < grade 2 (e.g., painless edema). In certain embodiments, if the method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 3 (e.g., erythema with associated symptoms) infusion site extravasation, the method of administration comprises resuming at 50% of the previous rate after a vascular access away from the initial affected site is established concurrently with continuous observation. In certain embodiments, if the method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 3 (e.g., erythema with associated symptoms) infusion site extravasation, the method of administration is discontinued (e.g., permanently).

In certain embodiments, when a subject experiences a grade 4 infusion site extravasation, the method of administration of the expression repressor disclosed herein comprises stopping infusion (e.g., immediately), e.g., permanently. In certain embodiments, if the method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 4 infusion site extravasation, the subject receives one or both of: 1) an infectious disease consultation, and 2) a plastic surgery consultation.

In some embodiments, when a subject experiences a grade 1 ocular adverse event (e.g., uveitis, eye pain, blurred vision), the subject receives an ophthalmology consultation. In certain embodiments, when a subject experiences a grade 1 ocular adverse event (e.g., uveitis, eye pain, blurred vision), the method of administration of the expression repressor disclosed herein is continued.

In certain embodiments, when a subject experiences a grade 2 ocular adverse event (e.g., uveitis, eye pain, blurred vision), the method of administration of the expression repressor disclosed herein comprises stopping infusion. In some embodiments, when a subject experiences a grade 2 ocular adverse event (e.g., uveitis, eye pain, blurred vision), the subject receives an urgent ophthalmology consultation. In certain embodiments, when the method of administration (e.g., IV infusion) is interrupted due to the subject experiencing a grade 2 ocular adverse event (e.g., uveitis, eye pain, blurred vision), the method of administration of the expression repressor disclosed herein comprises resuming administration without dose reduction upon resolution of the ocular adverse event (e.g., uveitis, eye pain, blurred vision) to < grade 1 within 7 days and in consultation with ophthalmology. In certain embodiments, when the method of administration (e.g., IV infusion) is interrupted due to the subject experiencing a grade 2 ocular adverse event (e.g., uveitis, eye pain, blurred vision), the method of administration of the expression repressor disclosed herein comprises resuming administration at a dose reduction (e.g., the next lower dose) upon resolution of the ocular adverse event (e.g., uveitis, eye pain, blurred vision) to < grade 1 greater than 7 days but less than 14 days and in consultation with ophthalmology. In some embodiments, when the method of administration (e.g., IV infusion) is interrupted due to the subject experiencing a grade 2 ocular adverse event (e.g., uveitis, eye pain, blurred vision), the method of administration of the expression repressor disclosed herein comprises discontinuing administration (e.g., permanently) when a grade 2 treatment-related ocular adverse event (e.g., uveitis, eye pain, blurred vision) does not respond to topical therapy and does not improve to < grade 1 within 14 days.

In certain embodiments, when a subject experiences a grade 3 or 4 ocular adverse event (e.g., uveitis, eye pain, blurred vision), the method of administration of the expression repressor disclosed herein comprises stopping administration (e.g., permanently). In some embodiments, when a subject experiences a grade 3 or 4 ocular adverse event (e.g., uveitis, eye pain, blurred vision), the subject receives an urgent ophthalmology consultation.

In some embodiments, when a subject experiences a grade 1 (e.g., symptomatic; clinical or diagnostic observations only; intervention not indicated) pneumonitis, the method of administration of the expression repressor disclosed herein comprises either continuing administration or stopping administration. In one embodiment, when a subject experiences a grade 1 (e.g., symptomatic; clinical or diagnostic observations only; intervention not indicated) pneumonitis, the subject receives one or both of: 1) a pulmonary consultation, and 2) a pulmonary and infection workup. In certain embodiments, if the method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 1 (e.g., symptomatic; clinical or diagnostic observations only; intervention not indicated) pneumonitis, the method of administration of the expression repressor disclosed herein may be resumed, e.g., upon radiographic evidence of improvement.

In certain embodiments, when a subject experiences a grade 2 (e.g., symptomatic; medical intervention indicated; limiting instrumental activities of daily living (ADL)) pneumonitis, the method of administration of the expression repressor disclosed herein comprises stopping administration. In one embodiment, when a subject experiences a grade 2 (e.g., symptomatic; medical intervention indicated; limiting instrumental ADL) pneumonitis, the subject receives one or both of: 1) a pulmonary consultation, and 2) a pulmonary and infection workup. In certain embodiments, if the method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 1 (e.g., symptomatic; clinical or diagnostic observations only; intervention not indicated) pneumonitis, the method of administration of the expression repressor disclosed herein may be resumed upon resolution to < grade 1. In certain embodiments, if the method of administration (e.g., IV infusion) is interrupted due to a subject experiencing a grade 1 (e.g., symptomatic; clinical or diagnostic observations only; intervention not indicated) pneumonitis, the method of administration of the expression repressor disclosed herein may be resumed at the next lower dose.

In certain embodiments, when a subject experiences a grade 3 (e.g., severe symptoms; limiting self-care ADL; oxygen indicated) or grade 4 pneumonitis, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently, hi one embodiment, when a subject experiences a grade 3 (e.g., severe symptoms; limiting self-care ADL; oxygen indicated) or grade 4 pneumonitis, the subject receives one or both of: 1) a pulmonary consultation, and 2) a pulmonary and infection workup.

In some embodiments, when a subject experiences a grade 2 (e.g., symptomatic, no intervention needed) claudication, the method of administration of the expression repressor disclosed herein comprises continuation without modification. In certain embodiments, when a subject experiences a grade 3 or grade 4 (e.g., disabling) claudication, the method of administration of the expression repressor disclosed herein comprises permanent discontinuation.

In some embodiments, when a subject experiences a grade 2 hypertension (e.g., systolic BP 140 - 159 mmHg or diastolic BP 90 - 99 mmHg if previously within normal limits (WNL); change in baseline medical intervention indicated; recurrent or persistent (>24 hrs); symptomatic increase by >20 mmHg (diastolic) or to >140/90 mmHg; monotherapy indicated initiated) hypertension, antihypertensive therapy may be administered. In some embodiments, when a subject experiences a grade 2 hypertension (e.g., systolic BP 140 - 159 mmHg or diastolic BP 90 - 99 mmHg if previously WNL; change in baseline medical intervention indicated; recurrent or persistent (>24 hrs); symptomatic increase by >20 mmHg (diastolic) or to >140/90 mmHg; monotherapy indicated initiated) hypertension, the method of administration of the expression repressor disclosed herein comprises continuation without modification.

In some embodiments, when a subject experiences a grade 3 (e.g., systolic BP >160 mmHg or diastolic BP >100 mmHg; medical intervention indicated; more than one drug or more intensive therapy than previously used indicated) hypertension, the method of administration of the expression repressor disclosed herein comprises stopping administration if grade 3 hypertension persists > 7 days despite optimal hypertensive therapy. In some embodiments, when a subject experiences a grade 3 (e.g., systolic BP >160 mmHg or diastolic BP >100 mmHg; medical intervention indicated; more than one drug or more intensive therapy than previously used indicated) hypertension, the method of administration of the expression repressor disclosed herein may be resumed upon resolution to < grade 2. In some embodiments, when a subject experiences agrade 3 (e.g., systolic BP >160 mmHg or diastolic BP >100 mmHg; medical intervention indicated; more than one drug or more intensive therapy than previously used indicated) hypertension, the method of administration of the expression repressor disclosed herein may be resumed at a lower dose.

In some embodiments, when a subject experiences a grade 4 hypertension, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In some embodiments, when a subject experiences a grade 4 hypertension, the subject receives a cardiology consultation.

In some embodiments, when a subject experiences a grade 2 (Average QTc 450 - 480 ms) ECG QTc interval prolongation, the method of administration of the expression repressor disclosed herein comprises stopping administration until resolved or < grade 1 or baseline (e.g., ECG(s) collected prior to administration). In certain embodiments, when a subject experiences a grade 2 (Average QTc 450 - 480 ms) ECG QTc interval prolongation, the method of administration of the expression repressor disclosed herein may be resumed without modification. In certain embodiments, when a subject experiences a grade 3 (average QTc 481 - 500 ms) ECG QTc interval prolongation, the method of administration of the expression repressor disclosed herein comprises stopping administration. In some embodiments, when a subject experiences a grade 3 (average QTc 481 - 500 ms) ECG QTc interval prolongation, the subject receives a cardiology consultation. In certain embodiments, when a subject experiences a grade 3 (average QTc 481 - 500 ms) ECG QTc interval prolongation, the method of administration of the expression repressor disclosed herein comprises resuming administration upon resolution to grade < 1 or < 30 ms difference from baseline QTc with < 7 days. In certain embodiments, when a subject experiences a grade 3 (average QTc 481 - 500 ms) ECG QTc interval prolongation, the method of administration of the expression repressor disclosed herein comprises resuming administration at a lower dose.

In certain embodiments, when a subject experiences a grade 4 (Torsade de pointes; polymorphic ventricular tachycardia; signs/symptoms of serious arrhythmia) ECG QTc interval prolongation, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, when a subject experiences a grade 4 (Torsade de pointes; polymorphic ventricular tachycardia; signs/symptoms of serious arrhythmia) ECG QTc interval prolongation, the subject receives a cardiology consultation.

In some embodiments, when a subject experiences a grade 2 or above myocarditis or cardiac ischemia/infarction, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, when a subject experiences a grade 2 or above myocarditis or cardiac ischemia/infarction, the subject receives a cardiology consultation.

In some embodiments, when a subject experiences any grade of arterial thromboembolic event, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In some embodiments, when a subject experiences a grade 2 cardiovascular disorder, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 2 cardiovascular disorder, the method of administration of the expression repressor disclosed herein comprises resuming administration at a lower dose upon resolution to grade < 1 or baseline. In certain embodiments, when a subject experiences a grade 2 cardiovascular disorder, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently if the grade 2 cardiovascular disorder does not resolve to grade < 1 or baseline.

In some embodiments, when a subject experiences a grade 3 or greater cardiovascular disorder, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, when a subject experiences a grade 3 cardiovascular disorder, the subject receives a cardiology consultation.

In some embodiments, when a subject experiences diarrhea or colitis, the subject is administered anti-diarrheal medication at the first sign of abdominal cramping, loose stools, or overt diarrhea. In one embodiments, when a subject experiences a grade 1 (e.g., increase of < 4 stools per day over baseline; mild increase in ostomy output compared to baseline) diarrhea or colitis, the method of administration of the expression repressor disclosed herein comprises continuation without modification.

In certain embodiments, when a subject experiences a grade 2 (e.g., increase of 4 - 6 stools per day over baseline; moderate increase in ostomy output compared to baseline; limiting instrumental ADL) diarrhea or colitis, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 2 (e.g., increase of 4 - 6 stools per day over baseline; moderate increase in ostomy output compared to baseline; limiting instrumental ADL) diarrhea or colitis, the method of administration of the expression repressor disclosed herein comprises resuming administration without dose modification upon resolution to grade < 1.

In some embodiments, when a subject experiences a grade 3 (e.g., increase of >7 stools per day over baseline; hospitalization indicated; severe increase in ostomy output compared to baseline; limiting self-care ADL) diarrhea or colitis, the method of administration of the expression repressor disclosed herein comprises stopping administration. In some embodiments, when a subject experiences a grade 3 (e.g., increase of >7 stools per day over baseline; hospitalization indicated; severe increase in ostomy output compared to baseline; limiting self-care ADL) diarrhea or colitis, the subject receives a GI consultation. In certain embodiments, when a subject experiences a grade 3 (e.g., increase of >7 stools per day over baseline; hospitalization indicated; severe increase in ostomy output compared to baseline; limiting self-care ADL) diarrhea or colitis, the method of administration of the expression repressor disclosed herein comprises resuming administration without dose modification upon resolution to grade < 1. In certain embodiments, when a subject experiences a grade 3 (e.g., increase of >7 stools per day over baseline; hospitalization indicated; severe increase in ostomy output compared to baseline; limiting self- care ADL) diarrhea or colitis, the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose. In certain embodiments, when a subject experiences a grade 3 (e.g., increase of >7 stools per day over baseline; hospitalization indicated; severe increase in ostomy output compared to baseline; limiting self-care ADL) diarrhea or colitis, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently if the grade 3 diarrhea or colitis does not resolve to grade < 1.

In some embodiments, when a subject experiences a grade 4 diarrhea or colitis, the method of administration of tire expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, when a subject experiences a grade 4 diarrhea or colitis, the subject receives a GI consultation.

In some embodiments, when a subject experiences a grade 1 gastrointestinal perforation, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 1 gastrointestinal perforation, the subject receives one of both of: 1) a GI consultation, and 2) a general surgery consultation. In certain embodiments, when a subject experiences a grade 1 gastrointestinal perforation, the method of administration of the expression repressor disclosed herein comprises resuming administration upon resolution of the grade 1 gastrointestinal perforation.

In some embodiments, when a subject experiences a grade 2, 3, or 4 gastrointestinal perforation, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In some embodiments, when a subject experiences a grade 2 (e.g., medical intervention indicated) hemorrhage or bleeding, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 2 (e.g., medical intervention indicated) hemorrhage or bleeding, the method of administration of the expression repressor disclosed herein comprises resuming administration without dose modification upon resolution to grade < I . In certain embodiments, when a subject experiences a grade 2 (e.g., medical intervention indicated) hemorrhage or bleeding, the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose. In certain embodiments, when a subject experiences a grade 2 (e.g., medical intervention indicated) hemorrhage or bleeding, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently if the grade 2 hemorrhage or bleeding does not resolve to grade < 1.

In certain embodiments, when a subject experiences a grade 3 (e.g., transfusion indicated) or grade 4 hemorrhage or bleeding, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In some embodiments, when a subject experiences a grade 2 AST and/or ALT elevation (e.g., > 3.0 - 5.0 x ULN if baseline was normal; > 3.0 - 5.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 2 AST and/or ALT elevation (e.g., > 3.0 - 5.0 x ULN if baseline was normal; > 3.0 - 5.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises resuming administration without a modification (e.g., in the first or second occurrence) upon resolution to < grade 1 . In certain embodiments, when a subject experiences a grade 2 AST and/or ALT elevation (e.g., > 3.0 - 5.0 x ULN if baseline was normal; > 3.0 - 5.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises resuming administration at a lower dose. In certain embodiments, when a subject experiences a grade 2 AST and/or ALT elevation (e.g., > 3.0 - 5.0 * ULN if baseline was normal;

> 3.0 - 5.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises resuming administration without a modification (e.g., at the third occurrence).

In certain embodiments, when the subject experiences a grade 3 AST and/or ALT elevation (e.g.,

> 5.0 - 20.0 x ULN if baseline was normal; > 5.0 - 20.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 3 AST and/or ALT elevation (e.g., > 5.0 - 20.0 x ULN if baseline was normal; > 5.0 - 20.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose (e.g., in the first occurrence) upon resolution to < grade 1 or baseline within 7 days. In certain embodiments, when a subject experiences a second occurrence of a grade 3 AST and/or ALT elevation (e.g., > 5.0 - 20.0 x ULN if baseline was normal; > 5.0 - 20.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In some embodiments, when the subject experiences a grade 4 AST and/or ALT elevation (> 20.0 x ULN if baseline was normal; > 20.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In some embodiments, when a subject experiences a grade 2 total bilirubin elevation (e.g., > 1.5 - 3.0 x ULN if baseline was normal; > 1.5 - 3.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 2 total bilirubin elevation (e.g., > 1.5 - 3.0 x ULN if baseline was normal; > 1.5 - 3.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose (e.g., in the first occurrence) upon resolution to < grade 1. In certain embodiments, when a subject experiences a second occurrence of a grade 2 total bilirubin elevation (e.g., > 1.5 - 3.0 x ULN if baseline was normal; > 1.5 - 3.0 x baseline if baseline was abnormal), the method of administration of tire expression repressor disclosed herein comprises stopping administration permanently .

In certain embodiments, when a subject experiences agrade 3 total bilirubin elevation (e.g., > 3.0 - 10.0 x ULN if baseline was normal; > 3.0 - 10.0 x baseline if baseline was abnormal) or grade 4 (> 10.0 x ULN if baseline was normal; > 10.0 x baseline if baseline was abnormal), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, if grade 3 or grade 4 hypcr-bilirubincmia is due to the indirect (nonconjugated) component only, and hemolysis as the etiology has been ruled out as per institutional guidelines (e.g., review of peripheral blood smear and haptoglobin determination), the method of administration of the expression repressor disclosed herein comprises stopping administration until the grade 3 or grade 4 hyper-bilirubinemia is resolved < grade 1, and optionally resume administration thereafter. In certain embodiments, the management of simultaneous AST/ALT and total bilirubin elevation should be based on the higher grade of the two.

In some embodiments, when a subject experiences a grade 3 asymptomatic amylase and/or lipase elevation (e.g., > 5.0 x upper limit of normal (ULN); not associated with symptoms or clinical manifestations of pancreatitis), the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 3 asymptomatic amylase and/or lipase elevation, the subject receives a GI consultation. In certain embodiments, when a subject experiences a grade 3 asymptomatic amylase and/or lipase elevation (e.g., > 5.0 x upper limit of normal (ULN); not associated with symptoms or clinical manifestations of pancreatitis), the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon resolution to < grade 2 within 14 days. In certain embodiments, when a subject experiences a grade 3 asymptomatic amylase and/or lipase elevation (e.g., > 5.0 x upper limit of normal (ULN); not associated with symptoms or clinical manifestations of pancreatitis), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently if the grade 3 asymptomatic amylase and/or lipase elevation does not resolve to < grade 2 within 14 days. In certain embodiments, a subject suspected of experiencing, or identified as experiencing, a > Grade 3 amylase and/or lipase receives a CT scan or other imaging study to assess the pancreas, liver, and gallbladder within 1 week of the first occurrence.

In some embodiments, when a subject experiences a grade 2 (e.g., confirmed by radiologic evidence) pancreatitis, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 2 (e.g., confirmed by radiologic evidence) pancreatitis, the subject receives a GI consultation. In some embodiments, when a subject experiences a grade 2 (e.g., confinned by radiologic evidence) pancreatitis, the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon resolution to grade 1. In certain embodiments, when a subject experiences a grade 2 (e g., confirmed by radiologic evidence) pancreatitis, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently if the grade 2 pancreatitis does not resolve to grade 1. In certain embodiments, when a subject experiences a grade 3 or 4 pancreatitis, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, when a subject experiences a grade 3 or 4 pancreatitis, the subject receives a GI consultation.

In some embodiments, when a subject experiences a grade 2 serum creatinine (e.g., > 1.5 - 3.0 x baseline; > 1.5 - 3.0 x ULN), the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 2 serum creatinine (e.g., > 1.5 - 3.0 x baseline; > 1.5 - 3.0 x ULN), the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon resolution to < grade 1. In certain embodiments, when a subject experiences a grade 2 serum creatinine (e.g., > 1.5 - 3.0 x baseline; > 1.5 - 3.0 x ULN), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently if the grade 2 serum creatinine does not resolve to grade 1.

In certain embodiments, when a subject experiences agrade 3 serum creatinine (e.g., > 3.0 x baseline; > 3.0 - 6.0 x ULN) or grade 4 serum creatinine (> 6.0 x ULN), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In some embodiments, when a subject experiences a grade 2 or grade 3 musculoskeletal, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences agrade 2 or grade 3 musculoskeletal, the subject receives a rheumatology consultation. In some embodiments, the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon resolution to < grade 1. In certain embodiments, when a subject experiences a grade 2 or grade 3 musculoskeletal, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently if the grade 2 or grade 3 musculoskeletal does not resolve to < grade 1.

In certain embodiments, when a subject experiences agrade 4 musculoskeletal, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 4 musculoskeletal, the subject receives a rheumatology consultation. In some embodiments, when a subject experiences a grade 4 musculoskeletal, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, when a subject experiences a grade 4 musculoskeletal, the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon consultation with a rheumatologist. In some embodiments, when a subject experiences a grade 2 hypothyroidism or hyperthyroidism, the method of administration of the expression repressor disclosed herein comprises continuing administration without dose modification.

In certain embodiments, when a subject experiences a grade 3 hypothyroidism or hyperthyroidism, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 3 hypothyroidism or hyperthyroidism, the subject receives an endocrinology consultation. In some embodiments, when a subject experiences a grade 3 hypothyroidism or hyperthyroidism, the method of administration of the expression repressor disclosed herein comprises resuming administration without dose modification upon resolution to < grade 1 with appropriate management.

In some embodiments, when a subject experiences a grade 4 hypothyroidism or hyperthyroidism, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In some embodiments, when a subject experiences a grade 2 or grade 3 endocrine disorder (e.g., thyroiditis, hypothyroidism, hypophysitis, insulin-deficient diabetes, primary adrenal insufficiency), the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 2 or grade 3 endocrine disorder (e.g., thyroiditis, hypothyroidism, hypophysitis, insulin-deficient diabetes, primary adrenal insufficiency), the subject receives an endocrinology consultation. In some embodiments, when a subject experiences a grade 2 or grade 3 endocrine disorder (e g., thyroiditis, hypothyroidism, hypophysitis, insulin-deficient diabetes, primary' adrenal insufficiency), the method of administration of the expression repressor disclosed herein comprises resuming administration without dose modification upon resolution to < grade 1 with appropriate management.

In certain embodiments, when a subject experiences a grade 4 endocrine disorder, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, when a subject experiences a grade 4 endocrine disorder, the subject receives an endocrinology consultation. In some embodiments, when a subject experiences a grade 4 endocrine disorder (e.g., adrenal insufficiency, ACTH deficiency or glucose intolerance), which resolve or are adequately controlled with physiologic hormone replacement (e.g., corticosteroids or thyroid hormones) or glucose-controllmg agents, respectively, may continue treatment with the expression repressor disclosed herein.

In some embodiments, when a subject experiences a grade 1 neurology adverse event, the method of administration of the expression repressor disclosed herein comprises stopping administration. In some embodiments, when a subject experiences a grade 1 neurology adverse event, the subject receives a neurology consultation. In certain embodiments, when a subject is suspected of having one or more of Guillain-Barre syndrome, encephalitis, aseptic meningitis, transverse myelitis, or peripheral neuropathy, the method of administration of the expression repressor disclosed herein comprises stopping administration. In some embodiments, when a subject is suspected of having one or more of Guillain- Barre syndrome, encephalitis, aseptic meningitis, transverse myelitis, or peripheral neuropathy, the subject receives a neurology consultation. In certain embodiments, when a subject experiences a grade 1 neurology adverse event, the method of administration of the expression repressor disclosed herein comprises continuing administration without dose modification. In some embodiments, when a subject experiences a grade 1 neurology adverse event, the method of administration of the expression repressor disclosed herein comprises and the administration is stopped, the method comprises resume administration without a dose modification upon resolution of the adverse event.

In certain embodiments, when a subject experiences a grade 2 neurology adverse event, the method of administration of the expression repressor disclosed herein comprises stopping administration. In some embodiments, when a subject experiences a grade 2 neurology adverse event, the subject receives a neurology consultation. In certain embodiments, the method of administration of the expression repressor disclosed herein comprises, after halting administration in response to a grade 2 neurology adverse event, resuming administration at the next lower dose. In certain embodiments, when a subject experiences a grade 2 neurology adverse event, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In certain embodiments, when a subject experiences a grade 3 or grade 4 neurology adverse event, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In some embodiments, when a subject experiences a grade 3 or grade 4 neurology adverse event, the subject receives a neurology consultation.

In some embodiments, when a subject experiences a grade 1 dermatology adverse event, the method of administration of the expression repressor disclosed herein comprises continuing administration without dose modification. In certain embodiments, when a subject experiences a grade 1 dermatology adverse event, the subject receives a topical therapy.

In certain embodiments, when a subject experiences a grade 2 dermatology adverse event (e.g., in the first occurrence), the method of administration of the expression repressor disclosed herein comprises continuing administration without a dose modification. In certain embodiments, when a subject experiences a grade 2 dermatology adverse event (e.g., in the first occurrence), the subject receives a topical therapy. In certain embodiments, when a subject experiences a grade 2 dermatology adverse event (e.g., in the first occurrence) that does not resolve within 7 days after topical therapy, the method of administration of the expression repressor disclosed herein comprises stopping administration. In some embodiments, when a subject experiences a grade 2 dermatology adverse event (e.g., in the second or third occurrence), the method of administration of the expression repressor disclosed herein comprises stopping administration. In some embodiments, when a subject experiences a grade 2 dermatology adverse event (e.g., in the second or third occurrence), the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon resolution to < grade 1. In some embodiments, when a subject experiences a grade 2 dermatology adverse event (e.g., in the fourth occurrence), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In some embodiments, when a subject experiences a grade 3 dermatology adverse event, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 3 dermatology adverse event, the subject receives a topical therapy and/or systemic therapy. In certain embodiments, when a subject experiences a grade 2 dermatology adverse event (e.g., in the first occurrence), the subject receives a topical therapy. In certain embodiments, when a subject experiences a grade 3 dermatology adverse event, the subject receives a dermatology consultation. In some embodiments, when a subject experiences a grade 3 dermatology adverse event (e.g., in the first or second occurrence), the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon resolution to < grade 1. In some embodiments, when a subject experiences a grade 3 dermatology adverse event (e.g., in the third occurrence), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently .

In some embodiments, when a subject experiences a grade 4 dermatology adverse event, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In some embodiments, when a subject experiences a grade 4 dermatology adverse event, the subject receives a dermatology consultation.

In some embodiments, when a subject experiences a grade 1 or grade 2 bullous dermatitis, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 1 or grade 2 bullous dermatitis, the subject receives a dermatology consultation. In certain embodiments, tire method of administration of the expression repressor disclosed herein comprises resuming administration. In certain embodiments, the method of administration of the expression repressor disclosed herein does not comprise resuming administration.

In certain embodiments, when a subject experiences a grade 3 or grade 4 bullous dermatitis, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, when a subject experiences a grade 3 or grade 4 bullous dermatitis, the subject receives a dermatology consultation.

In some embodiments, when a subject experiences any grade of Stevens-Johnson Syndrome (SJS), Lyell syndrome/toxic epidermal necrolysis (TEN), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In certain embodiments, when a subject experiences any grade of Stevens- Johnson Syndrome (SJS), Lyell syndrome/toxic epidermal necrolysis (TEN), the subject receives a dermatology consultation.

In some embodiments, when a subject experiences a grade 3 or grade 4 neutropenia (e.g., absolute neutrophil count (ANC)), the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 3 or grade 4 neutropenia, the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon resolution of the grade 3 or grade 4 neutropenia to < grade 1 or baseline within 7 days.

In some embodiments, when a subject experiences a grade 3 febrile neutropenia (e.g., NC < 1000/mm³ with a single temperature of > 38.3°C (101°F) or a sustained temperature of > 38°C (100.4°F) for more than 1 hour), the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 3 or grade 4 neutropenia, the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon resolution of the fever and improvement of the grade 3 febrile neutropenia (e.g., NC < 1000/mm³ with a single temperature of > 38.3°C (101°F) or a sustained temperature of > 38°C (100.4°F) for more than 1 hour), to < grade 1 or baseline.

In certain embodiments, when a subject experiences a grade 4 febrile neutropenia (e.g., wherein the subject experiences ANC < 500/mm3 with a single temperature of > 38.5°C (101°F) or a sustained temperature of > 38°C (100.4°F) for more than 1 hour), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

In some embodiments, when a subject experiences a grade 3 thrombocytopenia (e.g., < 50,000 - 25,000/mm³), the method of administration of the expression repressor disclosed herein comprises stopping administration, hr some embodiments, when a subject experiences a grade 3 thrombocytopenia (e.g., < 50,000 - 25,000/mm³), the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose upon resolution of the grade 3 thrombocytopenia (e.g., < 50,000 - 25,000/mm³) to < grade 2 or baseline.

In certain embodiments, when a subject experiences a grade 4 thrombocytopenia (e.g., < 25,000/mm³), the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In some embodiments, when a subject experiences a grade 3 (< 8.0 g/dL) or grade 4 anemia, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 3 (< 8.0 g/dL) or grade 4 anemia, the method of administration of the expression repressor disclosed herein comprises resuming administration, without dose modification, upon resolution of the grade 3 (< 8.0 g/dL) or grade 4 anemia to < grade 2 or baseline < 7 days.

In some embodiments, when a subject experiences any grade lymphopenia, the method of administration of the expression repressor disclosed herein comprises continuing administration without dose modification.

In some embodiments, when a subject experiences a grade 3 adverse event not disclosed herein, the method of administration of the expression repressor disclosed herein comprises stopping administration. In certain embodiments, when a subject experiences a grade 3 adverse event not disclosed herein, the method of administration of the expression repressor disclosed herein comprises resuming administration, without dose modification, upon resolution of the adverse event to < grade 1. In certain embodiments, when a subject experiences a grade 3 adverse event not disclosed herein, the method of administration of the expression repressor disclosed herein comprises resuming administration at the next lower dose.

In certain embodiments, when a subject experiences a grade 4 adverse event not disclosed herein, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In some embodiments, when a subject experiences an isolated grade 4 electrolyte abnormality not associated with clinical sequelae and corrected after appropriate management within 72 hours of their onset do not require stopping administration. In some embodiments, when a subject experiences an isolated grade 4 electrolyte abnormality that does not resolve to < grade 1 within 72 hours after appropriate management, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently. In some embodiments, when a subject experiences an isolated grade 4 electrolyte abnormality associated with clinical sequelae, the method of administration of the expression repressor disclosed herein comprises stopping administration permanently.

The person of ordinary skill will realize that a variety of factors, such as age, sex, weight, severity of disorder to be treated may be considered in selecting a dosage of a modulating agent or a combination of modulating agents as disclosed herein, and that the dosage and/or frequency of administration may be increased or decreased during the course of therapy. The dosage may be repeated as needed, with evidence of reduction of tumor volume observed after as few as 2 to 8 doses. The dosages and schedules of administration disclosed herein show minimal effect on overall weight of the subject compared to cisplatin, sorafenib, or a small molecule comparator. The subject methods may include use of CT and/or PET/CT, or MRI, to measure tumor response at regular intervals. Blood levels of tumor markers may also be monitored. Dosages and/or administration schedules may be adjusted as needed, according to the results of imaging and/or marker blood levels.

In some embodiments, the compositions disclosed herein may be administered in combination with one or more therapeutic agents or methods chosen from immune checkpoint inhibitor polypeptides (e.g., an antibody molecule (e.g., an anti-PD-1, an anti-PD-Ll, an anti-CTLA-4, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) or peptide), surgical resection, orthotopic liver transplantation, radiofrequency ablation, immunotherapy, immune checkpoint plus anti-vascular- endothelial -growth-factor combination therapy, photodynamic therapy (PDT), laser therapy, brachytherapy, radiation therapy, trans-catheter arterial chemo- or radio-embolization, stereotactic radiation therapy, chemotherapy, and/or systemic chemotherapy, or any combination thereof to treat a disease or disorder.

In some embodiments, an expression repressor disclosed herein may be administered in combination with an immune checkpoint inhibitor polypeptide (e.g., an antibody molecule (e.g., an anti- PD-1, an anti-PD-Ll, an anti-CTLA-4, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) or peptide), and one or more of tyrosine kinase inhibitors (TKIs), e.g., sorafenib, bromodomain inhibitors, e.g., BET inhibitors, e.g., JQ1, e.g., BET672, e.g., birabresib, MEK inhibitors, (e.g., Trametinib). In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-PD-1 antibody molecule and atyrosine kinase inhibitor (TKI), e.g., sorafenib. In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-PD-1 antibody molecule and a bromodomain inhibitor, e.g., BET inhibitor, e.g., JQ1, e.g., BET672, e.g., birabresib. In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-PD-1 antibody molecule and a MEK inhibitor, (e.g., Trametinib). In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-PD-Ll antibody molecule and atyrosine kinase inhibitor (TKI), e.g., sorafenib. In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-PD-Ll antibody molecule and a bromodomain inhibitor, e.g., BET inhibitors, e.g., JQ1, e.g., BET672, e.g., birabresib. In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-PD-Ll antibody molecule and a MEK inhibitor, (e.g., Trametinib). In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-CTLA-4 antibody molecule and atyrosine kinase inhibitor (TKI), e.g., sorafenib. In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-CTLA-4 antibody molecule and a bromodomain inhibitor, e.g., BET inhibitor, e.g., JQ1, e.g., BET672, e.g., birabresib. In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-CTLA-4 antibody molecule and a MEK inhibitor, (e.g., Trametinib). In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-LAG3 antibody molecule and a tyrosine kinase inhibitor (TKI), e.g., sorafenib. In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-LAG3 antibody molecule and a bromodomain inhibitor, e.g., BET inhibitor, e.g., JQ1, e.g., BET672, e.g., birabresib. In some embodiments, an expression repressor disclosed herein may be administered in combination with an anti-LAG3 antibody molecule and a MEK inhibitor, (e.g., Trametinib).

Exemplary antibody molecules are disclosed in Section “Combination Therapies”, for example, portions of the antibody molecules are disclosed in Tables 18-26.

In some embodiments, the composition is administered in combination with an immune checkpoint inhibitor polypeptide (e.g., an antibody molecule (e.g., an anti-PD-1, an anti-PD-Ll, an anti- CTLA-4, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) or peptide). In certain embodiments, the immune checkpoint inhibitor polypeptide is an antibody molecule (e.g., anti-PD-1, anti- PD-Ll, anti-CTLA-4, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule).

In certain embodiments, the antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered by injection (e.g., subcutaneously or intravenously), for instance, at a dose of about 1 to 30 mg/kg, about 1 to about 20 mg/kg, about 1 to about 15 mg/kg, about 1 to about 10 mg/kg, about 1 to about 5 mg/kg, about 2 to 30 mg/kg, about 2 to about 20 mg/kg, about 2 to about 15 mg/kg, about 2 to about 10 mg/kg, about 2 to about 5 mg/kg, about 5 to about 30 mg/kg, about 5 to about 20 mg/kg, about 5 to about 15 mg/kg, about 5 to about 10 mg/kg, about 10 to about 30 mg/kg, about 10 to about 20 mg/kg, about 10 to about 15 mg/kg, about 3 mg/kg, or about 2 mg/kg). The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks. In one embodiment, the antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered at a dose from about 10 to 20 mg/kg every other week, hr one embodiment, tire antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered at a dose from about 10 to 20 mg/kg every third week. In one embodiment, the dose is about 1 to 5 mg/kg, e.g., every 2 weeks, every 3 weeks, or every 4 weeks. In one embodiment, the dose is less than 5 mg/kg, less than 4 mg/kg, less than 3 mg/kg, less than 2 mg/kg, or less than 1 mg/kg, e.g., every 2 weeks, every 3 weeks, or every 4 weeks. In one embodiment, the dose is about 2 mg/kg, e.g., every 2 weeks, every 3 weeks, or every 4 weeks.

In some embodiments, the dose of an antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule), is a flat dose. In some embodiments, the antibody molecule (e.g., an anti- PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered by injection (e.g., subcutaneously or intravenously), for instance, at a dose (e.g., a flat dose) of about 100 mg to 2000 mg, (e.g., about 100 mg to about 1900 mg, about 100 mg to about 1800 mg, about 100 mg to about 1700 mg, about 100 mg to about 1600 mg, about 100 mg to about 1500 mg, about 100 mg to about 1400 mg, about 100 mg to about 1300 mg, about 100 mg to about 1200 mg, about 100 mg to about 1100 mg, about 100 mg to about 1000 mg, about 100 mg to about 900 mg, about 100 mg to about 800 mg, about 100 mg to about 700 mg, about 100 mg to about 600 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 200 mg, about 200 mg to about 20000 mg, about 200 mg to about 1900 mg, about 200 mg to about 1800 mg, about 200 mg to about 1700 mg, about 200 mg to about 1600 mg, about 200 mg to about 1500 mg, about 200 mg to about 1400 mg, about 200 mg to about 1300 mg, about 200 mg to about 1200 mg, about 200 mg to about 1100 mg, about 200 mg to about 1000 mg, about 200 mg to about 900 mg, about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 200 mg to about 500 mg, about 200 mg to about 400 mg, about 200 mg to about 300 mg, about 300 mg to about 2000 mg, about 300 mg to about 1900 mg, about 300 mg to about 1800 mg, about 300 mg to about 1700 mg, about 300 mg to about 1600 mg, about 300 mg to about 1500 mg, about 300 mg to about 1400 mg, about 300 mg to about 1300 mg, about 300 mg to about 1200 mg, about 300 mg to about 1100 mg, about 300 mg to about 1000 mg, about 300 mg to about 900 mg, about 300 mg to about 800 mg, about 300 mg to about 700 mg, about 300 mg to about 600 mg, about 300 mg to about 500 mg, about 300 mg to about 400 mg, about 400 mg to about 2000 mg, about 400 mg to about 1900 mg, about 400 mg to about 1800 mg, about 400 mg to about 1700 mg, about 400 mg to about 1600 mg, about 400 mg to about 1500 mg, about 400 mg to about 1400 mg, about 400 mg to about 1300 mg, about 400 mg to about 1200 mg, about 400 mg to about 1100 mg, about 400 mg to about 1000 mg, about 400 mg to about 900 mg, about 400 mg to about 800 mg, about 400 mg to about 700 mg, about 400 mg to about 600 mg, about 400 mg to about 500 mg, about 500 mg to about 2000 mg, about 500 mg to about 1900 mg, about 500 mg to about 1800 mg, about 500 mg to about 1700 mg, about 500 mg to about 1600 mg, about 500 mg to about 1500 mg, about 500 mg to about 1400 mg, about 500 mg to about 1300 mg, about 500 mg to about 1200 mg, about 500 mg to about 1100 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 600 mg to about 2000 mg, about 600 mg to about 1900 mg, about 600 mg to about 1800 mg, about 600 mg to about 1700 mg, about 600 mg to about 1600 mg, about 600 mg to about 1500 mg, about 600 mg to about 1400 mg, about 600 mg to about 1300 mg, about 600 mg to about 1200 mg, about 600 mg to about 1100 mg, about 600 mg to about 1000 mg, about 600 mg to about 900 mg, about 600 mg to about 800 mg, about 600 mg to about 700 mg, about 700 mg to about 2000 mg, about 700 mg to about 1900 mg, about 700 mg to about 1800 mg, about 700 mg to about 1700 mg, about 700 mg to about 1600 mg, about 700 mg to about 1500 mg, about 700 mg to about 1400 mg, about 700 mg to about 1300 mg, about 700 mg to about 1200 mg, about 700 mg to about 1100 mg, about 700 mg to about 1000 mg, about 700 mg to about 900 mg, about 700 mg to about 800 mg, about 800 mg to about 2000 mg, about 800 mg to about 1900 mg, about 800 mg to about 1800 mg, about 800 mg to about 1700 mg, about 800 mg to about 1600 mg, about 800 mg to about 1500 mg, about 800 mg to about 1400 mg, about 800 mg to about 1300 mg, about 800 mg to about 1200 mg, about 800 mg to about 1100 mg, about 800 mg to about 1000 mg, about 800 mg to about 900 mg, about 900 mg to about 1900 mg, about 900 mg to about 1800 mg, about 900 mg to about 1700 mg, about 900 mg to about 1600 mg, about 900 mg to about 1500 mg, about 900 mg to about 1400 mg, about 900 mg to about 1300 mg, about 900 mg to about 1200 mg, about 900 mg to about 1100 mg, about 900 mg to about 1000 mg, about 1000 mg to about 2000 mg, about 1000 mg to about 1900 mg, about 1000 mg to about 1800 mg, about 1000 mg to about 1700 mg, about 1000 mg to about 1600 mg, about 1000 mg to about 1500 mg, about 1000 mg to about 1400 mg, about 1000 mg to about 1300 mg, about 1000 mg to about 1200 mg, about 1000 mg to about 1100 mg, about 1100 mg to about 1900 mg, about 1100 mg to about 1800 mg, about 1100 mg to about 1700 mg, about 1100 mg to about 1600 mg, about 1100 mg to about 1500 mg, about 1100 mg to about 1400 mg, about 1100 mg to about 1300 mg, about 1100 mg to about 1200 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 1900 mg, about 1300 mg to about 1800 mg, about 1300 mg to about 1700 mg, about 1300 mg to about 1600 mg, about 1300 mg to about 1500 mg, about 1300 mg to about 1400 mg, about 1400 mg to about 2000 mg, about 1400 mg to about 1900 mg, about 1400 mg to about 1800 mg, about 1400 mg to about 1700 mg, about 1400 mg to about 1600 mg, about 1400 mg to about 1500 mg, about 1500 mg to about 2000 mg, about 1500 mg to about 1900 mg, about 1500 mg to about 1800 mg, about 1500 mg to about 1700 mg, about 1500 mg to about 1600 mg, about 1600 mg to about 2000 mg, about 1600 mg to about 1900 mg, about 1600 mg to about 1800 mg, about 1600 mg to about 1700 mg, or about 1800 mg to about 1900 mg). The dosing schedule (e.g., flat dosing schedule) can vary from, e.g., once a week to once every 2, 3, 4, 5, or 6 weeks. The dosing schedule (e.g,, flat dosing schedule) can vary from, e.g., 1, 2, 3, 4, 5, 6, 7, or more times a week for 1, 2, 3, 4, 5, 6, or more weeks. In one embodiment, the antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered at a dose from about 200 mg, for instance, once every three weeks or once every four weeks. In one embodiment, the antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti- LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti- KIR antibody molecule, or an anti-BTLA antibody molecule) is administered at a dose from about 300 mg to 400 mg, for instance, once every three weeks or once every four weeks. In one embodiment, the antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered at a dose from about 300 mg, for instance, once every three weeks, e.g., by way of intravenous infusion. In one embodiment, the antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered at a dose from about 200 mg, for instance once every three weeks, e.g., by way of intravenous infusion. In one embodiment, the antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered at a dose from about 400 mg, for instance, once every four weeks, e.g., by way of intravenous infusion. In one embodiment, the antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered at a dose from about 300 mg, for instance once every four weeks, e.g., by way of intravenous infusion. In one embodiment, tire antibody molecule (e.g., an anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, anti-CTLA-4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, and anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule) is administered at a dose from about 400 mg, for instance, once every three weeks, e.g., by way of intravenous infusion.

In some embodiments, the anti-PD-1 antibody molecule comprises cemiplimab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

The dose may be within a defined range. For example, the dose may be at about 35-500 mg, about 35 mg to about 450 mg, about 35 mg to about 400 mg, about 35 mg to about 350 mg, about 35 mg to about 300 mg, about 35 mg to about 250 mg, about 35 mg to about 200 mg, about 35 mg to about 150 mg, about 35 mg to about 100 mg, about 35 mg to about 50 mg, about 40 mg to about 500 mg, about 40 mg to about 450 mg, about 40 mg to about 400 mg, about 40 mg to about 350 mg, about 40 mg to about 300 mg, about 40 mg to about 250 mg, about 40 mg to about 200 mg, about 40 mg to about 150 mg, about 40 mg to about 100 mg, or about 40 mg to about 50 mg, about 75 mg to about 500 mg, about 75 mg to about 450 mg, about 75 mg to about 400 mg, about 75 mg to about 350 mg, about 75 mg to about 300 mg, about 75 mg to about 250 mg, about 75 mg to about 200 mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 450 mg, about 100 mg to about 400 mg, about 100 mg to about 350 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, about 100 mg to about 150 mg, about 250 mg to about 500 mg, about 250 mg to about 450 mg, about 250 mg to about 400 mg, about 250 mg to about 350 mg, about 250 mg to about 300 mg, about 300 mg to about 500 mg, about 300 mg to about 450 mg, about 300 mg to about 400 mg, about 300 mg to about 350 mg, about 350 mg to about 500 mg, about 350 mg to about 450 mg, about 350 mg to about 400 mg, or about 400 mg to about 500 mg, or about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 mg).

The dose may be a defined value. For example, the dose may be about 35 mg, about 40 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg.

Hie dose may be administered for a defined dosing period (e.g., the length of time taken to administer a single infusion). For example, the dosing period may be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, or about 6 hours.

Multiple doses may be administered at defined intervals. For example, the interval between doses may be about 1 days, about 2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, about 10 weeks, or about 12 weeks.

In some embodiments, the anti-PD-1 antibody molecule comprises cemiplimab and is administered as an intravenous infusion (e.g., over 30 minutes) every 3 weeks. In some embodiments, the anti-PD-1 antibody molecule is cemiplimab administered at 350 mg as an intravenous infusion over 30 minutes every 3 weeks.

In some embodiments, the anti-PD-1 antibody molecule comprises dostarlimab-gxly and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

The dose may be within a defined range. For example, the dose may be about 50-600 mg, about 75 mg to about 600 mg, about 75 mg to about 550 mg, about 75 mg to about 500 mg, about 75 mg to about 450 mg, about 75 mg to about 400 mg, about 75 mg to about 350 mg, about 75 mg to about 300 mg, about 75 mg to about 250 mg, about 75 mg to about 200 mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about 100 mg to about 600 mg, about 100 mg to about 550 mg, about 100 mg to about 500 mg, about 100 mg to about 450 mg, about 100 mg to about 400 mg, about 100 mg to about 350 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, about 100 mg to about 150 mg, about 150 mg to about 600 mg, about 150 mg to about 550 mg, about 150 mg to about 500 mg, about 150 mg to about 450 mg, about 150 mg to about 400 mg, about 150 mg to about 350 mg, about 150 mg to about 300 mg, about 150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg to about 600 mg, about 200 mg to about 550 mg, about 200 mg to about 500 mg, about 200 mg to about 450 mg, about 200 mg to about 400 mg, about 200 mg to about 350 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, about 250 mg to about 600 mg, about 250 mg to about 550 mg, about 250 mg to about 500 mg, about 250 mg to about 450 mg, about 250 mg to about 400 mg, about 250 mg to about 350 mg, about 250 mg to about 300 mg, about 300 mg to about 600 mg, about 300 mg to about 550 mg, about 300 mg to about 500 mg, about 300 mg to about 450 mg, about 300 mg to about 400 mg, about 300 mg to about 350 mg, about 350 mg to about 600 mg, about 350 mg to about 550 mg, about 350 mg to about 500 mg, about 350 mg to about 450 mg, about 350 mg to about 400 mg, about 400 mg to about 600 mg, about 400 mg to about 550 mg, about 400 mg to about 500 mg, about 400 mg to about 450 mg, about 450 mg to about 600 mg, about 450 mg to about 550 mg, about 450 mg to about 500 mg, about 500 mg to about 600 mg, or about 500 mg to about 550 mg, or about 100-1500 mg, about 100 mg to about 1400 mg, about 100 mg to about 1350 mg, about 100 mg to about 1300 mg, about 100 mg to about 1250 mg, about 100 mg to about 1200 mg, about 100 mg to about 1150 mg, about 100 mg to about 1100 mg, about 100 mg to about 1050 mg, about 100 mg to about 1000 mg, about 100 mg to about 950 mg, about 100 mg to about 900 mg, about 100 mg to about 850 mg, about 100 mg to about 800 mg, about 100 mg to about 750 mg, about 100 mg to about 700 mg, about 100 mg to about 650 mg, about 100 mg to about 600 mg, about 100 mg to about 550 mg, about 100 mg to about 500 mg, about 100 mg to about 450 mg, about 100 mg to about 400 mg, about 100 mg to about 350 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, about 100 mg to about 150 mg, about 200 mg to about 1500 mg, about 200 mg to about 1450 mg, about 200 mg to about 1400 mg, about 200 mg to about 1350 mg, about 200 mg to about 1300 mg, about 200 mg to about 1250 mg, about 200 mg to about 1200 mg, about 200 mg to about 1150 mg, about 200 mg to about 1100 mg, about 200 mg to about 1050 mg, about 200 mg to about 1000 mg, about 200 mg to about 950 mg, about 200 mg to about 900 mg, about 200 mg to about 850 mg, about 200 mg to about 800 mg, about 200 mg to about 750 mg, about 200 mg to about 700 mg, about 200 mg to about 650 mg, about 200 mg to about 600 mg, about 200 mg to about 550 mg, about 200 mg to about 500 mg, about 200 mg to about 450 mg, about 200 mg to about 400 mg, about 200 mg to about 350 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, about 300 mg to about 1500 mg, about 300 mg to about 1450 mg, about 300 mg to about 1400 mg, about 300 mg to about 1350 mg, about 300 mg to about 1300 mg, about 300 mg to about 1250 mg, about 300 mg to about 1200 mg, about 300 mg to about 1150 mg, about 300 mg to about 1100 mg, about 300 mg to about 1050 mg, about 300 mg to about 1000 mg, about 300 mg to about 950 mg, about 300 mg to about 900 mg, about 300 mg to about 850 mg, about 300 mg to about 800 mg, about 300 mg to about 750 mg, about 300 mg to about 700 mg, about 300 mg to about 650 mg, about 300 mg to about 600 mg, about 300 mg to about 550 mg, about 300 mg to about 500 mg, about 300 mg to about 450 mg, about 300 mg to about 400 mg, about 300 mg to about 350 mg, about 400 mg to about 1500 mg, about 400 mg to about 1450 mg, about 400 mg to about 1400 mg, about 400 mg to about 1350 mg, about 400 mg to about 1300 mg, about 400 mg to about 1250 mg, about 400 mg to about 1200 mg, about 400 mg to about 1150 mg, about 400 mg to about 1100 mg, about 400 mg to about 1050 mg, about 400 mg to about 1000 mg, about 400 mg to about 950 mg, about 400 mg to about 900 mg, about 400 mg to about 850 mg, about 400 mg to about 800 mg, about 400 mg to about 750 mg, about 400 mg to about 700 mg, about 400 mg to about 650 mg, about 400 mg to about 600 mg, about 400 mg to about 550 mg, about 400 mg to about 500 mg, about 400 mg to about 450 mg, about 500 mg to about 1500 mg, about 500 mg to about 1450 mg, about 500 mg to about 1400 mg, about 500 mg to about 1350 mg, about 500 mg to about 1300 mg, about 500 mg to about 1250 mg, about 500 mg to about 1200 mg, about 500 mg to about 1150 mg, about 500 mg to about 1100 mg, about 500 mg to about 1050 mg, about 500 mg to about 1000 mg, about 500 mg to about 950 mg, about 500 mg to about 900 mg, about 500 mg to about 850 mg, about 500 mg to about 800 mg, about 500 mg to about 750 mg, about 500 mg to about 700 mg, about 500 mg to about 650 mg, about 500 mg to about 600 mg, about 500 mg to about 550 mg, about 600 mg to about 1500 mg, about 600 mg to about 1450 mg, about 600 mg to about 1400 mg, about 600 mg to about 1350 mg, about 600 mg to about 1300 mg, about 600 mg to about 1250 mg, about 600 mg to about 1200 mg, about 600 mg to about 11 0 mg, about 600 mg to about 1100 mg, about 600 mg to about 1050 mg, about 600 mg to about 1000 mg, about 600 mg to about 950 mg, about 600 mg to about 900 mg, about 600 mg to about 850 mg, about 600 mg to about 800 mg, about 600 mg to about 750 mg, about 600 mg to about 700 mg, about 600 mg to about 650 mg, about 700 mg to about 1500 mg, about 700 mg to about 1450 mg, about 700 mg to about 1400 mg, about 700 mg to about 1350 mg, about 700 mg to about 1300 mg, about 700 mg to about 1250 mg, about 700 mg to about 1200 mg, about 700 mg to about 1150 mg, about 700 mg to about 1100 mg, about 700 mg to about 1050 mg, about 700 mg to about 1000 mg, about 700 mg to about 950 mg, about 700 mg to about 900 mg, about 700 mg to about 850 mg, about 700 mg to about 800 mg, about 700 mg to about 750 mg, about 800 mg to about 1500 mg, about 800 mg to about 1450 mg, about 800 mg to about 1400 mg, about 800 mg to about 1350 mg, about 800 mg to about 1300 mg, about 800 mg to about 1250 mg, about 800 mg to about 1200 mg, about 800 mg to about 1150 mg, about 800 mg to about 1100 mg, about 800 mg to about 1050 mg, about 800 mg to about 1000 mg, about 800 mg to about 950 mg, about 800 mg to about 900 mg, about 800 mg to about 850 mg, about 900 mg to about 1500 mg, about 900 mg to about 1450 mg, about 900 mg to about 1400 mg, about 900 mg to about 1350 mg, about 900 mg to about 1300 mg, about 900 mg to about 1250 mg, about 900 mg to about 1200 mg, about 900 mg to about 1150 mg, about 900 mg to about 1100 mg, about 900 mg to about 1050 mg, about 900 mg to about 1000 mg, about 900 mg to about 950 mg, about 1000 mg to about 1500 mg, about 1000 mg to about 1450 mg, about 1000 mg to about 1400 mg, about 1000 mg to about 1350 mg, about 1000 mg to about 1300 mg, about 1000 mg to about 1250 mg, about 1000 mg to about 1200 mg, about 1000 mg to about 1150 mg, about 1000 mg to about 1100 mg, about 1000 mg to about 1050 mg, about 1100 mg to about 1500 mg, about 1100 mg to about 1450 mg, about 1100 mg to about 1400 mg, about 1100 mg to about 1350 mg, about 1100 mg to about 1300 mg, about 1100 mg to about 1250 mg, about 1100 mg to about 1200 mg, about 1100 mg to about 1150 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1450 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1350 mg, about 1200 mg to about 1300 mg, about 1200 mg to about 1250 mg, about 1300 mg to about 1500 mg, about 1300 mg to about 1450 mg, about 1300 mg to about 1400 mg, about 1300 mg to about 1350 mg, about 1400 mg to about 1500 mg, or about 1400 mg to about 1450 mg.

The dose may be a defined value. For example, the dose may be about 50 mg, about 100 mg, about 200, about 300, about 400, about 500, about 600 mg, about 700, about 800, about 900, about 100, about 1200, about 1300, about 1400, or about 1500 mg).

The dose may be administered within a defined dosing period. For example, the dosing period may be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, or about 6 hours.

In some embodiments, the anti-PD-1 antibody molecule comprises dostarlimab-gxly and is administered as an intravenous infusion (e.g., over 30 minutes) every 3 weeks for the first 4 doses, and is administered as an intravenous infusion (e.g., over 30 minutes) every 6 weeks for subsequent doses (e.g., dose 5 beginning 3 weeks after dose 4). In some embodiments, the anti-PD-1 antibody molecule comprises dostarlimab-gxly and is administered at 500 mg as an intravenous infusion over 30 minutes every 3 weeks for the first 4 doses, and is administered at 1,000 mg as an intravenous infusion over 30 minutes every 6 weeks for subsequent doses (e.g., dose 5 administered 3 weeks after dose 4). In some embodiments, the anti-PD-1 antibody molecule is administered at about 100, about 200, about 300, about 400, about 500, or about 600 mg) for 4 doses (e.g., 1 dose every three weeks), and subsequent doses (e.g., dose 5 onwards) is administered at about 100-1500 mg.

In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

The dose may be within a defined range. For example, the dose may be about 24-600 mg, about 24 mg to about 550 mg, about 24 mg to about 500 mg, about 24 mg to about 450 mg, about 24 mg to about 400 mg, about 24 mg to about 350 mg, about 24 mg to about 300 mg, about 24 mg to about 250 mg, about 24 mg to about 200 mg, about 24 mg to about 150 mg, about 24 mg to about 100 mg, about 24 mg to about 50 mg, about 50 mg to about 600 mg, about 50 mg to about 550 mg, about 50 mg to about 500 mg, about 50 mg to about 450 mg, about 50 mg to about 400 mg, about 50 mg to about 350 mg, about 50 mg to about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 100 mg to about 600 mg, about 100 mg to about 550 mg, about 100 mg to about 500 mg, about 100 mg to about 450 mg, about 100 mg to about 400 mg, about 100 mg to about 350 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, about 100 mg to about 150 mg, about 150 mg to about 600 mg, about 150 mg to about 550 mg, about 150 mg to about 500 mg, about 150 mg to about 450 mg, about 150 mg to about 400 mg, about 150 mg to about 350 mg, about 150 mg to about 300 mg, about 150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg to about 600 mg, about 200 mg to about 550 mg, about 200 mg to about 500 mg, about 200 mg to about 450 mg, about 200 mg to about 400 mg, about 200 mg to about 350 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, about 250 mg to about 600 mg, about 250 mg to about 550 mg, about 250 mg to about 500 mg, about 250 mg to about 450 mg, about 250 mg to about 400 mg, about 250 mg to about 350 mg, about 250 mg to about 300 mg, about 300 mg to about 600 mg, about 300 mg to about 550 mg, about 300 mg to about 500 mg, about 300 mg to about 450 mg, about 300 mg to about 400 mg, about 300 mg to about 350 mg, about 350 mg to about 600 mg, about 350 mg to about 550 mg, about 350 mg to about 500 mg, about 350 mg to about 450 mg, about 350 mg to about 400 mg, about 400 mg to about 600 mg, about 400 mg to about 550 mg, about 400 mg to about 500 mg, about 400 mg to about 450 mg, about 450 mg to about 600 mg, about 450 mg to about 550 mg, or about 450 mg to about 500 mg, about 0.5-5 mg/kg (e.g., about 0.5 mg/kg to about 4.8 mg/kg, about 0.5 mg/kg to about 4.3 mg/kg, about 0.5 mg/kg to about 3.8 mg/kg, about 0.5 mg/kg to about 3.3 mg/kg, about 0.5 mg/kg to about 2.8 mg/kg, about 0.5 mg/kg to about 2.3 mg/kg, about 0.5 mg/kg to about 1.8 mg/kg, about 0.5 mg/kg to about 1.3 mg/kg, about 0.5 mg/kg to about 0.8 mg/kg, about 0.75 mg/kg to about 5 mg/kg, about 0.75 mg/kg to about 4.5 mg/kg, about 0.75 mg/kg to about 4 mg/kg, about 0.75 mg/kg to about 3.5 mg/kg, about 0.75 mg/kg to about 3 mg/kg, about 0.75 mg/kg to about 2.5 mg/kg, about 0.75 mg/kg to about 2 mg/kg, about 0.75 mg/kg to about 1 .5 mg/kg, about 0.75 mg/kg to about 1 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 4.5 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3.5 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 2.5 mg/kg, about 1 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.5 mg/kg, about 1.25 mg/kg to about 5 mg/kg, about 1 .25 mg/kg to about

4.5 mg/kg, about 1.25 mg/kg to about 4 mg/kg, about 1.25 mg/kg to about 3.5 mg/kg, about 1.25 mg/kg to about 3 mg/kg, about 1.25 mg/kg to about 2.5 mg/kg, about 1 .25 mg/kg to about 2 mg/kg, about 1.25 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 4.5 mg/kg, about

1.5 mg/kg to about 4 mg/kg, about 1.5 mg/kg to about 3.5 mg/kg, about 1 .5 mg/kg to about 3 mg/kg, about 1 .5 mg/kg to about 2.5 mg/kg, about 1.5 mg/kg to about 2 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2 mg/kg to about 4.5 mg/kg, about 2 mg/kg to about 4 mg/kg, about 2 mg/kg to about 3.5 mg/kg, about 2 mg/kg to about 3 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2.5 mg/kg to about 5 mg/kg, about 2.5 mg/kg to about 4.5 mg/kg, about 2.5 mg/kg to about 4 mg/kg, about 2.5 mg/kg to about 3.5 mg/kg, about 2.5 mg/kg to about 3 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 4.5 mg/kg, about 3 mg/kg to about 4 mg/kg, about 3 mg/kg to about 3.5 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 3.5 mg/kg to about 4.5 mg/kg, about 3.5 mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, or about 4 mg/kg to about 4.5 mg/kg.

The dose may be a defined value. For example, the dose may be about 50, about 100, about 150, about 200, about 240, about 300, about 350, about 400, about 450, about 480, about 500, about 550, or about 600 mg, or about 0.8 mg/kg, about 1 .0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5 mg/kg, about 4.0 mg/kg, about 4.5 mg/kg, or about 5.0 mg/kg. The dose may be administered for a defined dosing period (e.g., the length of time taken to administer a single infusion). For example, the dosing period may be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, or about 6 hours.

In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab and is administered as an intravenous infusion (e.g., over 30 minutes) every 2 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab and is administered as an intravenous infusion (e.g., over 30 minutes) cvcrv 3 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab and is administered as an intravenous infusion (e.g., over 30 minutes) every 4 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab and is administered at 240 as an intravenous infusion (e.g., over 30 minutes) every 2 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab and is administered at 360 mg as an intravenous infusion (e.g., over 30 minutes) every 3 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab and is administered at 480 mg as an intravenous infusion (e.g., over 30 minutes) every 4 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab and is administered at 3 mg/kg as an intravenous infusion (e g., over 30 minutes) every 2 weeks. In some embodiments, the anti-PD-1 antibody molecule is nivolumab administered at 3 mg/kg as an intravenous infusion (e.g., over 30 minutes) every 3 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab and is administered at 1 mg/kg as an intravenous infusion (e.g., over 30 minutes) every 3 weeks.

In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

Hie dose may be within a defined range. For example, the dose may be about 20 mg to about 500 mg, about 20 mg to about 450 mg, about 20 mg to about 400 mg, about 20 mg to about 350 mg, about 20 mg to about 300 mg, about 20 mg to about 250 mg, about 20 mg to about 200 mg, about 20 mg to about 150 mg, about 20 mg to about 100 mg, about 20 mg to about 50 mg, about 50 mg to about 500 mg, about 50 mg to about 450 mg, about 50 mg to about 400 mg, about 50 mg to about 350 mg, about 50 mg to about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 450 mg, about 100 mg to about 400 mg, about 100 mg to about 350 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, about 100 mg to about 150 mg, about 150 mg to about 500 mg, about 150 mg to about 450 mg, about 150 mg to about 400 mg, about 150 mg to about 350 mg, about 150 mg to about 300 mg, about 150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg to about 500 mg, about 200 mg to about 450 mg, about 200 mg to about 400 mg, about 200 mg to about 350 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, about 250 mg to about 500 mg, about 250 mg to about 450 mg, about 250 mg to about 400 mg, about 250 mg to about 350 mg, about 250 mg to about 300 mg, about 300 mg to about 500 mg, about 300 mg to about 450 mg, about 300 mg to about 400 mg, about 300 mg to about 350 mg, about 350 mg to about 500 mg, about 350 mg to about 450 mg, about 350 mg to about 400 mg, or about 400 mg to about 500 mg, or about 400 mg to about 450 mg, or about 0.5-5 mg/kg (e.g., about 0.5 mg/kg to about 4.8 mg/kg, about 0.5 mg/kg to about 4.3 mg/kg, about 0.5 mg/kg to about 3.8 mg/kg, about 0.5 mg/kg to about 3.3 mg/kg, about 0.5 mg/kg to about 2.8 mg/kg, about 0.5 mg/kg to about 2.3 mg/kg, about 0.5 mg/kg to about 1.8 mg/kg, about 0.5 mg/kg to about 1.3 mg/kg, about 0.5 mg/kg to about 0.8 mg/kg, about 0.75 mg/kg to about 5 mg/kg, about 0.75 mg/kg to about 4.5 mg/kg, about 0.75 mg/kg to about 4 mg/kg, about 0.75 mg/kg to about 3.5 mg/kg, about 0.75 mg/kg to about 3 mg/kg, about 0.75 mg/kg to about 2.5 mg/kg, about 0.75 mg/kg to about 2 mg/kg, about 0.75 mg/kg to about 1.5 mg/kg, about 0.75 mg/kg to about 1 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 4.5 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3.5 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 2.5 mg/kg, about 1 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.5 mg/kg, about 1 .25 mg/kg to about 5 mg/kg, about 1.25 mg/kg to about 4.5 mg/kg, about 1 .25 mg/kg to about 4 mg/kg, about 1.25 mg/kg to about 3.5 mg/kg, about 1.25 mg/kg to about 3 mg/kg, about 1.25 mg/kg to about 2.5 mg/kg, about 1.25 mg/kg to about 2 mg/kg, about 1.25 mg/kg to about 1.5 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 4.5 mg/kg, about 1.5 mg/kg to about 4 mg/kg, about 1.5 mg/kg to about 3.5 mg/kg, about 1 .5 mg/kg to about 3 mg/kg, about 1 .5 mg/kg to about 2.5 mg/kg, about 1.5 mg/kg to about 2 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2 mg/kg to about 4.5 mg/kg, about 2 mg/kg to about 4 mg/kg, about 2 mg/kg to about 3.5 mg/kg, about 2 mg/kg to about 3 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2.5 mg/kg to about 5 mg/kg, about 2.5 mg/kg to about 4.5 mg/kg, about 2.5 mg/kg to about 4 mg/kg, about 2.5 mg/kg to about 3.5 mg/kg, about 2.5 mg/kg to about 3 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 4.5 mg/kg, about 3 mg/kg to about 4 mg/kg, about 3 mg/kg to about 3.5 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 3.5 mg/kg to about 4.5 mg/kg, about 3.5 mg/kg to about 4 mg/kg, about 4 mg/kg to about 5 mg/kg, or about 4 mg/kg to about 4.5 mg/kg.

The dose may be a defined value. For example, the dose may be about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 mg, or about 0.8 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5 mg/kg, about 4.0 mg/kg, about 4.5 mg/kg, or about 5.0 mg/kg. In some embodiments, the dose may be about 0.5- 5 mg/kg up to 200 mg.

In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered as an intravenous infusion (e.g., over 30 minutes) every 3 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered as an intravenous infusion (e.g., over 30 minutes) every 6 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered at 200 mg as an intravenous infusion (e g., over 30 minutes) every 3 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered at 400 mg as an intravenous infusion (e.g., over 30 minutes) every 6 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered as an intravenous infusion (e.g., over 30 minutes (-5/+10 minutes)) about every 3 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered as an intravenous infusion (e.g., over 30 minutes (-5/+10 minutes)) about every 6 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered at 200 mg as an intravenous infusion (e.g., over 30 minutes (-5/+10 minutes)) every 3 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered at 400 mg as an intravenous infusion (e.g., over 30 minutes (-5/+10 minutes)) every 6 weeks. In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab and is administered at 2 mg/kg (e.g., up to 200 mg) as an intravenous infusion (e.g., over 30 minutes) every 3 weeks. In some embodiments, when bicistronic ZF9-MQ1 ZF3-KRAB and pembrolizumab are both administered, pembrolizumab infusion will be administered at least 1 hour (and optionally less than 3 hours) after completion of the bicistronic ZF9-MQ1 ZF3-KRAB infusion.

In some embodiments, when a subject experiences an immune-related adverse event related to pembrolizumab, the subject will be graded by the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE), version 5.

In some embodiments, a subject experiencing an immune-related adverse event related to pembrolizumab receives one or more of: systemic corticosteroids (e g., 1-2 mg/day, e.g., of prednisone or equivalent), e.g., until improvement to grade 1 or less. In some embodiments, a subject receiving systemic corticosteroids is tapered over at least 1 month upon improvement to grade 1 or less. In some embodiments, administration of pembrolizumab may be resumed once the subject is clinically stable, the adverse event has resolved to < grade 1, and prednisone is administered at 10 mg/day (or equivalent) or lower. In some embodiments, when a subject experiences grade 2 pneumonitis (e.g., an immune-related adverse event), the method of administration of pembrolizumab is withheld. In some embodiments, when a subject experiences grade 2 pneumonitis, the method of administration of pembrolizumab is withheld until complete or partial resolution (e.g., grades 0 or 1) of the pneumonitis and after corticosteroid taper. In some embodiments, the method of administration of pembrolizumab is discontinued in subjects where no complete or partial resolution of the adverse event occurs within about 12 weeks of initiating steroids. In some embodiments, the method of administration of pembrolizumab is discontinued in subjects experiencing grade 2 pneumonitis having the inability to reduce prednisone to 10 mg/day or less (or equivalent) within 12 weeks of initiating steroids. In some embodiments, when a subject experiences grade 3 or 4 pneumonitis (e.g., an immune-related adverse event), the method of administration of pembrolizumab is discontinued (e.g., permanently).

In some embodiments, when a subject experiences grade 2 or 3 colitis (e.g., an immune-related adverse event), the method of administration of pembrolizumab is withheld. In some embodiments, when a subject experiences grade 2 or 3 colitis, the method of administration of pembrolizumab is withheld until complete or partial resolution (e.g., grades 0 or 1) of the colitis and after corticosteroid taper. In some embodiments, the method of administration of pembrolizumab is discontinued in subjects where no complete or partial resolution of the adverse event occurs within about 12 weeks of initiating steroids. In some embodiments, the method of administration of pembrolizumab is discontinued in subjects experiencing grade 2 or 3 colitis having the inability to reduce prednisone to 10 mg/day or less (or equivalent) within 12 weeks of initiating steroids. In some embodiments, when a subject experiences grade 4 colitis (e.g., an immune -related adverse event), the method of administration of pembrolizumab is discontinued (e.g., permanently).

In some embodiments, when a subject experiences grade 3 or 4 endocrinopathy (e.g., an immune- related adverse event), the method of administration of pembrolizumab is withheld. In some embodiments, when a subject experiences grade 3 or 4 endocrinopathy (e.g., an immune-related adverse event), the method of administration of pembrolizumab is withheld until the subject is clinically stable.

In some embodiments, when a subject experiences nephritis with renal dysfunction with grade 2 or 3 increased blood creatine, the method of administration of pembrolizumab is withheld. In some embodiments, when a subject experiences nephritis with renal dysfunction with grade 2 or 3 increased blood creatine, the method of administration of pembrolizumab is withheld until complete or partial resolution (e.g., grades 0 or 1) of the grade 2 or 3 increased blood creatine, and after corticosteroid taper. In some embodiments, the method of administration of pembrolizumab is discontinued in subjects where no complete or partial resolution of the adverse event occurs within about 12 weeks of initiating steroids. In some embodiments, tire method of administration of pembrolizumab is discontinued in subjects experiencing nephritis with renal dysfunction with grade 2 or 3 increased blood creatine, having the inability to reduce prednisone to 10 mg/day or less (or equivalent) within 12 weeks of initiating steroids. In some embodiments, when a subject experiences nephritis with renal dysfunction with grade 4 increased blood creatine, the method of administration of pembrolizumab is permanently discontinued.

In some embodiments, when a subject is suspected of experiencing or is identified as experiencing an exfoliative dermatologic condition (e.g., suspected Stevens-Johnson Syndrome (SJS), toxic epidermal necrolysis (TEN), or drug rash with Eosinophlia and systemic symptoms (DRESS)), the method of administration of pembrolizumab is withheld. In some embodiments, when a subject is suspected of experiencing an exfoliative dermatologic condition (e.g., SJS, TEN, or DRESS), the method of administration of pembrolizumab is withheld until complete or partial resolution (e g., grades 0 or 1) of the suspected exfoliative dermatologic condition (e.g., SJS, TEN, or DRESS), and after corticosteroid taper. In some embodiments, the method of administration of pembrolizumab is discontinued in subjects where no complete or partial resolution of the suspected adverse event occurs within about 12 weeks of initiating steroids. In some embodiments, the method of administration of pembrolizumab is discontinued in subjects suspected of experiencing an exfoliative dermatologic condition (e.g., SJS, TEN, or DRESS), having the inability to reduce prednisone to 10 mg/day or less (or equivalent) within 12 weeks of initiating steroids. In some embodiments, when a subject has or is identified as having an exfoliative dermatologic condition (e.g., SJS, TEN, or DRESS), the method of administration of pembrolizumab is permanently discontinued.

In some embodiments, when a subject is suspected of experiencing grade 2, 3, or 4 myocarditis, the method of administration of pembrolizumab is permanently discontinued.

In some embodiments, when a subject experiences grade 2 a neurological toxicity, the method of administration of pembrolizumab is withheld. In some embodiments, when a subject experiences grade 2 neurological toxicity, the method of administration of pembrolizumab is withheld until complete or partial resolution (e.g., grades 0 or 1) of the neurological toxicity and after corticosteroid taper. In some embodiments, the method of administration of pembrolizumab is discontinued in subjects where no complete or partial resolution of the adverse event occurs within about 12 weeks of initiating steroids. In some embodiments, the method of administration of pembrolizumab is discontinued in subjects experiencing grade 2 neurological toxicity having the inability to reduce prednisone to 10 mg/day or less (or equivalent) within 12 weeks of initiating steroids. In some embodiments, when a subject experiences grade 3 or 4 a neurological toxicity, the method of administration of pembrolizumab is discontinued (e.g., permanently).

In some embodiments, when a subject experiences a grade 1 or 2 infusion-related reaction, the method of administration of pembrolizumab can be interrupted and/or infusion time may be extended (e.g., the rate of infusion is reduced). In certain embodiments, when a subject experiences a grade 3 or 4 infusion-related reaction, the method of administration of pembrolizumab is discontinued (e.g., permanently).

In some embodiments, when a subject experiences a grade 2 serum creatinine (e.g., > 1.5 - 3.0 * baseline, > 1.5 - 3.0 x ULN), the method of administration of pembrolizumab comprises stopping administration. In certain embodiments, when a subject experiences a grade 2 serum creatinine (e.g., > 1.5 - 3.0 x baseline; > 1.5 - 3.0 x ULN), the method of administration of pembrolizumab comprises resuming administration without dose modulation upon resolution to < grade 1. In certain embodiments, when a subject experiences a grade 2 serum creatinine (e.g., > 1.5 - 3.0 x baseline; > 1.5 - 3.0 x ULN), the method of administration of the pembrolizumab comprises stopping administration permanently if the grade 2 serum creatinine does not resolve to grade 1.

In some embodiments, the anti-PD-Ll antibody molecule comprises atezolizumab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

The dose may be within a defined range. For example, the dose may be about 80 mg to about 2000 mg, about 100 mg to about 1900 mg, about 100 mg to about 1800 mg, about 100 mg to about 1700 mg, about 100 mg to about 1600 mg, about 100 mg to about 1500 mg, about 100 mg to about 1400 mg, about 100 mg to about 1300 mg, about 100 mg to about 1200 mg, about 100 mg to about 1100 mg, about 100 mg to about 1000 mg, about 100 mg to about 900 mg, about 100 mg to about 800 mg, about 100 mg to about 700 mg, about 100 mg to about 600 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 200 mg, about 200 mg to about 20000 mg, about 200 mg to about 1900 mg, about 200 mg to about 1800 mg, about 200 mg to about 1700 mg, about 200 mg to about 1600 mg, about 200 mg to about 1500 mg, about 200 mg to about 1400 mg, about 200 mg to about 1300 mg, about 200 mg to about 1200 mg, about 200 mg to about 1100 mg, about 200 mg to about 1000 mg, about 200 mg to about 900 mg, about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 200 mg to about 500 mg, about 200 mg to about 400 mg, about 200 mg to about 300 mg, about 300 mg to about 2000 mg, about 300 mg to about 1900 mg, about 300 mg to about 1800 mg, about 300 mg to about 1700 mg, about 300 mg to about 1600 mg, about 300 mg to about 1500 mg, about 300 mg to about 1400 mg, about 300 mg to about 1300 mg, about 300 mg to about 1200 mg, about 300 mg to about 1100 mg, about 300 mg to about 1000 mg, about 300 mg to about 900 mg, about 300 mg to about 800 mg, about 300 mg to about 700 mg, about 300 mg to about 600 mg, about 300 mg to about 500 mg, about 300 mg to about 400 mg, about 400 mg to about 2000 mg, about 400 mg to about 1900 mg, about 400 mg to about 1800 mg, about 400 mg to about 1700 mg, about 400 mg to about 1600 mg, about 400 mg to about 1500 mg, about 400 mg to about 1400 mg, about 400 mg to about 1300 mg, about 400 mg to about 1200 mg, about 400 mg to about 1100 mg, about 400 mg to about 1000 mg, about 400 mg to about 900 mg, about 400 mg to about 800 mg, about 400 mg to about 700 mg, about 400 mg to about 600 mg, about 400 mg to about 500 mg, about 500 mg to about 2000 mg, about 500 mg to about 1900 mg, about 500 mg to about 1800 mg, about 500 mg to about 1700 mg, about 500 mg to about 1600 mg, about 500 mg to about 1500 mg, about 500 mg to about 1400 mg, about 500 mg to about 1300 mg, about 500 mg to about 1200 mg, about 500 mg to about 1100 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 600 mg to about 2000 mg, about 600 mg to about 1900 mg, about 600 mg to about 1800 mg, about 600 mg to about 1700 mg, about 600 mg to about 1600 mg, about 600 mg to about 1500 mg, about 600 mg to about 1400 mg, about 600 mg to about 1300 mg, about 600 mg to about 1200 mg, about 600 mg to about 1100 mg, about 600 mg to about 1000 mg, about 600 mg to about 900 mg, about 600 mg to about 800 mg, about 600 mg to about 700 mg, about 700 mg to about 2000 mg, about 700 mg to about 1900 mg, about 700 mg to about 1800 mg, about 700 mg to about 1700 mg, about 700 mg to about 1600 mg, about 700 mg to about 1500 mg, about 700 mg to about 1400 mg, about 700 mg to about 1300 mg, about 700 mg to about 1200 mg, about 700 mg to about 1100 mg, about 700 mg to about 1000 mg, about 700 mg to about 900 mg, about 700 mg to about 800 mg, about 800 mg to about 2000 mg, about 800 mg to about 1900 mg, about 800 mg to about 1800 mg, about 800 mg to about 1700 mg, about 800 mg to about 1600 mg, about 800 mg to about 1500 mg, about 800 mg to about 1400 mg, about 800 mg to about 1300 mg, about 800 mg to about 1200 mg, about 800 mg to about 1100 mg, about 800 mg to about 1000 mg, about 800 mg to about 900 mg, about 900 mg to about 1900 mg, about 900 mg to about 1800 mg, about 900 mg to about 1700 mg, about 900 mg to about 1600 mg, about 900 mg to about 1500 mg, about 900 mg to about 1400 mg, about 900 mg to about 1300 mg, about 900 mg to about 1200 mg, about 900 mg to about 1100 mg, about 900 mg to about 1000 mg, about 1000 mg to about 2000 mg, about 1000 mg to about 1900 mg, about 1000 mg to about 1800 mg, about 1000 mg to about 1700 mg, about 1000 mg to about 1600 mg, about 1000 mg to about 1500 mg, about 1000 mg to about 1400 mg, about 1000 mg to about 1300 mg, about 1000 mg to about 1200 mg, about 1000 mg to about 1100 mg, about 1100 mg to about 1900 mg, about 1100 mg to about 1800 mg, about 1100 mg to about 1700 mg, about 1100 mg to about 1600 mg, about 1100 mg to about 1500 mg, about 1100 mg to about 1400 mg, about 1100 mg to about 1300 mg, about 1100 mg to about 1200 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 1900 mg, about 1300 mg to about 1800 mg, about 1300 mg to about 1700 mg, about 1300 mg to about 1600 mg, about 1300 mg to about 1500 mg, about 1300 mg to about 1400 mg, about 1400 mg to about 2000 mg, about 1400 mg to about 1900 mg, about 1400 mg to about 1800 mg, about 1400 mg to about 1700 mg, about 1400 mg to about 1600 mg, about 1400 mg to about 1500 mg, about 1500 mg to about 2000 mg, about 1500 mg to about 1900 mg, about 1500 mg to about 1800 mg, about 1500 mg to about 1700 mg, about 1500 mg to about 1600 mg, about 1600 mg to about 2000 mg, about 1600 mg to about 1900 mg, about 1600 mg to about 1800 mg, about 1600 mg to about 1700 mg, or about 1800 mg to about 1900 mg. The dose may be a defined value. For example, the dose may be about 80 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1600 mg, about 1800 mg, or about 2000 mg.

In some embodiments, the anti-PD-Ll antibody molecule comprises atezolizumab and is administered (e.g., at about 840 mg) as an intravenous infusion (e.g., over about 30 minutes or over about 60 minutes) about every 2 weeks. In some embodiments, the anti-PD-Ll antibody molecule comprises atezolizumab and is administered (e.g., at about 1200 mg) as an intravenous infusion (e.g., over about 30 minutes or over about 60 minutes) about every 3 weeks. In some embodiments, the anti-PD-Ll antibody molecule comprises atezolizumab and is administered (e.g., at about 1680 mg) as an intravenous infusion (e.g., over about 30 minutes or over about 60 minutes) about every 4 weeks. In some embodiments, the anti-PD-Ll antibody molecule comprises atezolizumab and is administered (e.g., at 840 mg) as an intravenous infusion (e.g., over 30 minutes or over 60 minutes) every 2 weeks. In some embodiments, the anti-PD-Ll antibody molecule comprises atezolizumab and is administered (e.g., at 1200 mg) as an intravenous infusion (e.g., over 30 minutes or over 60 minutes) every 3 weeks, hr some embodiments, the anti-PD-Ll antibody molecule comprises atezolizumab and is administered (e.g., at 1680 mg) as an intravenous infusion (e.g., over 30 minutes or over 60 minutes) every 4 weeks.

In some embodiments, the anti-PD-Ll antibody molecule comprises avelumab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals. The dose may be within a defined range. For example, the dose may be about 80 mg to about 1000 mg, about 100 mg to about 1000 mg, about 100 mg to about 900 mg, about 100 mg to about 800 mg, about 100 mg to about 700 mg, about 100 mg to about 600 mg, about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300 mg, about 100 mg to about 200 mg, about 200 mg to about 1000 mg, about 200 mg to about 900 mg, about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 200 mg to about 500 mg, about 200 mg to about 400 mg, about 200 mg to about 300 mg, about 300 mg to about 1000 mg, about 300 mg to about 900 mg, about 300 mg to about 800 mg, about 300 mg to about 700 mg, about 300 mg to about 600 mg, about 300 mg to about 500 mg, about 300 mg to about 400 mg, about 400 mg to about 1000 mg, about 400 mg to about 900 mg, about 400 mg to about 800 mg, about 400 mg to about 700 mg, about 400 mg to about 600 mg, about 400 mg to about 500 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 600 mg to about 1000 mg, about 600 mg to about 900 mg, about 600 mg to about 800 mg, about 600 mg to about 700 mg, about 700 mg to about 1000 mg, about 700 mg to about 900 mg, about 700 mg to about 800 mg, about 800 mg to about 1000 mg, or about 800 mg to about 900 mg.

The dose may be a defined value. For example, the dose may be about 80 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or about 1000 mg.

The dose may be administered for a defined dosing period (e.g., the length of time taken to administer a single infusion). For example, the dosing period may be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, or about 6 hours.

In some embodiments, tire anti-PD-Ll antibody molecule comprises avelumab and is administered as an intravenous infusion (e.g., over 60 minutes) every 2 weeks. In some embodiments, the anti-PD-Ll antibody molecule comprises avelumab and is administered at 800 mg as an intravenous infusion over 60 minutes every 2 weeks.

In some embodiments, the anti-PD-Ll antibody molecule comprises durvalumab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals. The dose may be within a defined range. For example, the dose may be about 150 mg to about 2000 mg, about 150 mg to about 1900 mg, about 150 mg to about 1800 mg, about 150 mg to about 1700 mg, about 150 mg to about 1600 mg, about 150 mg to about 1500 mg, about 150 mg to about 1400 mg, about 150 mg to about 1300 mg, about 150 mg to about 1200 mg, about 150 mg to about 1100 mg, about 150 mg to about 1000 mg, about 150 mg to about 900 mg, about 150 mg to about 800 mg, about 150 mg to about 700 mg, about 150 mg to about 600 mg, about 150 mg to about 500 mg, about 150 mg to about 400 mg, about 150 mg to about 300 mg, about 150 mg to about 200 mg, about 200 mg to about 20000 mg, about 200 mg to about 1900 mg, about 200 mg to about 1800 mg, about 200 mg to about 1700 mg, about 200 mg to about 1600 mg, about 200 mg to about 1500 mg, about 200 mg to about 1400 mg, about 200 mg to about 1300 mg, about 200 mg to about 1200 mg, about 200 mg to about 1100 mg, about 200 mg to about 1000 mg, about 200 mg to about 900 mg, about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 200 mg to about 500 mg, about 200 mg to about 400 mg, about 200 mg to about 300 mg, about 300 mg to about 2000 mg, about 300 mg to about 1900 mg, about 300 mg to about 1800 mg, about 300 mg to about 1700 mg, about 300 mg to about 1600 mg, about 300 mg to about 1500 mg, about 300 mg to about 1400 mg, about 300 mg to about 1300 mg, about 300 mg to about 1200 mg, about 300 mg to about 1100 mg, about 300 mg to about 1000 mg, about 300 mg to about 900 mg, about 300 mg to about 800 mg, about 300 mg to about 700 mg, about 300 mg to about 600 mg, about 300 mg to about 500 mg, about 300 mg to about 400 mg, about 400 mg to about 2000 mg, about 400 mg to about 1900 mg, about 400 mg to about 1800 mg, about 400 mg to about 1700 mg, about 400 mg to about 1600 mg, about 400 mg to about 1500 mg, about 400 mg to about 1400 mg, about 400 mg to about 1300 mg, about 400 mg to about 1200 mg, about 400 mg to about 1100 mg, about 400 mg to about 1000 mg, about 400 mg to about 900 mg, about 400 mg to about 800 mg, about 400 mg to about 700 mg, about 400 mg to about 600 mg, about 400 mg to about 500 mg, about 500 mg to about 2000 mg, about 500 mg to about 1900 mg, about 500 mg to about 1800 mg, about 500 mg to about 1700 mg, about 500 mg to about 1600 mg, about 500 mg to about 1500 mg, about 500 mg to about 1400 mg, about 500 mg to about 1300 mg, about 500 mg to about 1200 mg, about 500 mg to about 1100 mg, about 500 mg to about 1000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg, about 500 mg to about 700 mg, about 500 mg to about 600 mg, about 600 mg to about 2000 mg, about 600 mg to about 1900 mg, about 600 mg to about 1800 mg, about 600 mg to about 1700 mg, about 600 mg to about 1600 mg, about 600 mg to about 1500 mg, about 600 mg to about 1400 mg, about 600 mg to about 1300 mg, about 600 mg to about 1200 mg, about 600 mg to about 1100 mg, about 600 mg to about 1000 mg, about 600 mg to about 900 mg, about 600 mg to about 800 mg, about 600 mg to about 700 mg, about 700 mg to about 2000 mg, about 700 mg to about 1900 mg, about 700 mg to about 1800 mg, about 700 mg to about 1700 mg, about 700 mg to about 1600 mg, about 700 mg to about 1500 mg, about 700 mg to about 1400 mg, about 700 mg to about 1300 mg, about 700 mg to about 1200 mg, about 700 mg to about 1100 mg, about 700 mg to about 1000 mg, about 700 mg to about 900 mg, about 700 mg to about 800 mg, about 800 mg to about 2000 mg, about 800 mg to about 1900 mg, about 800 mg to about 1800 mg, about 800 mg to about 1700 mg, about 800 mg to about 1600 mg, about 800 mg to about 1500 mg, about 800 mg to about 1400 mg, about 800 mg to about 1300 mg, about 800 mg to about 1200 mg, about 800 mg to about 1100 mg, about 800 mg to about 1000 mg, about 800 mg to about 900 mg, about 900 mg to about 1900 mg, about 900 mg to about 1800 mg, about 900 mg to about 1700 mg, about 900 mg to about 1600 mg, about 900 mg to about 1500 mg, about 900 mg to about 1400 mg, about 900 mg to about 1300 mg, about 900 mg to about 1200 mg, about 900 mg to about 1100 mg, about 900 mg to about 1000 mg, about 1000 mg to about 2000 mg, about 1000 mg to about 1900 mg, about 1000 mg to about 1800 mg, about 1000 mg to about 1700 mg, about 1000 mg to about 1600 mg, about 1000 mg to about 1500 mg, about 1000 mg to about 1400 mg, about 1000 mg to about 1300 mg, about 1000 mg to about 1200 mg, about 1000 mg to about 1100 mg, about 1100 mg to about 1900 mg, about 1100 mg to about 1800 mg, about 1100 mg to about 1700 mg, about 1100 mg to about 1600 mg, about 1100 mg to about 1500 mg, about 1100 mg to about 1400 mg, about 1100 mg to about 1300 mg, about 1100 mg to about 1200 mg, about 1200 mg to about 2000 mg, about 1200 mg to about 1900 mg, about 1200 mg to about 1800 mg, about 1200 mg to about 1700 mg, about 1200 mg to about 1600 mg, about 1200 mg to about 1500 mg, about 1200 mg to about 1400 mg, about 1200 mg to about 1300 mg, about 1300 mg to about 1900 mg, about 1300 mg to about 1800 mg, about 1300 mg to about 1700 mg, about 1300 mg to about 1600 mg, about 1300 mg to about 1500 mg, about 1300 mg to about 1400 mg, about 1400 mg to about 2000 mg, about 1400 mg to about 1900 mg, about 1400 mg to about 1800 mg, about 1400 mg to about 1700 mg, about 1400 mg to about 1600 mg, about 1400 mg to about 1500 mg, about 1500 mg to about 2000 mg, about 1500 mg to about 1900 mg, about 1500 mg to about 1800 mg, about 1500 mg to about 1700 mg, about 1500 mg to about 1600 mg, about 1600 mg to about 2000 mg, about 1600 mg to about 1900 mg, about 1600 mg to about 1800 mg, about 1600 mg to about 1700 mg, or about 1800 mg to about 1900 mg, or about 1 to about 30 mg/kg, about 1 to about 20 mg/kg, about 1 to about 15 mg/kg, about 1 to about 10 mg/kg, about 1 to about 5 mg/kg, about 2 to 30 mg/kg, about 2 to about 20 mg/kg, about 2 to about 15 mg/kg, about 2 to about 10 mg/kg, about 2 to about 5 mg/kg, about 5 to about 30 mg/kg, about 5 to about 20 mg/kg, about 5 to about 15 mg/kg, about 5 to about 10 mg/kg, about 10 to about 30 mg/kg, about 10 to about 20 mg/kg, or about 10 to about 15 mg/kg.

The dose may be a defined value. For example, the dose may be about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1400 mg, about 1600 mg, about 1800 mg, or about 2000 mg, or about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 30 mg/kg. The dose may be administered for a defined dosing period (e.g., the length of time taken to administer a single infusion). For example, the dosing period may be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, or about 6 hours.

In some embodiments, the anti-PD-Ll antibody molecule comprises durvalumab and is administered (e.g., at 1500 mg) as an intravenous infusion (e.g., over 60 minutes) every 4 weeks. In some embodiments, the anti-PD-Ll antibody molecule comprises durvalumab and is administered as an intravenous infusion (e.g., over 60 minutes) every 2 weeks. In some embodiments, the anti-PD-Ll antibody molecule comprises durvalumab and is administered as an intravenous infusion (e.g., over 60 minutes) every 3 weeks. In some embodiments, the anti-PD-Ll antibody molecule comprises durvalumab and is administered at 10 mg/kg as an intravenous infusion (e.g., over 60 minutes) every 2 weeks. In some embodiments, the anti-PD-Ll antibody molecule comprises durvalumab and is administered at 20 mg/kg as an intravenous infusion (e.g., over 60 minutes) every 3 weeks.

In some embodiments, the anti-CTLA-4 antibody molecule comprises ipilimumab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

The dose may be within a defined range. For example, the dose may be about 1 to about 30 mg/kg, about 1 to about 20 mg/kg, about 1 to about 15 mg/kg, about 1 to about 10 mg/kg, about 1 to about 5 mg/kg, about 2 to 30 mg/kg, about 2 to about 20 mg/kg, about 2 to about 15 mg/kg, about 2 to about 10 mg/kg, about 2 to about 5 mg/kg, about 5 to about 30 mg/kg, about 5 to about 20 mg/kg, about 5 to about 15 mg/kg, about 5 to about 10 mg/kg, about 10 to about 30 mg/kg, about 10 to about 20 mg/kg, or about 10 to about 15 mg/kg.

The dose may be a defined value. For example, the dose may be about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 30 mg/kg.

In some embodiments, the anti-CTLA-4 antibody molecule comprises ipilimumab and is administered as an intravenous infusion (e.g., over 90 minutes) every 3 weeks (e.g., for 4 doses) followed by administration every 12 weeks (e.g., up to 3 years). In some embodiments, the anti-CTLA-4 antibody molecule comprises ipilimumab and is administered at 10 mg/kg as an intravenous infusion (e.g., over 90 minutes) every 3 weeks (e.g., for 4 doses) followed by administration at 10 mg/kg as an intravenous infusion every 12 weeks (e.g., up to 3 years).

In some embodiments, the anti-LAG3 antibody molecule comprises relatlimab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

The dose may be within a defined range. For example, the dose may be about 30 mg to about 300 mg, about 30 mg to about 290 mg, about 30 mg to about 250 mg, about 30 mg to about 200 mg, about 30 mg to about 150 mg, about 30 mg to about 100 mg, about 30 mg to about 50 mg, about 50 mg to about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, about 100 mg to about 150 mg, about 150 mg to about 300 mg, about 150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, or about 250 mg to about 300 mg.

The dose may be a defined value. For example, the dose may be about 30 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, or about 300 mg.

In some embodiments, the anti-LAG3 antibody molecule comprises relatlimab and is administered as an intravenous infusion (e.g., over 30 minutes) every 4 weeks. In some embodiments, the anti-LAG3 antibody molecule comprises relatlimab and is administered at 160 mg as an intravenous infusion (e.g., over 30 minutes) every 4 weeks. In some embodiments, the anti-LAG3 antibody molecule comprises relatlimab and is administered as an intravenous infusion (e.g., over 30 minutes) every 4 weeks in further combination with an anti-PD-1 antibody molecule (e.g., nivolumab (e.g., at 480 mg)).

In some embodiments, the anti-LAG3 antibody molecule comprises ieramilimab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

The dose may be within a defined range. For example, the dose may be about 40 mg to about 900 mg, about 40 mg to about 890 mg, about 40 mg to about 865 mg, about 40 mg to about 840 mg, about 40 mg to about 815 mg, about 40 mg to about 790 mg, about 40 mg to about 765 mg, about 40 mg to about 740 mg, about 40 mg to about 715 mg, about 40 mg to about 690 mg, about 40 mg to about 665 mg, about 40 mg to about 640 mg, about 40 mg to about 615 mg, about 40 mg to about 590 mg, about 40 mg to about 565 mg, about 40 mg to about 540 mg, about 40 mg to about 515 mg, about 40 mg to about 490 mg, about 40 mg to about 465 mg, about 40 mg to about 440 mg, about 40 mg to about 415 mg, about 40 mg to about 390 mg, about 40 mg to about 365 mg, about 40 mg to about 340 mg, about 40 mg to about 315 mg, about 40 mg to about 290 mg, about 40 mg to about 265 mg, about 40 mg to about 240 mg, about 40 mg to about 215 mg, about 40 mg to about 190 mg, about 40 mg to about 165 mg, about 40 mg to about 140 mg, about 40 mg to about 115 mg, about 40 mg to about 90 mg, about 40 mg to about 65 mg, about 40 mg to about 50 mg, about 50 mg to about 900 mg, about 50 mg to about 850 mg, about 50 mg to about 800 mg, about 50 mg to about 750 mg, about 50 mg to about 700 mg, about 50 mg to about 650 mg, about 50 mg to about 600 mg, about 50 mg to about 550 mg, about 50 mg to about 500 mg, about 50 mg to about 450 mg, about 50 mg to about 400 mg, about 50 mg to about 350 mg, about 50 mg to about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 100 mg to about 900 mg, about 100 mg to about 850 mg, about 100 mg to about 800 mg, about 100 mg to about 750 mg, about 100 mg to about 700 mg, about 100 mg to about 650 mg, about 100 mg to about 600 mg, about 100 mg to about 550 mg, about 100 mg to about 500 mg, about 100 mg to about 450 mg, about 100 mg to about 400 mg, about 100 mg to about 350 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, about 100 mg to about 150 mg, about 150 mg to about 900 mg, about 150 mg to about 850 mg, about 150 mg to about 800 mg, about 150 mg to about 750 mg, about 150 mg to about 700 mg, about 150 mg to about 650 mg, about 150 mg to about 600 mg, about 150 mg to about 550 mg, about 150 mg to about 500 mg, about 150 mg to about 450 mg, about 150 mg to about 400 mg, about 150 mg to about 350 mg, about 150 mg to about 300 mg, about 150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg to about 900 mg, about 200 mg to about 850 mg, about 200 mg to about 800 mg, about 200 mg to about 750 mg, about 200 mg to about 700 mg, about 200 mg to about 650 mg, about 200 mg to about 600 mg, about 200 mg to about 550 mg, about 200 mg to about 500 mg, about 200 mg to about 450 mg, about 200 mg to about 400 mg, about 200 mg to about 350 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, about 250 mg to about 900 mg, about 250 mg to about 850 mg, about 250 mg to about 800 mg, about 250 mg to about 750 mg, about 250 mg to about 700 mg, about 250 mg to about 650 mg, about 250 mg to about 600 mg, about 250 mg to about 550 mg, about 250 mg to about 500 mg, about 250 mg to about 450 mg, about 250 mg to about 400 mg, about 250 mg to about 350 mg, about 250 mg to about 300 mg, about 300 mg to about 900 mg, about 300 mg to about 850 mg, about 300 mg to about 800 mg, about 300 mg to about 750 mg, about 300 mg to about 700 mg, about 300 mg to about 650 mg, about 300 mg to about 600 mg, about 300 mg to about 550 mg, about 300 mg to about 500 mg, about 300 mg to about 450 mg, about 300 mg to about 400 mg, about 300 mg to about 350 mg, about 350 mg to about 900 mg, about 350 mg to about 850 mg, about 350 mg to about 800 mg, about 350 mg to about 750 mg, about 350 mg to about 700 mg, about 350 mg to about 650 mg, about 350 mg to about 600 mg, about 350 mg to about 550 mg, about 350 mg to about 500 mg, about 350 mg to about 450 mg, about 350 mg to about 400 mg, about 400 mg to about 900 mg, about 400 mg to about 850 mg, about 400 mg to about 800 mg, about 400 mg to about 750 mg, about 400 mg to about 700 mg, about 400 mg to about 650 mg, about 400 mg to about 600 mg, about 400 mg to about 550 mg, about 400 mg to about 500 mg, about 400 mg to about 450 mg, about 450 mg to about 900 mg, about 450 mg to about 850 mg, about 450 mg to about 800 mg, about 450 mg to about 750 mg, about 450 mg to about 700 mg, about 450 mg to about 650 mg, about 450 mg to about 600 mg, about 450 mg to about 550 mg, about 450 mg to about 500 mg, about 500 mg to about 900 mg, about 500 mg to about 850 mg, about 500 mg to about 800 mg, about 500 mg to about 750 mg, about 500 mg to about 700 mg, about 500 mg to about 650 mg, about 500 mg to about 600 mg, about 500 mg to about 550 mg, about 550 mg to about 900 mg, about 550 mg to about 850 mg, about 550 mg to about 800 mg, about 550 mg to about 750 mg, about 550 mg to about 700 mg, about 550 mg to about 650 mg, about 550 mg to about 600 mg, about 600 mg to about 900 mg, about 600 mg to about 850 mg, about 600 mg to about 800 mg, about 600 mg to about 750 mg, about 600 mg to about 700 mg, about 600 mg to about 650 mg, about 650 mg to about 900 mg, about 650 mg to about 850 mg, about 650 mg to about 800 mg, about 650 mg to about 750 mg, about 650 mg to about 700 mg, about 700 mg to about 900 mg, about 700 mg to about 850 mg, about 700 mg to about 800 mg, about 700 mg to about 750 mg, about 750 mg to about 900 mg, about 750 mg to about 850 mg, about 750 mg to about 800 mg, about 800 mg to about 900 mg, about 800 mg to about 850 mg, or about 850 mg to about 900 mg.

The dose may be a defined value. For example, the dose may be about 40 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, or about 900 mg. The dose may be administered for a defined dosing period (e.g., the length of time taken to administer a single infusion). For example, the dosing period may be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, or about 6 hours.

In some embodiments, the anti- LAG3 antibody molecule comprises ieramilimab and is administered as an intravenous infusion (e.g., over about 30 minutes to about 90 minutes) every 3 weeks. In some embodiments, the anti- LAG3 antibody molecule comprises ieramilimab and is administered at 400 mg as an intravenous infusion (e.g., over about 30 minutes to about 90 minutes) every 3 weeks. In some embodiments, the anti- LAG3 antibody molecule comprises ieramilimab and is administered as an intravenous infusion (e.g., over about 30 minutes to about 90 minutes) every' 3 weeks in further combination with an anti-PD-1 antibody molecule. In some embodiments, the anti- LAG3 antibody molecule comprises ieramilimab and is administered as an intravenous infusion (e.g., over about 30 minutes to about 90 minutes) every 4 weeks. In some embodiments, the anti- LAG3 antibody molecule comprises ieramilimab and is administered at 800 mg as an intravenous infusion (e.g., over about 30 minutes to about 90 minutes) every 4 weeks. In some embodiments, the anti- LAG3 antibody molecule comprises ieramilimab and is administered as an intravenous infusion (e.g., over 30 minutes) every 4 weeks in further combination with an anti-PD-1 antibody molecule.

In some embodiments, the anti-LAG3 antibody molecule comprises favezelimab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

The dose may be within a defined range. For example, the dose may be about 40 mg to about 900 mg, about 40 mg to about 890 mg, about 40 mg to about 865 mg, about 40 mg to about 840 mg, about 40 mg to about 815 mg, about 40 mg to about 790 mg, about 40 mg to about 765 mg, about 40 mg to about 740 mg, about 40 mg to about 715 mg, about 40 mg to about 690 mg, about 40 mg to about 665 mg, about 40 mg to about 640 mg, about 40 mg to about 615 mg, about 40 mg to about 590 mg, about 40 mg to about 565 mg, about 40 mg to about 540 mg, about 40 mg to about 515 mg, about 40 mg to about 490 mg, about 40 mg to about 465 mg, about 40 mg to about 440 mg, about 40 mg to about 415 mg, about 40 mg to about 390 mg, about 40 mg to about 365 mg, about 40 mg to about 340 mg, about 40 mg to about 315 mg, about 40 mg to about 290 mg, about 40 mg to about 265 mg, about 40 mg to about 240 mg, about 40 mg to about 215 mg, about 40 mg to about 190 mg, about 40 mg to about 165 mg, about 40 mg to about 140 mg, about 40 mg to about 115 mg, about 40 mg to about 90 mg, about 40 mg to about 65 mg, about 40 mg to about 50 mg, about 50 mg to about 900 mg, about 50 mg to about 850 mg, about 50 mg to about 800 mg, about 50 mg to about 750 mg, about 50 mg to about 700 mg, about 50 mg to about 650 mg, about 50 mg to about 600 mg, about 50 mg to about 550 mg, about 50 mg to about 500 mg, about 50 mg to about 450 mg, about 50 mg to about 400 mg, about 50 mg to about 350 mg, about 50 mg to about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 100 mg to about 900 mg, about 100 mg to about 850 mg, about

100 mg to about 800 mg, about 100 mg to about 750 mg, about 100 mg to about 700 mg, about 100 mg to about 650 mg, about 100 mg to about 600 mg, about 100 mg to about 550 mg, about 100 mg to about 500 mg, about 100 mg to about 450 mg, about 100 mg to about 400 mg, about 100 mg to about 350 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, about 100 mg to about 150 mg, about 150 mg to about 900 mg, about 150 mg to about 850 mg, about 150 mg to about 800 mg, about 150 mg to about 750 mg, about 150 mg to about 700 mg, about 150 mg to about 650 mg, about 150 mg to about 600 mg, about 150 mg to about 550 mg, about 150 mg to about 500 mg, about 150 mg to about 450 mg, about 150 mg to about 400 mg, about 150 mg to about 350 mg, about 150 mg to about 300 mg, about 150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg to about 900 mg, about 200 mg to about 850 mg, about 200 mg to about 800 mg, about 200 mg to about 750 mg, about 200 mg to about 700 mg, about 200 mg to about 650 mg, about 200 mg to about 600 mg, about 200 mg to about 550 mg, about 200 mg to about 500 mg, about 200 mg to about 450 mg, about 200 mg to about 400 mg, about 200 mg to about 350 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, about 250 mg to about 900 mg, about 250 mg to about 850 mg, about 250 mg to about 800 mg, about 250 mg to about 750 mg, about 250 mg to about 700 mg, about 250 mg to about 650 mg, about 250 mg to about 600 mg, about 250 mg to about 550 mg, about 250 mg to about 500 mg, about 250 mg to about 450 mg, about 250 mg to about 400 mg, about 250 mg to about 350 mg, about 250 mg to about 300 mg, about 300 mg to about 900 mg, about 300 mg to about 850 mg, about 300 mg to about 800 mg, about 300 mg to about 750 mg, about 300 mg to about 700 mg, about 300 mg to about 650 mg, about 300 mg to about 600 mg, about 300 mg to about 550 mg, about 300 mg to about 500 mg, about 300 mg to about 450 mg, about 300 mg to about 400 mg, about 300 mg to about 350 mg, about 350 mg to about 900 mg, about 350 mg to about 850 mg, about 350 mg to about 800 mg, about 350 mg to about 750 mg, about 350 mg to about 700 mg, about 350 mg to about 650 mg, about 350 mg to about 600 mg, about 350 mg to about 550 mg, about 350 mg to about 500 mg, about 350 mg to about 450 mg, about 350 mg to about 400 mg, about 400 mg to about 900 mg, about 400 mg to about 850 mg, about 400 mg to about 800 mg, about 400 mg to about 750 mg, about 400 mg to about 700 mg, about 400 mg to about 650 mg, about 400 mg to about 600 mg, about 400 mg to about 550 mg, about 400 mg to about 500 mg, about 400 mg to about 450 mg, about 450 mg to about 900 mg, about 450 mg to about 850 mg, about 450 mg to about 800 mg, about 450 mg to about 750 mg, about 450 mg to about 700 mg, about 450 mg to about 650 mg, about 450 mg to about 600 mg, about 450 mg to about 550 mg, about 450 mg to about 500 mg, about 500 mg to about 900 mg, about 500 mg to about 850 mg, about 500 mg to about 800 mg, about 500 mg to about 750 mg, about 500 mg to about 700 mg, about 500 mg to about 650 mg, about 500 mg to about 600 mg, about 500 mg to about 550 mg, about 550 mg to about 900 mg, about 550 mg to about 850 mg, about 550 mg to about 800 mg, about 550 mg to about 750 mg, about 550 mg to about 700 mg, about 550 mg to about 650 mg, about 550 mg to about 600 mg, about 600 mg to about 900 mg, about 600 mg to about 850 mg, about 600 mg to about 800 mg, about 600 mg to about 750 mg, about 600 mg to about 700 mg, about 600 mg to about 650 mg, about 650 mg to about 900 mg, about 650 mg to about 850 mg, about 650 mg to about 800 mg, about 650 mg to about 750 mg, about 650 mg to about 700 mg, about 700 mg to about 900 mg, about 700 mg to about 850 mg, about 700 mg to about 800 mg, about 700 mg to about 750 mg, about 750 mg to about 900 mg, about 750 mg to about 850 mg, about 750 mg to about 800 mg, about 800 mg to about 900 mg, about 800 mg to about 850 mg, or about 850 mg to about 900 mg.

The dose may be a defined value. For example, the dose may be about 40 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, or about 900 mg.

In some embodiments, the anti- LAG3 antibody molecule comprises favezelimab and is administered as an intravenous infusion (e.g., over about 30 minutes to about 90 minutes) every 3 weeks. In some embodiments, the anti- LAG3 antibody molecule comprises favezelimab and is administered at 800 mg as an intravenous infusion (e.g., over about 30 minutes to about 90 minutes) every 3 weeks. In some embodiments, the anti- LAG3 antibody molecule comprises favezelimab and is administered as an intravenous infusion (e.g., over about 30 minutes to about 90 minutes) every 3 weeks in further combination with an anti-PD-1 antibody molecule. In some embodiments, the anti-KIR antibody molecule comprises lirilumab and is administered according to a dosing regimen comprising a defined dose administered over a defined period periodically at defined intervals.

The dose may be within a defined range. For example, the dose may be about 0.003-15 mg/kg, about 0.003 mg/kg to about 14.75 mg/kg, about 0.003 mg/kg to about 14 mg/kg, about 0.003 mg/kg to about 13 mg/kg, about 0.003 mg/kg to about 12 mg/kg, about 0.003 mg/kg to about 11 mg/kg, about 0.003 mg/kg to about 10 mg/kg, about 0.003 mg/kg to about 9 mg/kg, about 0.003 mg/kg to about 8 mg/kg, about 0.003 mg/kg to about 7 mg/kg, about 0.003 mg/kg to about 6 mg/kg, about 0.003 mg/kg to about 5 mg/kg, about 0.003 mg/kg to about 4 mg/kg, about 0.003 mg/kg to about 3 mg/kg, about 0.003 mg/kg to about 2 mg/kg, about 0.003 mg/kg to about 1 mg/kg, about 0.003 mg/kg to about 0.95 mg/kg, about 0.003 mg/kg to about 0.9 mg/kg, about 0.003 mg/kg to about 0.85 mg/kg, about 0.003 mg/kg to about 0.8 mg/kg, about 0.003 mg/kg to about 0.75 mg/kg, about 0.003 mg/kg to about 0.7 mg/kg, about 0.003 mg/kg to about 0.65 mg/kg, about 0.003 mg/kg to about 0.6 mg/kg, about 0.003 mg/kg to about 0.55 mg/kg, about 0.003 mg/kg to about 0.5 mg/kg, about 0.003 mg/kg to about 0.45 mg/kg, about 0.003 mg/kg to about 0.4 mg/kg, about 0.003 mg/kg to about 0.35 mg/kg, about 0.003 mg/kg to about 0.3 mg/kg, about 0.003 mg/kg to about 0.25 mg/kg, about 0.003 mg/kg to about 0.2 mg/kg, about 0.003 mg/kg to about 0.15 mg/kg, about 0.003 mg/kg to about 0.1 mg/kg, about 0.003 mg/kg to about 0.05 mg/kg, about 0.015 mg/kg to about 15 mg/kg, about 0.015 mg/kg to about 14 mg/kg, about 0.015 mg/kg to about 13 mg/kg, about 0.015 mg/kg to about 12 mg/kg, about 0.015 mg/kg to about 11 mg/kg, about 0.015 mg/kg to about 10 mg/kg, about 0.015 mg/kg to about 9 mg/kg, about 0.015 mg/kg to about 8 mg/kg, about 0.015 mg/kg to about 7 mg/kg, about 0.015 mg/kg to about 6 mg/kg, about 0.015 mg/kg to about 5 mg/kg, about 0.015 mg/kg to about 4 mg/kg, about 0.015 mg/kg to about 3 mg/kg, about 0.015 mg/kg to about 2 mg/kg, about 0.015 mg/kg to about 1 mg/kg, about 0.015 mg/kg to about 0.95 mg/kg, about 0.015 mg/kg to about 0.9 mg/kg, about 0.015 mg/kg to about 0.85 mg/kg, about 0.015 mg/kg to about 0.8 mg/kg, about 0.015 mg/kg to about 0.75 mg/kg, about 0.015 mg/kg to about 0.7 mg/kg, about 0.015 mg/kg to about 0.65 mg/kg, about 0.015 mg/kg to about 0.6 mg/kg, about 0.015 mg/kg to about 0.55 mg/kg, about 0.015 mg/kg to about 0.5 mg/kg, about 0.015 mg/kg to about 0.45 mg/kg, about 0.015 mg/kg to about 0.4 mg/kg, about 0.015 mg/kg to about 0.35 mg/kg, about 0.015 mg/kg to about 0.3 mg/kg, about 0.015 mg/kg to about 0.25 mg/kg, about 0.015 mg/kg to about 0.2 mg/kg, about 0.1 mg/kg to about 15 mg/kg, about 0. 1 mg/kg to about 14 mg/kg, about 0.1 mg/kg to about 13 mg/kg, about 0. 1 mg/kg to about 12 mg/kg, about 0. 1 mg/kg to about 11 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about

0. 1 mg/kg to about 9 mg/kg, about 0. 1 mg/kg to about 8 mg/kg, about 0.1 mg/kg to about 7 mg/kg, about

0. 1 mg/kg to about 6 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 4 mg/kg, about

0. 1 mg/kg to about 3 mg/kg, about 0. 1 mg/kg to about 2 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0. 1 mg/kg to about 0.95 mg/kg, about 0.1 mg/kg to about 0.9 mg/kg, about 0.1 mg/kg to about 0.85 mg/kg, about 0.1 mg/kg to about 0.8 mg/kg, about 0.1 mg/kg to about 0.75 mg/kg, about 0.1 mg/kg to about 0.7 mg/kg, about 0.1 mg/kg to about 0.65 mg/kg, about 0.1 mg/kg to about 0.6 mg/kg, about 0.1 mg/kg to about 0.55 mg/kg, about 0.1 mg/kg to about 0.5 mg/kg, about 0.1 mg/kg to about 0.45 mg/kg, about 0.1 mg/kg to about 0.4 mg/kg, about 0.1 mg/kg to about 0.35 mg/kg, about 0.1 mg/kg to about 0.3 mg/kg, about 0.1 mg/kg to about 0.25 mg/kg, about 0.1 mg/kg to about 0.2 mg/kg, about 0.1 mg/kg to about 0.15 mg/kg, about 0.3 mg/kg to about 15 mg/kg, about 0.3 mg/kg to about 14 mg/kg, about 0.3 mg/kg to about 13 mg/kg, about 0.3 mg/kg to about 12 mg/kg, about 0.3 mg/kg to about 11 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 9 mg/kg, about 0.3 mg/kg to about 8 mg/kg, about 0.3 mg/kg to about 7 mg/kg, about 0.3 mg/kg to about 6 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about

0.3 mg/kg to about 4 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 0.3 mg/kg to about 2 mg/kg, about

0.3 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 0.95 mg/kg, about 0.3 mg/kg to about 0.9 mg/kg, about 0.3 mg/kg to about 0.85 mg/kg, about 0.3 mg/kg to about 0.8 mg/kg, about 0.3 mg/kg to about 0.75 mg/kg, about 0.3 mg/kg to about 0.7 mg/kg, about 0.3 mg/kg to about 0.65 mg/kg, about 0.3 mg/kg to about 0.6 mg/kg, about 0.3 mg/kg to about 0.55 mg/kg, about 0.3 mg/kg to about 0.5 mg/kg, about 0.3 mg/kg to about 0.45 mg/kg, about 0.3 mg/kg to about 0.4 mg/kg, about 0.8 mg/kg to about 15 mg/kg, about 0.8 mg/kg to about 14 mg/kg, about 0.8 mg/kg to about 13 mg/kg, about 0.8 mg/kg to about 12 mg/kg, about 0.8 mg/kg to about 11 mg/kg, about 0.8 mg/kg to about 10 mg/kg, about 0.8 mg/kg to about 9 mg/kg, about 0.8 mg/kg to about 8 mg/kg, about 0.8 mg/kg to about 7 mg/kg, about 0.8 mg/kg to about 6 mg/kg, about 0.8 mg/kg to about 5 mg/kg, about 0.8 mg/kg to about 4 mg/kg, about 0.8 mg/kg to about 3 mg/kg, about 0.8 mg/kg to about 2 mg/kg, about 0.8 mg/kg to about 1 mg/kg, about 0.8 mg/kg to about 0.95 mg/kg, about 0.8 mg/kg to about 0.9 mg/kg, about 0.8 mg/kg to about 0.85 mg/kg, about 1 mg/kg to about 15 mg/kg, about 1 mg/kg to about 14 mg/kg, about 1 mg/kg to about 13 mg/kg, about 1 mg/kg to about 12 mg/kg, about 1 mg/kg to about 11 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 9 mg/kg, about 1 mg/kg to about 8 mg/kg, about 1 mg/kg to about 7 mg/kg, about 1 mg/kg to about 6 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 2 mg/kg, about 5 mg/kg to about 15 mg/kg, about 5 mg/kg to about 14 mg/kg, about 5 mg/kg to about 13 mg/kg, about 5 mg/kg to about 12 mg/kg, about 5 mg/kg to about 11 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 9 mg/kg, about 5 mg/kg to about 8 mg/kg, about 5 mg/kg to about 7 mg/kg, about 5 mg/kg to about 6 mg/kg, about 8 mg/kg to about 15 mg/kg, about 8 mg/kg to about 14 mg/kg, about 8 mg/kg to about 13 mg/kg, about 8 mg/kg to about 12 mg/kg, about 8 mg/kg to about 11 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8 mg/kg to about 9 mg/kg, about 12 mg/kg to about 15 mg/kg, about 12 mg/kg to about 14 mg/kg, or about 12 mg/kg to about 13 mg/kg. The dose may be a defined value. For example, the dose may be about 0.003 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, about 10 mg/kg, about 10.5 mg/kg, about 11 mg/kg, about 11.5 mg/kg, about 12 mg/kg, about 12.5 mg/kg, about 13 mg/kg, about 13.5 mg/kg, or about 14 mg/kg.

In some embodiments, the anti-BTLA antibody molecule comprises TAB004 and is administered at about 0.1 mg/kg to about 15 mg/kg, about 0. 1 mg/kg to about 14.75 mg/kg, about 0. 1 mg/kg to about 14 mg/kg, about 0.1 mg/kg to about 13 mg/kg, about 0.1 mg/kg to about 12 mg/kg, about 0.1 mg/kg to about 11 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 9 mg/kg, about 0. 1 mg/kg to about 8 mg/kg, about 0.1 mg/kg to about 7 mg/kg, about 0. 1 mg/kg to about 6 mg/kg, about 0. 1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 4 mg/kg, about 0. 1 mg/kg to about 3 mg/kg, about 0. 1 mg/kg to about 2 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 0.95 mg/kg, about 0.1 mg/kg to about 0.9 mg/kg, about 0. 1 mg/kg to about 0.85 mg/kg, about 0. 1 mg/kg to about 0.8 mg/kg, about O.l mg/kg to about 0.75 mg/kg, about O.l mg/kg to about 0.7 mg/kg, about O. l mg/kg to about 0.65 mg/kg, about 0.1 mg/kg to about 0.6 mg/kg, about 0.1 mg/kg to about 0.55 mg/kg, about 0.1 mg/kg to about 0.5 mg/kg, about 0.1 mg/kg to about 0.45 mg/kg, about 0.1 mg/kg to about 0.4 mg/kg, about 0.1 mg/kg to about 0.35 mg/kg, about 0.1 mg/kg to about 0.3 mg/kg, about 0. 1 mg/kg to about 0.25 mg/kg, about 0.1 mg/kg to about 0.2 mg/kg, about 0.1 mg/kg to about 0.15 mg/kg, about 0.3 mg/kg to about 15 mg/kg, about 0.3 mg/kg to about 14 mg/kg, about 0.3 mg/kg to about 13 mg/kg, about 0.3 mg/kg to about 12 mg/kg, about 0.3 mg/kg to about 11 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 9 mg/kg, about 0.3 mg/kg to about 8 mg/kg, about 0.3 mg/kg to about 7 mg/kg, about 0.3 mg/kg to about 6 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 4 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 0.3 mg/kg to about 2 mg/kg, about 0.3 mg/kg to about 1 mg/kg, about 0.3 mg/kg to about 0.95 mg/kg, about 0.3 mg/kg to about 0.9 mg/kg, about 0.3 mg/kg to about 0.85 mg/kg, about 0.3 mg/kg to about 0.8 mg/kg, about 0.3 mg/kg to about 0.75 mg/kg, about 0.3 mg/kg to about 0.7 mg/kg, about 0.3 mg/kg to about 0.65 mg/kg, about 0.3 mg/kg to about 0.6 mg/kg, about 0.3 mg/kg to about 0.55 mg/kg, about 0.3 mg/kg to about 0.5 mg/kg, about 0.3 mg/kg to about 0.45 mg/kg, about 0.3 mg/kg to about 0.4 mg/kg, about 0.8 mg/kg to about 15 mg/kg, about 0.8 mg/kg to about 14 mg/kg, about 0.8 mg/kg to about 13 mg/kg, about 0.8 mg/kg to about 12 mg/kg, about 0.8 mg/kg to about 11 mg/kg, about 0.8 mg/kg to about 10 mg/kg, about 0.8 mg/kg to about 9 mg/kg, about 0.8 mg/kg to about 8 mg/kg, about 0.8 mg/kg to about 7 mg/kg, about 0.8 mg/kg to about 6 mg/kg, about 0.8 mg/kg to about 5 mg/kg, about 0.8 mg/kg to about 4 mg/kg, about 0.8 mg/kg to about 3 mg/kg, about 0.8 mg/kg to about 2 mg/kg, about 0.8 mg/kg to about 1 mg/kg, about 0.8 mg/kg to about 0.95 mg/kg, about 0.8 mg/kg to about 0.9 mg/kg, about 0.8 mg/kg to about 0.85 mg/kg, about 1 mg/kg to about 15 mg/kg, about 1 mg/kg to about 14 mg/kg, about 1 mg/kg to about 13 mg/kg, about 1 mg/kg to about 12 mg/kg, about 1 mg/kg to about 11 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 9 mg/kg, about 1 mg/kg to about 8 mg/kg, about 1 mg/kg to about 7 mg/kg, about 1 mg/kg to about 6 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 2 mg/kg, about 5 mg/kg to about 15 mg/kg, about 5 mg/kg to about 14 mg/kg, about 5 mg/kg to about 13 mg/kg, about 5 mg/kg to about 12 mg/kg, about 5 mg/kg to about 11 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 9 mg/kg, about 5 mg/kg to about 8 mg/kg, about 5 mg/kg to about 7 mg/kg, about 5 mg/kg to about 6 mg/kg, about 8 mg/kg to about 15 mg/kg, about 8 mg/kg to about 14 mg/kg, about 8 mg/kg to about 13 mg/kg, about 8 mg/kg to about 12 mg/kg, about 8 mg/kg to about 11 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8 mg/kg to about 9 mg/kg, about 12 mg/kg to about 15 mg/kg, about 12 mg/kg to about 14 mg/kg, or about 12 mg/kg to about 13 mg/kg, or about 1 mg to about 700 mg, about 1 mg to about 690 mg, about 1 mg to about 650 mg, about 1 mg to about 600 mg, about 1 mg to about 550 mg, about 1 mg to about 500 mg, about 1 mg to about 450 mg, about 1 mg to about 400 mg, about 1 mg to about 350 mg, about 1 mg to about 300 mg, about 1 mg to about 250 mg, about 1 mg to about 200 mg, about 1 mg to about 150 mg, about 1 mg to about 100 mg, about 1 mg to about 50 mg, about 1 mg to about 25 mg, about 15 mg to about 700 mg, about 15 mg to about 650 mg, about 15 mg to about 600 mg, about 15 mg to about 550 mg, about 15 mg to about 500 mg, about 15 mg to about 450 mg, about 15 mg to about 400 mg, about 15 mg to about 350 mg, about 15 mg to about 300 mg, about 15 mg to about 250 mg, about 15 mg to about 200 mg, about 15 mg to about 150 mg, about 15 mg to about 100 mg, about 15 mg to about 50 mg, about 50 mg to about 0 mg, about 50 mg to about 700 mg, about 50 mg to about 650 mg, about 50 mg to about 600 mg, about 50 mg to about 550 mg, about 50 mg to about 500 mg, about 50 mg to about 450 mg, about 50 mg to about 400 mg, about 50 mg to about 350 mg, about 50 mg to about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about 100 mg to about 700 mg, about 100 mg to about 650 mg, about 100 mg to about 600 mg, about 100 mg to about 550 mg, about 100 mg to about 500 mg, about 100 mg to about 450 mg, about 100 mg to about 400 mg, about 100 mg to about 350 mg, about 100 mg to about 300 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, about 100 mg to about 150 mg, about 150 mg to about 700 mg, about 150 mg to about 650 mg, about 150 mg to about 600 mg, about 150 mg to about 550 mg, about 150 mg to about 500 mg, about 150 mg to about 450 mg, about 150 mg to about 400 mg, about 150 mg to about 350 mg, about 150 mg to about 300 mg, about 150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg to about 700 mg, about 200 mg to about 650 mg, about 200 mg to about 600 mg, about 200 mg to about 550 mg, about 200 mg to about 500 mg, about 200 mg to about 450 mg, about 200 mg to about 400 mg, about 200 mg to about 350 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, about 250 mg to about 700 mg, about 250 mg to about 650 mg, about 250 mg to about 600 mg, about 250 mg to about 550 mg, about 250 mg to about 500 mg, about 250 mg to about 450 mg, about 250 mg to about 400 mg, about 250 mg to about 350 mg, about 250 mg to about 300 mg, about 300 mg to about 700 mg, about 300 mg to about 650 mg, about 300 mg to about 600 mg, about 300 mg to about 550 mg, about 300 mg to about 500 mg, about 300 mg to about 450 mg, about 300 mg to about 400 mg, about 300 mg to about 350 mg, about 350 mg to about 700 mg, about 350 mg to about 650 mg, about 350 mg to about 600 mg, about 350 mg to about 550 mg, about 350 mg to about 500 mg, about 350 mg to about 450 mg, about 350 mg to about 400 mg, about 400 mg to about 700 mg, about 400 mg to about 650 mg, about 400 mg to about 600 mg, about 400 mg to about 550 mg, about 400 mg to about 500 mg, about 400 mg to about 450 mg, about 450 mg to about 700 mg, about 450 mg to about 650 mg, about 450 mg to about 600 mg, about 450 mg to about 550 mg, about 450 mg to about 500 mg, about 500 mg to about 700 mg, about 500 mg to about 650 mg, about 500 mg to about 600 mg, about 500 mg to about 550 mg, about 550 mg to about 700 mg, about 550 mg to about 650 mg, about 550 mg to about 600 mg, about 600 mg to about 700 mg, about 600 mg to about 650 mg, or about 650 mg to about 700 mg.

The dose may be a defined value. For example, the dose may be about 0. 1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg, about 10 mg/kg, about 10.5 mg/kg, about 11 mg/kg, about 11.5 mg/kg, about 12 mg/kg, about 12.5 mg/kg, about 13 mg/kg, about 13.5 mg/kg, or about 14 mg/kg, or about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg.

In some embodiments, the anti-BTLA antibody molecule comprises TAB004 and is administered as an intravenous infusion (e.g., over about 30 minutes to about 60 minutes). In certain embodiments, the anti-BTLA antibody molecules comprises TAB004 and is administered at 0.3 mg/kg as an intravenous infusion (e.g., over about 30 minutes to about 60 minutes) (e.g., every 2, 3, 4, 5, or 6 weeks). In certain embodiments, the anti-BTLA antibody molecules comprises TAB004 and is administered at 1 mg/kg as an intravenous infusion (e g., over about 30 minutes to about 60 minutes) (e.g., every 2, 3, 4, 5, or 6 weeks). In certain embodiments, the anti-BTLA antibody molecules comprises TAB004 and is administered at 3 mg/kg as an intravenous infusion (e.g., over about 30 minutes to about 60 minutes) (e.g., every 2, 3, 4, 5, or 6 weeks). In certain embodiments, the anti-BTLA antibody molecules comprises TAB004 and is administered at 10 mg/kg as an intravenous infusion (e.g., over about 30 minutes to about 60 minutes) (e.g., every 2, 3, 4, 5, or 6 weeks). In certain embodiments, the anti-BTLA antibody molecules comprises TAB004 and is administered at 10 mg as an intravenous infusion (e.g., over about 30 minutes to about 60 minutes) (e.g., every 2, 3, 4, 5, or 6 weeks). In certain embodiments, the anti- BTLA antibody molecules comprises TAB004 and is administered at 20 mg as an intravenous infusion (e.g., over about 30 minutes to about 60 minutes) (e.g., every 2, 3, 4, 5, or 6 weeks). In certain embodiments, the anti-BTLA antibody molecules comprises TAB004 and is administered at 70 mg as an intravenous infusion (e.g., over about 30 minutes to about 60 minutes) (e.g., every 2, 3, 4, 5, or 6 weeks). In certain embodiments, the anti-BTLA antibody molecules comprises TAB004 and is administered at 200 mg as an intravenous infusion (e.g., over about 30 minutes to about 60 minutes) (e.g., every 2, 3, 4, 5, or 6 weeks). In certain embodiments, the anti-BTLA antibody molecules comprises TAB004 and is administered at 500 mg as an intravenous infusion (e.g., over about 30 minutes to about 60 minutes) (e.g., every 2, 3, 4, 5, or 6 weeks).

In some embodiments, the subject will be administered a premedication prior to each dose (e.g., prior to the start of infusion) of expression repressor. In certain embodiments, the premedication is provided at least 60 minutes prior to the start of the infusion. In some embodiments, the premedication is selected from one or more of oral acetaminophen (e.g., 500 mg), intravenous Hl blocker (e.g., diphenhydramine 50 mg or equivalent), and intravenous corticosteroids (e.g., dexamethasone 10 mg or equivalent). In some embodiments, the intravenous corticosteroid (e.g., dexamethasone 10 mg or equivalent) is provided at least 60 minutes prior to the start of the infusion.

In some embodiments, a method of treatment described herein comprises imaging a tumor of the subject. In some embodiments, a computed tomography (CT) scan, e.g., with contrast or magnetic resonance imaging (MRI), is performed to image a tumor of the subject. A CT scan may be performed, e.g., every 6 weeks (± 1 week) or every 3 months (± 3 weeks). In some embodiments, a subject’s response is assessed according to modified Response Evaluation Criteria in Solid Tumors (mRECIST) for HCC or by RECIST 1. 1 for a non-HCC solid tumor. In some embodiments, a subject is considered to have achieved a complete response (CR) based on 2 tumor imaging assessments conducted at least 4 weeks apart.

In some embodiments, a method of treatment described herein comprises determining the incidence or seventy of adverse events (including SAEs), laboratory abnormalities, electrocardiogram (ECG) changes, cytokines, C-reactive protein (CRP), and complement. In some embodiments, a method of treatment described herein comprises collecting plasma samples, blood samples, or tumor biopsy samples.

Pharmaceutical compositions according to the present disclosure may be delivered in a therapeutically effective amount. A precise therapeutically effective amount is an amount of a composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to characteristics of a therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), physiological condition of a subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), nature of a pharmaceutically acceptable carrier or carriers in a formulation, and/or route of administration.

In some aspects, the present disclosure provides methods of delivering a therapeutic comprising administering a composition as described herein to a subject, wherein a modulating agent is a therapeutic and/or wherein delivery of a therapeutic causes changes in gene expression relative to gene expression in absence of a therapeutic.

Methods as provided in various embodiments herein may be utilized in any some aspects delineated herein. In some embodiments, one or more compositions is/are targeted to specific cells, or one or more specific tissues.

For example, in some embodiments one or more compositions is/are targeted to hepatic, epithelial, connective, muscular, reproductive, and/or nervous tissue or cells. In some embodiments a composition is targeted to a cell or tissue of a particular organ system, e g., cardiovascular system (heart, vasculature); digestive system (esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum and anus); endocrine system (hypothalamus, pituitary gland, pineal body or pineal gland, thyroid, parathyroids, adrenal glands); excretory system (kidneys, ureters, bladder); lymphatic system (lymph, lymph nodes, lymph vessels, tonsils, adenoids, thymus, spleen); integumentary system (skin, hair, nails); muscular system (e.g., skeletal muscle); nervous system (brain, spinal cord, nerves): reproductive system (ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate); respiratory system (pharynx, larynx, trachea, bronchi, lungs, diaphragm); skeletal system (bone, cartilage); and/or combinations thereof.

In some embodiments, a composition of the present disclosure crosses a blood-brain-barrier, a placental membrane, or a blood-testis barrier.

In some embodiments, a pharmaceutical composition as provided herein is administered systemically.

In some embodiments, administration is non-parenteral and a therapeutic is a parenteral therapeutic.

Methods and compositions provided herein may comprise a pharmaceutical composition administered by a regimen sufficient to alleviate a symptom of a disease, disorder, and/or condition. In some aspects, the present disclosure provides methods of delivering a therapeutic by administering compositions as described herein.

Pharmaceutical uses of the present disclosure may include compositions (e.g., modulating agents, e.g., disrupting agents) as described herein.

In some embodiments, a pharmaceutical composition of the present disclosure has improved PK/PD, e.g., increased pharmacokinetics or pharmacodynamics, such as improved targeting, absorption, ortransport (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% improved or more) as compared to an active agent alone. In some embodiments, a pharmaceutical composition has reduced undesirable effects, such as reduced diffusion to a nontarget location, off-target activity, or toxic metabolism, as compared to a therapeutic alone (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more reduced, as compared to an active agent alone). In some embodiments, a composition increases efficacy and/or decreases toxicity of a therapeutic (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more) as compared to an active agent alone.

Pharmaceutical compositions described herein may be formulated for example including a carrier, such as a pharmaceutical carrier and/or a polymeric carrier, e.g., a nanoparticle, a liposome or vesicle, and delivered by known methods to a subject in need thereof (e.g., a human or non-human agricultural or domestic animal, e.g., cattle, dog, cat, horse, poultry). Such methods include transfection (e.g., lipid- mediated, cationic polymers, calcium phosphate); electroporation or other methods of membrane disruption (e.g., nucleofection) and viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV). Methods of delivery are also described, e.g., in Gori et al., Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy. July 2015, 26(7): 443-451. Doi: 10.1089/hum.2015.074; and Zuris et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct 30;33(l):73-80.

Lipid Nanoparticles

Expression repressors or expression repression systems as described herein can be delivered using any biological delivery system/formulation including a particle, for example, a nanoparticle delivery system. Nanoparticles include particles with a dimension (e.g. diameter) between about 1 and about 1000 nanometers, between about 1 and about 500 nanometers in size, between about 1 and about 100 nm, between about 30 nm and about 200 nm, between about 50 nm and about 300 nm, between about 75 nm and about 200 nm, between about 100 nm and about 200 nm, and any range therebetween. A nanoparticle has a composite structure of nanoscale dimensions. In some embodiments, nanoparticles are typically spherical although different morphologies are possible depending on the nanoparticle composition. The portion of the nanoparticle contacting an environment external to the nanoparticle is generally identified as the surface of the nanoparticle. In some embodiments, nanoparticles have a greatest dimension ranging between 25 nm and 200 nm. Nanoparticles as described herein comprise delivery systems that may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal nanoparticles. A nanoparticle delivery system may include but not limited to lipid-based systems, liposomes, micelles, micro-vesicles, exosomes, or gene gun. In one embodiment, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the LNP is a particle that comprises a plurality of lipid molecules physically associated with each other by intermolecular forces.

In some embodiments, an LNP may comprise multiple components, e.g., 3-4 components. In one embodiment, the expression repressor or a pharmaceutical composition comprising said expression repressor (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repressor nucleic acid) is encapsulated in an LNP. In one embodiment, the expression repression system or a pharmaceutical composition comprising said expression repression system (or a nucleic acid encoding tire same, or pharmaceutical composition comprising said expression repression system nucleic acid) is encapsulated in an LNP. In some embodiments, the nucleic acid encoding the first expression repressor and the nucleic acid encoding the second expression repressor are present in same LNP. In some embodiments, the nucleic acid encoding the first expression repressor and the nucleic acid encoding the second expression repressor are present in different LNPs. Preparation of LNPs and the modulating agent encapsulation may be used/and or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011). hi some embodiments, lipid nanoparticle compositions disclosed herein are useful for expression of protein encoded by mRNA. In some embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.

In some embodiments, the LNP formulations may include a CCD lipid, a neutral lipid, and/or a helper lipid. In some embodiments, the LNP formulation comprises an ionizable lipid. In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, or an amine- containing lipid that can be readily protonated. In some embodiments, the lipid is a cationic lipid that can exist in a positively charged or neutral form depending on pH. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. In some embodiments, the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine -containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids.

In some embodiments, LNP formulation (e.g., MC3 and/or SSOP) includes cholesterol, PEG, and/or a helper lipid. The LNPs may be, e.g., microspheres (including unilamellar and multilamellar vesicles, lamellar phase lipid bilayers that, in some embodiments, are substantially spherical.

In some embodiments, the LNP can comprise an aqueous core, e.g., comprising a nucleic acid encoding an expression repressor or a system as disclosed herein. In some embodiments of the present disclosure, the cargo for the LNP formulation includes at least one guide RNA. In some embodiments, the cargo, e.g., a nucleic acid encoding an expression repressor, or a system as disclosed herein, may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the cargo, e.g., a nucleic acid encoding an expression repressor, or a system as disclosed herein may be associated with the LNP. In some embodiments, the cargo, e.g., a nucleic acid encoding an expression repressor, or a system as disclosed herein, may be encapsulated, e.g., fully encapsulated and/or partially encapsulated in an LNP.

In some embodiments, an LNP comprising a cargo may be administered for systemic delivery , e.g., delivery of a therapeutically effective dose of cargo that can result in a broad exposure of an active agent within an organism. Systemic delivery of lipid nanoparticles can be by any means known in the art including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery. In some embodiments, an LNP comprising a cargo may be administered for local delivery, e.g., delivery of an active agent directly to a target site within an organism. In some embodiments, an LNP may be locally delivered into a disease site, e.g., a tumor, other target site, e.g., a site of inflammation, or to a target organ, e.g., the liver, lung, stomach, colon, pancreas, uterus, breast, lymph nodes, and the like. In some embodiments, an LNP as disclosed herein may be locally delivered to a specific cell, e.g., hepatocytes, stellate cells, Kupffer cells, endothelial, alveolar, and/or epithelial cells. In some embodiments, an LNP as disclosed herein may be locally delivered to a specific tumor site, e.g., subcutaneous, orthotopic.

The LNPs may be formulated as a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. In some embodiments, the LNPs are biodegradable. In some embodiments, the LNPs do not accumulate to cytotoxic levels or cause toxicity in vivo at a therapeutically effective dose. In some embodiments, the LNPs do not accumulate to cytotoxic levels or cause toxicity in vivo after repeat administrations at a therapeutically effective dose. In some embodiments, the LNPs do not cause an innate immune response that leads to a substantially adverse effect at a therapeutically effective dose.

In some embodiments, the LNP used, comprises the formula (6Z,9Z,28Z,3 lZ)-heptatriacont- 6,9,28,31 -tetraene- 19-yl 4-(dimethylamino)butanoate or ssPalmO-phenyl-P4C2 (ssPalmO-Phe, SS-OP). In some embodiments, the LNP formulation comprises the formula, (6Z,9Z,28Z,31Z)-heptatriacont- 6,9,28,31 -tetraene- 19-yl 4-(dimethylamino)butanoate (MC3), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), Cholesterol, l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 (PEG2k-DMG), e.g., MC3 LNP or ssPalmO-phenyl-P4C2 (ssPalmO-Phe, SS-OP), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), Cholesterol, l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG), e.g., SSOP-LNP.

Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral, or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. Doi: 10.1155/2011/469679 for review).

Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Vesicles may comprise without limitation DOTMA, DOTAP, DOTIM, DDAB, alone or together with cholesterol to yield DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, tire teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. Doi:10. 1155/2011/469679 for review). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.

Uses

The present disclosure is further directed to uses of the expression repressors or expression repressor systems disclosed herein. Among other things, in some embodiments such provided technologies may be used to achieve modulation, e.g., repression, of target gene, e.g., MYC expression and, for example, enable control of target gene, e.g., MYC activity, delivery, and penetrance, e.g., in a cell. In some embodiments, a cell is a mammalian, e.g., human, cell. In some embodiments, a cell is a somatic cell. In some embodiments, a cell is a primary cell. For example, in some embodiments, a cell is a mammalian somatic cell. In some embodiments, a mammalian somatic cell is a primary cell. In some embodiments, a mammalian somatic cell is a non-embryonic cell.

In some embodiments, the expression repressors or expression repressor systems disclosed herein can be used to treat cancer in a subject in need thereof. In some embodiments, the cancer is hepatocellular carcinoma (HCC), Fibrolamellar Hepatocellular Carcinoma (FHCC), cholangiocarcinoma, angiosarcoma, secondary liver cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, small cell lung cancer (SCLC), large cell (undifferentiated) carcinoma, triple negative breast cancer, gastric adenocarcinoma, endometrial carcinoma, or pancreatic carcinoma. In some embodiments, the cancer is metastatic, advanced (non-resectable, or recurrent solid tumor types known for association with the MYC oncogene (e.g., including, but not limited to, hepatocellular carcinoma (HCC), bladder, lung, pancreatic, ovarian, uterine, endometrial, gastric, esophageal, hepatobiliary, colorectal cancer, and neuroblastoma), which has progressed on, relapsed after, are refractory to, or intolerant of standard of care or no alternative treatment exists.

In some embodiments, the subject having a cancer (e.g., a cancer described herein), has progressed on, relapsed after, is refractory to, or intolerant of, at least 1 prior systemic therapy. In certain embodiments, there is no available standard of care for the subject.

In certain embodiments, the subject has at least 1 measurable lesion according to RECIST 1.1.

In some embodiments, the subject comprises an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, wherein Grade 0 = Fully active, able to carry on all pre-disease performance without restriction; Grade 1 = Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light housework or office work; Grade 2 = Ambulatory and capable of all self-care but unable to carry out any work activities, and/or up and about more than 50% of waking hours; Grade 3 = Capable of only limited self-care, and/or confined to bed or chair more than 50% of waking hours; Grade 4 = Completely disabled, and/or cannot carry on any self-care, and/or totally confined to bed or chair; Grade 5 = deceased.

In some embodiments, the subject has a predicted life expectancy of less than 3 months (e.g., less than 2 months, less than 1 month, less than 25 days, less than 20 days, less than 10 days).

In some embodiments, the subject has not received chemotherapy or immunotherapy, had surgery, or received locoregional therapy (TACE, TAE, HAI, radiation, radioembolization or ablation), in at least 28 days. In certain embodiments, the subject has recovered from any toxicity associated with prior treatments. In some embodiments, the subject has recovered from any toxicities related to prior therapies to < Grade 1 or baseline.

In certain embodiments, the subject has, or has been identified as having, chronic hepatitis B and must have received antiviral therapy for hepatitis B virus (HBV) for at least 12 weeks and HBV viral load must be < 500 lU/mL prior to first dose of study drug. In some embodiment, subjects on active HBV therapy with viral loads < 500 lU/mL are to stay on the same therapy throughout treatment.

In some embodiments, subjects having, or identified as having, hepatitis C virus (HCV) have not received anti-hepatitis C therapy. IN certain embodiments, subject having, or identified as having, HCV have received anti-hepatitis C therapy but have not reached seroconversion.

In certain embodiments, subject having, or identified as having, an HBV/HCV coinfection must fulfill treatment and viral load requirements for hepatitis B (e.g., must have received antiviral therapy for hepatitis B virus (HBV) for at least 12 weeks and HBV viral load must be < 500 lU/mL prior to first dose of study drug).

In some embodiments, the subject has adequate organ function. In some embodiments, adequate organ function includes, but is not limited to: a. Absolute neutrophil count (ANC) > 1500/mm3; b. Hemoglobin > 8.5 g/dL without transfusion or erythropoietin within 7 days of first dose of study drug; c. Platelet count > 75,000/mm3 without transfusion; d. Total bilirubin < 1.5 mg/dL and/or < 2.0 mg/dL; e. AST and ALT < 3 x ULN and/or < 5 x ULN; f. International normalized ratio (INR) < 1.5; g. Estimated glomerular filtration rate (eGFR) > 30 mL/min/1.73 m2 by Modification of Diet in Renal Disease (MDRD) equation (e.g., GFR (mL/min/1.73 m2) = 175 x (Scr)- 1.154 x (Age)-0.203 x (0.742 if female) * (1.212 if African American) (Levey et al. A new equation to estimate glomerular filtration rate, Ann Intern Med, 150: 604-12 (2009))).

In some embodiments, the subject has HCC. In certain embodiments, the HCC has been confirmed radiographically, histologically, or cytologically. In some embodiments, the HCC is BCLC Stage B (intermediate state) or C (advanced stage), e.g., a BCLC stage B or C cancerthat is not amenable to locoregional therapy, refractory to locoregional therapy, and/or not amenable to curative treatment. In certain embodiments, the subject has neurotrophic tyrosine receptor kinase (NTRK) fusion-positive HCC. In some embodiments, the subject having NTRK-HCC has progressed on, relapsed after, is refractory to, or intolerant of NTRK-directed therapy. In certain embodiments, the subject has hepatic cirrhosis (e.g., underlying hepatic cirrhosis). In some embodiments, the hepatic cirrhosis (e.g., underlying hepatic cirrhosis) is well compensated (e.g., C-P Class A: Score 5-6). In certain embodiments, the subject has no evidence of hepatic encephalopathy (e.g., confirmed within 7 days of administration of the expression repressor or expression repressor system.

In some embodiments, the subject has received at least one, but not more than 3, prior systemic therapies for HCC, without available subsequent standard of care. In certain embodiments, subjects with known NTRK fusion-positive.

In some embodiments, the subject has total triiodothyronine (T3) or free T3, and free thyroxine (T4) within normal limits. In some embodiments, the subject has preexisting hypothyroidism that is well controlled with thyroid replacement therapy.

In some embodiments, a subject does not have one or more conditions excluding the subject from treatment using the expression repressors or expression repressor systems described herein. In certain embodiment, the subject does not have a history of central nervous system (CNS) metastases or carcinomatous meningitis. In certain embodiments, the subject does not have a history of CNS metastases or carcinomatous meningitis. In certain embodiments, the subject has a history of CNS metastases or carcinomatous meningitis and have completed local therapy and discontinued the use of corticosteroids at least 4 weeks (e.g., at least 4 weeks, at least 5 weeks, at least 6 weeks, or more) prior to administration of tire expression repressor or expression repressor systems described herein. In some embodiment, the subject does not have a history of other malignancies within the last 3 years (e.g., within the last 2.5 years, within the last 2 years, within the last 1.5 years, within the last 1 year, or within the last 0.5 years).

In some embodiments, the subject does not have a history of non-melanoma skin cancer (squamous or basal cell), carcinoma in situ of the breast or cervix. In some embodiments, the subject has a history of non-melanoma skin cancer (squamous or basal cell), carcinoma in situ of the breast or cervix, which has been adequately treated.

In some embodiments, the subject is receiving anticancer hormonal therapy for a previously treated non-HCC cancer.

In certain embodiments, the subject does not have an active, uncontrolled bacterial, viral, or fungal infection requiring systemic therapy, or an active COVID-19 infection within 14 days prior to administration of a first dose of an expression repressor or expression repressor system described herein. In certain embodiments, the subject has not tested positive for SARS-CoV2 within 14 days prior to administration of a first dose of an expression repressor or expression repressor system described herein.

In some embodiments, the subject does not have, or has not been identified as having, human immunodeficiency virus (HIV). In certain embodiments, the subject does not have a history of prior solid organ transplantation. In certain embodiments, the subject does not have a history of hematopoietic stem cell transplantation (HSCT).

In certain embodiments, the subject does not have a history of congestive heart failure > Class II New York Heart Association (NYHA), active coronary artery disease (myocardial infarction, unstable angina, coronary artery bypass graft [CABG] or percutaneous transluminal coronary angioplasty [PTCA]) within the past 6 months, ventricular arrhythmias requiring treatment, uncontrolled hypertension (systolic blood pressure > 160 mmHg and/or diastolic blood pressure > 100 mmHg), or history of hypertensive crisis. In certain embodiments, the subject does not have a QTc interval of > 450 ms (male) or > 470 ms (female). In certain embodiments, the subject has a QTc interval of < 450 ms (male) or < 470 ms (female).

In certain embodiments, the subject has not received prior MYC-targeting therapy (e.g., MYC promotor bromodomain inhibitors, MY C mRNA-degrading therapies, MC proteasomal degradationtargeting therapies, and therapies specifically inhibiting the transcription, translation, or biosynthesis of MYC).

In certain embodiments, the subject has, or is identified as having, HCC, and the subject does not have, or has been identified as not having, a) mixed histology cholangiocarcinoma and HCC or fibrolamellar variant HCC; b) imaging finding with one or more of (e.g., one, two, or all of): i) hepatocellular carcinoma with > 50% liver occupation, ii) clear invasion into the bile duct, iii) portal vein invasion with Vp4 (e.g., thrombus involving the main trunk of the portal vein); c) clinically apparent ascites on physical exam; d) history of ascites requiring paracentesis within the past 3 months; e) esophageal or gastric variceal bleeding in the past 3 months; f) history of hepatic encephalopathy in the past 3 months. In certain embodiments, the subject does not receive or take rifaximin or lactulose to control hepatic encephalopathy. In some embodiments, the subject has not received live vaccine within 30 days of administration of the first dose of the expression repressor or expression repressor system described herein. In some embodiments, the subject has not received an mRNA vaccine within 14 days of administration of the first dose of the expression repressor or expression repressor system described herein.

In some embodiments, the subject has not previously received immunotherapy, including anti- PD-1 antibody molecule or anti-PD-Ll antibody molecule.

In certain embodiments, the subject does not have an active autoimmune disease requiring systemic treatment. In certain embodiments, subject have, or are identified as having, autoimmune hypothyroidism, diabetes mellitus, pituitary or adrenal insufficiency that requires replacement therapy may be enrolled.

In some embodiments, the subject does not have a history of allergic reactions/hypersensitivity to monoclonal antibodies that resulted in discontinuation of the therapy.

In certain embodiments, the subject receiving the therapy described herein does not experience one or more of: i) Any Grade 5 hematologic adverse event (AE); ii) Grade 4 neutropenia and thrombocytopenia >7 days; iii) Grade 3+ febrile neutropenia lasting more than 48 hours; or iv) Grade 3+ thrombocytopenia associated with bleeding / hemorrhage that require transfusion.

In some embodiments, the subject receiving the therapy described herein does not experience one or more of: i) Grade 2 eye pain or reduction of visual acuity that does not improve to Grade 1 within 7 days after giving topical therapy or requires systemic treatment; ii) Grade 2 pneumonitis that does not improve to Grade 1 within 7 days after giving corticosteroids; iii) Grade 2 myocarditis; iv) Any Grade 4 or Grade 5 non-hematologic AE; v) Grade 3 non-laboratory value toxicity lasting> 72 hours (excluding nausea, vomiting, diarrhea, and/or dennatological toxicities controlled by medical intervention, fatigue, fever, or chills); vi) Grade 3 hypertension not controlled by medication; vii) Grade 2 gastrointestinal (GI) tract perforation; viii) Grade 3 wound dehiscence that requires surgical intervention; ix) Grade 3 thromboembolic event; x) Any Grade 3 non-hematologic laboratory value that requires medical intervention or hospitalization; xi) Aspartate aminotransferase (AST)Zalanine aminotransferase (ALT) > 10 * upper limit of normal (ULN); or xii) Grade 3 hepatic failure.

In some embodiments, a subject receiving a therapy described herein receives one or more of: 1) a tumor assessments before the beginning of the therapy, 2) every 6 weeks (± 1 week), e.g., for the first year, and 3) every 3 months (± 3 weeks), e.g., beginning 1 year after the start of the treatment until disease progression.

In certain embodiments, a subject receiving, or expected to receive, a therapy described herein is analyzed for PK/PD, e.g., pharmacokinetics or pharmacodynamics, of the therapy. In some embodiments, the subject has a baseline AFP level of < 400 ng/mL. In certain embodiments, the subject has a baseline AFP level of >400 ng/mL.

In some embodiments, the subject receiving, or expected to receive, a therapy described herein has, or is identified as having, HCC caused by HBV. In some embodiments, the subject receiving, or expected to receive, a therapy described herein has, or is identified as having, HCC caused by HCV.

In some embodiments, after treatment with a therapy described herein, the subject experiences an improvement in score on the European Organisation for Research and Treatment of Cancer (EORTC) QLC-C30 or EORTC QLQ-HCC18.

In some embodiments, the subject does not produce a detectable level of anti-drug antibodies.

Combination Therapies

The present disclosure provides, for instance, combination therapies comprising an expression repressor described herein and an immune checkpoint inhibitor polypeptide.

In some aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject:

(1) a nucleic acid (e g., RNA, e.g., mRNA) encoding an expression repressor described herein, and

(2) an immune checkpoint inhibitor polypeptide (e.g., an antibody molecule or peptide).

In some embodiments, the immune checkpoint inhibitor polypeptide is an antibody molecule. In some embodiments, the antibody molecule is an anti-PD-1, anti-PD-Ll, anti-CTLA-4, anti-LAG3, anti PD-L2, anti-Tim3, anti- inhibitory killer IgG-like receptor (KIR), or anti- B- and T-lymphocyte attenuator (BTLA) antibody molecule. In some embodiments, the antibody molecule is an anti-PD-1, anti-PD-Ll, anti-CTLA-4, or anti-LAG3 antibody molecule.

In some embodiments, the checkpoint inhibitor polypeptide, e.g., an antibody molecule comprising a CDR of any antibody molecule amino acid sequence listed in Tables 18-26, is administered (e.g., intravenously) at least 1 hour (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours) after completion of administration of the nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor described herein. In some embodiments, the checkpoint inhibitor polypeptide, e.g., an antibody molecule comprising a CDR of any antibody molecule amino acid sequence listed in Tables 18-26, is administered (e.g., intravenously) at within at least 24 hours (e.g., at least 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour)) after completion of administration of the nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor described herein. In some embodiments, the checkpoint inhibitor polypeptide, e.g., an antibody molecule comprising a CDR of any antibody molecule amino acid sequence listed in Tables 18-26, is administered (e.g., intravenously) at between 1-3 hours after completion of administration of the nucleic acid (e.g., RNA, e g., mRNA) encoding an expression repressor described herein. In some embodiments, administration (e.g., intravenously) of the checkpoint inhibitor polypeptide, e.g., an antibody molecule comprising a CDR of any antibody molecule amino acid sequence listed in Tables 18-26, is initiated at least 1 hour (e.g., at least I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours) after completion of administration of the nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor described herein.

In some embodiments, the antibody molecule comprises an anti-PD-1 antibody molecule.

In one embodiment, the anti-PD-1 antibody molecule comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy' chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of any anti-PD-1 antibody molecule amino acid sequence listed in Table 18; and/or a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of any anti-PD-1 antibody molecule amino acid sequence listed in Table 18.

In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region comprising the amino acid sequence of any heavy chain variable region listed in Table 18, e.g., SEQ ID NOs: 208, 210, 211, 213, 214, 216, 217, or 219. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 18, e.g., SEQ ID NOs: 208, 210, 211, 213, 214, 216, 217, or 219. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 18, e.g., SEQ ID NOs: 208, 210, 211, 213, 214, 216, 217, or 219. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of any heavy chain listed in Table 18, e.g., SEQ ID Nos: 208, 211, 214, or 217. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any heavy chain listed in Table 18, e.g., SEQ ID Nos: 208, 211, 214, or 217. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain listed in Table 18, e.g., SEQ ID Nos: 208, 211, 214, or 217.

In one embodiment, the anti-PD-1 antibody molecule comprises a light chain variable region comprising the amino acid sequence of any light chain variable region listed in Table 18, e.g., SEQ ID NOs: 209, 210, 212, 213, 215, 216, 218, or 219. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain variable region comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 18, e.g., SEQ ID NOs: 209, 210, 212, 213, 215, 216, 218, or 219. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 18, e.g., SEQ ID NOs: 209, 210, 212, 213, 215, 216, 218, or 219.

In one embodiment, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence of any light chain listed in Table 18, e.g., SEQ ID Nos: 209, 212, 215, or 218. In one embodiment, the anti-PD-1 antibody molecule comprises a light chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any light chain listed in Table 18, e.g., SEQ ID Nos: 209, 212, 215, or 218. In one embodiment, the anti- PD-1 antibody molecule comprises a light chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence to any light chain listed in Table 18, e.g., SEQ ID Nos: 209, 212, 215, or 218.

In one embodiments, the anti-PD-1 antibody molecule comprises: i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 208 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 209; ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 211 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 212; iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 214 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 215; or iv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 217 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 218. In some embodiments, the anti-PD-1 antibody molecule is an anti-PD-1 antibody molecule described in WO2008156712A1, incorporated herein by reference in its entirety. In some embodiments, the anti-PD-1 antibody molecule is an anti-PD-1 antibody molecule described in W02004004771A1, incorporated herein by reference in its entirety. In some embodiments, the anti-PD-1 antibody molecule is an anti-PD-1 antibody molecule described in US Pat. 8,008,449 or W02006121168A1, each incorporated herein by reference in their entirety. In some embodiments, the anti-PD-1 antibody molecule is an anti-PD-1 antibody molecule described in US Pat. 9,987,500, e.g., antibody H4H7798N, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the anti-PD-1 antibody molecule is cemiplimab. Alternative names for cemiplimab are REGN2810 and Uibtayo. In some embodiments, the anti-PD-1 antibody molecule is cemiplimab (CAS Registry Number: 1801342-60-8). Cemiplimab and other antibodies that specifically bind to PD-1 are disclosed in US Pat. 9,987,500, e.g., antibody H4H7798N, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of cemiplimab. In some embodiments, the anti-PD-1 antibody molecule is an antibody that competes for binding with an anti-PD-1 antibody molecule disclosed in US Pat. 9,987,500, e.g., antibody H4H7798N, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the anti-PD-1 antibody molecule comprises nivolumab. Alternative names for nivolumab are ONO-4538, BMS-936558, MDX1106, and Opdivo®. In some embodiments, the anti-PD-1 antibody molecule is nivolumab (CAS Registry Number: 946414-94-4). Nivolumab and other antibodies that specifically bind to PD-1 are disclosed in W02004004771A1, US Pat. 8,008,449, W02006121168A1, each incorporated herein by reference in their entirety. In one embodiment, the anti- PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of nivolumab. In some embodiments, the anti-PD-1 antibody molecule is an antibody that competes for binding with an anti-PD-1 antibody molecule disclosed in W02004004771A1, US Pat. 8,008,449, W02006121168A1, each incorporated herein by reference in their entirety.

In some embodiments, the anti-PD-1 antibody molecule comprises pembrolizumab. Alternative names for pembrolizumab are MK-3475, lambrolizumab, and Keytruda®. In some embodiments, the anti-PD-1 antibody molecule is pembrolizumab (CAS Registry Number: 1374853-91-4). Pembrolizumab and other antibodies that specifically bind to PD-1 are disclosed in WO2008156712A1, incorporated herein by reference in its entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of pembrolizumab. In some embodiments, the anti-PD-1 antibody molecule is an antibody that competes for binding with an anti-PD- 1 antibody molecule disclosed in WO2008156712A1, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the anti-PD-1 antibody molecule comprises dostarlimab. Alternative names for dostarlimab are TSR-042, WBP-285, dostarlimab-gxly, and Jemperli®. In some embodiments, the anti-PD-1 antibody molecule is dostarlimab (CAS Registry Number: 2022215-59-2). Dostarlimab and other antibodies that specifically bind to PD-1 are disclosed in US Pat. US 11,155,624, the content of which is incorporated herein in its entirety. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of dostarlimab. In some embodiments, the anti-PD-1 antibody molecule is an antibody that competes for binding with dostarlimab. In some embodiments, the anti-PD-1 antibody molecule is an antibody that competes for binding with an anti-PD-1 antibody molecule disclosed in US Pat. US 11,155,624, the content of which is incorporated herein in its entirety.

In some embodiments, the anti-PD-1 antibody molecule comprises MIH4. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of MIH4. In some embodiments, the anti-PD-1 antibody molecule is an antibody that competes for binding with MIH4.

Table 18: Exemplary antibody molecules targeting PD-1

Table 19: Exemplary Kabat CDRs of exemplary antibody molecules targeting PD-1

Table 20: Exemplary CDRs of exemplary antibody molecules targeting PD-1

In some embodiments, the PD-1 inhibitor is an anti -PD-1 polypeptide.

In one embodiment, the anti-PD-1 polypeptide comprises a fusion protein that specifically binds to PD-1 . In some embodiments, the anti-PD-1 fusion protein comprises a targeting domain and an Fc region. In some embodiments, the targeting domain comprises a portion of the PD-L2 extracellular domain. In some embodiment, the Fc region comprises an Fc domain of human IgGl . In some embodiment, the anti- PD-1 fusion protein comprises AMP-224.

In some embodiments, the immune checkpoint inhibitor polypeptide comprises an anti-PD-Ll antibody molecule.

In some embodiments, the immune checkpoint inhibitor polypeptide is an anti-PD-Ll antibody molecule comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of any anti-PD-Ll antibody molecule amino acid sequence listed in Table 21; and/or a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of any anti-PD-Ll antibody molecule amino acid sequence listed in Table 21. In some embodiments, the immune checkpoint inhibitor polypeptide is an anti-PD-Ll antibody molecule comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of any anti-PD-Ll antibody molecule amino acid sequence listed in Table 22; and/or a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of any anti-PD-Ll antibody molecule amino acid sequence listed in Table 22.

In one embodiment, the anti-PD-Ll antibody molecule comprises a heavy chain variable region comprising the amino acid sequence of any heavy chain variable region listed in Table 21, e.g., SEQ ID NOs: 238, 240, 241, 243, 244, or 246. In one embodiment, the anti-PD-Ll antibody molecule comprises a heavy chain variable region comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 21, e.g., SEQ ID NOs: 238, 240, 241, 243, 244, or 246. hr one embodiment, the anti-PD-Ll antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 21, e.g., SEQ ID NOs: 238, 240, 241, 243, 244, or 246.

In one embodiment, the anti-PD-Ll antibody molecule comprises a heavy chain comprising the amino acid sequence of any heavy chain listed in Table 21, e.g., SEQ ID NOs: 238, 241, or 244. In one embodiment, the anti-PD-Ll antibody molecule comprises a heavy chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any heavy chain listed in Table 21, e.g., SEQ ID NOs: 238, 241, or 244. In one embodiment, the anti-PD- Ll antibody molecule comprises a heavy chain comprising an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain listed in Table 21, e.g., SEQ ID NOs: 238, 241, or

244.

In one embodiment, the anti-PD-Ll antibody molecule comprises a light chain variable region comprising the amino acid sequence of any light chain variable region listed in Table 21, e.g., SEQ ID NOs: 239, 240, 242, 243, 245, or 246. In one embodiment, the anti-PD-Ll antibody molecule comprises a light chain variable region comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 21, e.g., SEQ ID NOs: 239, 240, 242, 243, 245, or 246. In one embodiment, the anti-PD-Ll antibody molecule comprises a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 21, e.g., SEQ ID NOs: 239, 240, 242, 243, 245, or 246.

In one embodiment, the anti-PD-Ll antibody molecule comprises a light chain comprising the amino acid sequence of any light chain listed in Table 21, e.g., SEQ ID NOs: 239, 242, or 245. In one embodiment, the anti-PD-Ll antibody molecule comprises a light chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any light chain listed in Table 21, e.g., SEQ ID NOs: 239, 242, or 245. In one embodiment, the anti-PD- Ll antibody molecule comprises a light chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence to any light chain listed in Table 21, e g., SEQ ID NOs: 239, 242, or

245.

In one embodiments, the anti-PD-Ll antibody molecule comprises: i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 238 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 239; ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 241 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 242; or iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 244 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 245.

In some embodiments, the anti-PD-Ll antibody molecule comprises an anti-PD-Ll antibody molecule described in US Pat. 8,217,149, the content of which is incorporated herein by reference in its entirety. In some embodiments, the anti-PD-Ll antibody molecule comprises an anti-PD-Ll antibody molecule described in US Pat. 8,952,136, the content of which is incorporated herein by reference in its entirety. In some embodiments, the anti-PD-Ll antibody molecule comprises an anti-PD-Ll antibody molecule described in US Pat. 9,624,298, the content of which is incorporated herein by reference in its entirety. In some embodiments, the anti-PD-Ll antibody molecule comprises an anti-PD-Ll antibody molecule described in US Pat. 7,943,743, the content of which is incorporated herein by reference in its entirety.

In some embodiments, the anti-PD-Ll antibody molecule comprises atezolizumab. Alternative names for atezolizumab are MPDL3280A, RG7446, and Tecentriq®. In some embodiments, the anti-PD- Ll antibody molecule is atezolizumab (CAS Registry Number: 1380723-44-3). Atezolizumab and other antibodies that specifically bind to PD-L1 are disclosed in US Pat. 8,217,149, the content of which is incorporated herein by reference in its entirety. In one embodiment, the anti-PD-Ll antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of atezolizumab. In some embodiments, the anti-PD-1 antibody molecule comprises an antibody that competes for binding with an anti-PD-Ll antibody molecule disclosed in US Pat. 8,217,149, the content of which is incorporated herein by reference in its entirety.

In some embodiments, the immune checkpoint inhibitor polypeptide is an anti-PD-Ll antibody molecule comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising an amino acid sequence GFTFSDSWIH (SEQ ID NO: 259), a heavy chain complementarity determining region 2 (HC CDR2) comprising an amino acid sequence AWISPYGGSIYYADSVKG (SEQ ID NO: 260), and a heavy chain complementarity determining region 3 (HC CDR3) comprising an amino acid sequence RHWPGGFDY (SEQ ID NO: 261); and/or a light chain complementarity determining region 1 (LC CDR1) comprising an amino acid sequence RASQDVSTAVA (SEQ ID NO: 262), a light chain complementarity determining region 2 (LC CDR2) comprising an amino acid sequence SASFLYS (SEQ ID NO: 263), and a light chain complementarity determining region 3 (LC CDR3) comprising an amino acid sequence QQYLYHPAT (SEQ ID NO: 264). In some embodiments, the immune checkpoint inhibitor polypeptide is an anti-PD-Ll antibody molecule comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to tire amino acid sequence GFTFSDSWIH (SEQ ID NO: 259), a heavy chain complementarity determining region 2 (HC CDR2) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 260), and a heavy chain complementarity determining region 3 (HC CDR3) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence RHWPGGFDY (SEQ ID NO: 261); and/or a light chain complementarity determining region 1 (LC CDR1) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence RASQDVSTAVA (SEQ ID NO: 262), a light chain complementarity determining region 2 (LC CDR2) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence SASFLYS (SEQ ID NO: 263), and a light chain complementarity determining region 3 (LC CDR3) comprising an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence QQYLYHPAT (SEQ ID NO: 264).

In some embodiments, the anti-PD-Ll antibody molecule comprises avelumab. Alternative names for avelumab are MSB0010718C and Bavencio®. In some embodiments, the anti-PD-Ll antibody molecule is avelumab (CAS Registry Number: 1537032-82-8). Avelumab and other antibodies that specifically bind to PD-L1 are disclosed in US Pat. 9,624,298 and PCT Publication No WO2013/079174, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the anti- PD-Ll antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of avelumab. In one embodiment, the anti-PD-Ll antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of A09-246-2 as described in PCT Publication No. WO2013/079174, which is herein incorporated by reference in its entirety. In some embodiments, the antibody molecule is one described in US20180369377A1, which is herein incorporated by reference in its entirety. In some embodiments, the anti-PD-Ll antibody molecule is an antibody that competes for binding with an anti-PD-Ll antibody molecule disclosed in US Pat. 9,624,298 or PCT Publication No WO2013/079174, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the anti-PD-Ll antibody molecule comprises BMS-936559. An alternative name for BMS-936559 is MDX 1105. BMS-936559 and other antibodies that specifically bind to PD-L1 are disclosed in US Pat. 7,943,743, the content of which is incorporated herein by reference in its entirety. In one embodiment, the anti-PD-Ll antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of BMS-936559. In some embodiments, the anti- PD-1 antibody molecule is an antibody that competes for binding with an anti-PD-Ll antibody molecule disclosed in US Pat. 7,943,743, the content of which is incorporated herein by reference in its entirety. In some embodiments, the anti-PD-Ll antibody molecule comprises MKP1A07310. In one embodiment, the anti-PD-Ll antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of MKP1A07310. In some embodiments, the anti-PD-Ll antibody molecule is an antibody that competes for binding with MKP1A07310.

Table 21: Exemplary antibody molecules targeting PD-L1

Table 22: Exemplary CDRs of antibody molecules targeting PD-L1

In some embodiments, the immune checkpoint inhibitor polypeptide comprises an anti-CTLA-4 antibody molecule.

In some embodiments, the immune checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody molecule comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy' chain complementarity determining region 3 (HC CDR3) of any CTLA-4 antibody molecule amino acid sequence listed in Table 23; and/or a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of any CTLA- 4 antibody molecule amino acid sequence listed in Table 23.

In one embodiment, the anti-CTLA-4 antibody molecule comprises a heavy chain variable region comprising the amino acid sequence of any heavy chain variable region listed in Table 23, e.g., SEQ ID NOs: 247 or 249. In one embodiment, the anti-CTLA-4 antibody molecule comprises a heavy chain variable region comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 23, e.g., SEQ ID NOs: 247 or 249. In one embodiment, the anti-CTLA-4 antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 23, e.g., SEQ ID NOs: 247 or 249.

In one embodiment, the anti-CTLA-4 antibody molecule comprises a heavy chain comprising the amino acid sequence of any heavy chain listed in Table 23, e.g., SEQ ID NO: 247. In one embodiment, the anti-CTLA-4 antibody molecule comprises a heavy chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any heavy chain listed in Table 23, e.g., SEQ ID NO: 247. In one embodiment, the anti-CTLA-4 antibody molecule comprises a heavy chain comprising an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain listed in Table 23, e.g., SEQ ID NO: 247.

In one embodiment, the anti-CTLA-4 antibody molecule comprises a light chain variable region comprising the amino acid sequence of any light chain variable region listed in Table 23, e.g., SEQ ID NOs: 248 or 249. In one embodiment, the anti-CTLA-4 antibody molecule comprises a light chain variable region comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 23, e.g., SEQ ID NOs: 248 or 249. In one embodiment, the anti-CTLA-4 antibody molecule comprises a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 23, e.g., SEQ ID NOs: 248 or 249.

In one embodiment, the anti-CTLA-4 antibody molecule comprises a light chain comprising the amino acid sequence of any light chain listed in Table 23, e.g., SEQ ID NO: 248. In one embodiment, the anti-CTLA-4 antibody molecule comprises a light chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any light chain listed in Table 23, e.g., SEQ ID NO: 248. In one embodiment, the anti-CTLA-4 antibody molecule comprises a light chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence to any light chain listed in Table 23, e.g., SEQ ID NO: 248.

In some embodiments, the anti-CTLA-4 antibody molecule is an anti-CTLA-4 antibody molecule described in US Pat. 5,811,097, the content of which is incorporated herein by reference in its entirety. In some embodiments, the anti-CTLA-4 antibody molecule is an anti-CTLA-4 antibody molecule described in US Pat. 7,605,238, the content of which is incorporated herein by reference in its entirety.

In some embodiments, the anti-CTLA-4 antibody comprises ipilimumab. Alternative names for ipilimumab are BMS-734016, MDX-010, MDX-101, and Yervoy®. In some embodiments, the anti- CTLA-4 antibody is ipilimumab (CAS Registry Number: 477202-00-9). Ipilimumab and other antibodies that specifically bind to CTLA-4 are disclosed in US Pat. 5,811,097 and US Pat. 7,605,238, each of which are incorporated herein by reference in their entirety. In one embodiment, the anti-CTLA-4 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of ipilimumab. In some embodiments, the anti-CTLA-4 antibody is an antibody that competes for binding with an anti-CTLA-4 antibody disclosed in US Pat. 5,811,097 or US Pat. 7,605,238, each of which are incorporated herein by reference in their entirety.

In some embodiments, the anti-CTLA-4 antibody comprises tremelimumab. Alternative names for tremelimumab are ticilimumab and CP-675,206. In some embodiments, the anti-CTLA-4 antibody is tremelimumab (CAS Registry Number: 745013-59-6). Tremelimumab and other antibodies that specifically bind to CTLA-4 are disclosed in US Pat. 7,605,238, the content of which is incorporated herein by reference in its entirety. In one embodiment, the anti-CTLA-4 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of tremelimumab. In some embodiments, the anti-CTLA-4 antibody is an antibody that competes for binding with an anti-CTLA-4 antibody disclosed in US Pat. 7,605,238, the content of which is incorporated herein by reference in its entirety.

Table 23: Exemplary antibody molecules targeting CTLA-4

In some embodiments, the immune checkpoint inhibitor polypeptide comprises an anti-LAG3 antibody molecule.

In some embodiments, the immune checkpoint inhibitor polypeptide is an anti-LAG3 antibody molecule comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of any LAG3 antibody molecule amino acid sequence listed in Table 24; and/or a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of any LAG3 antibody molecule amino acid sequence listed in Table 24.

In one embodiment, the anti-LAG3 antibody molecule comprises a heavy chain variable region comprising the amino acid sequence of any heavy chain variable region listed in Table 24, e.g., SEQ ID NOs: 250, 252, 280, 282, 283, or 285. In one embodiment, the anti-LAG3 antibody molecule comprises a heavy chain variable region comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 24, e.g., SEQ ID NOs: 250, 252, 280, 282, 283, or 285. In one embodiment, the anti-LAG3 antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 24, e.g., SEQ ID NOs: 250, 252, 280, 282, 283, or 285.

In one embodiment, the anti-LAG3 antibody molecule comprises a heavy chain comprising the amino acid sequence of any heavy chain listed in Table 24, e.g., SEQ ID NOs: 250, 280, or 283. In one embodiment, the anti-LAG3 antibody molecule comprises a heavy chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any heavy chain listed in Table 24, e.g., SEQ ID NOs: 250, 280, or 283. hr one embodiment, tire anti- LAG3 antibody molecule comprises a heavy' chain comprising an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain listed in Table 24, e.g., SEQ ID NOs: 250, 280, or

283.

In one embodiment, the anti-LAG3 antibody molecule comprises a light chain variable region comprising the amino acid sequence of any light chain variable region listed in Table 24, e.g., SEQ ID NOs: 251, 252, 281, 282, 284, or 285. In one embodiment, the anti-LAG3 antibody molecule comprises a light chain variable region comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 24, e.g., SEQ ID NOs: 251, 252, 280, 282, 283, or 285. In one embodiment, the anti-LAG3 antibody molecule comprises a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 24, e.g., SEQ ID NOs: 251, 252, 280, 282, 283, or 285.

In one embodiment, the anti-LAG3 antibody molecule comprises a light chain comprising the amino acid sequence of any light chain listed in Table 24, e.g., SEQ ID NOs: 251, 281, or 284. In one embodiment, the anti-LAG3 antibody molecule comprises a light chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any light chain listed in Table 24, e.g., SEQ ID NOs: 251, 281, or 284. In one embodiment, the anti- LAG3 antibody molecule comprises a light chain comprising an ammo acid sequence having at least 95% identity to the amino acid sequence to any light chain listed in Table 24, e g., SEQ ID NOs: 251, 281, or

284.

In some embodiments, the anti-LAG3 antibody molecule is an anti-LAG3 antibody molecule described in WO2015042246A1, the content of which is incorporated herein by reference in its entirety. In some embodiments, the anti-LAG3 antibody molecule is an anti-LAG3 antibody molecule described in US Pat. 9,505,839, the content of which is incorporated herein by reference in its entirety.

In some embodiments, the anti-LAG3 antibody comprises relatlimab. Alternative names for relatlimab are BMS-986016 and relatlimab-rmbw. In some embodiments, the anti-LAG3 antibody is relatlimab (CAS Registry Number: 1673516-98-7). Relatlimab and other antibodies that specifically bind to LAG3 are disclosed in WO2015042246A1 and US Par. 9,505,839, each of which are incorporated herein by reference in their entirety. In one embodiment, the anti-LAG3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of relatlimab. In some embodiments, the anti-LAG3 antibody is an antibody that competes for binding with an anti-LAG3 antibody disclosed in WO2015042246A1 or US Pat. 9,505,839, each of which are incorporated herein by reference in their entirety. In some embodiments, the anti-LAG3 antibody comprises leramilimab. An alternative name for ieramilimab is LAG525. In some embodiments, the anti-LAG3 antibody is ieramilimab (2137049-37-5). In one embodiment, the anti-LAG3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of ieramilimab.

In some embodiments, the anti-LAG3 antibody comprises REGN3767. In one embodiment, the anti-LAG3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of REGN3767.

In some embodiments, the anti-LAG3 antibody comprises Sym022. In one embodiment, the anti- LAG3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of Sym022.

In some embodiments, the anti-LAG3 antibody comprises favezelimab. Alternative names for favezelimab are MK-4280 and mavezelimab. In some embodiments, the anti-LAG3 antibody is favezelimab (CAS Registry Number: 2231068-83-8). In one embodiment, the anti-LAG3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of favezelimab.

In some embodiments, the anti-LAG3 antibody is an antibody that competes for binding to LAG3 with an antibody comprising a complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of any LAG3 antibody molecule amino acid sequence listed in Table 24; and/or a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of any LAG3 antibody molecule amino acid sequence listed in Table 24.

Table 24: Exemplary antibody molecules targeting LAG3

Table 25: Exemplary Kabat CDRs of antibody molecules targeting LAG3

In some embodiments, the immune checkpoint inhibitor polypeptide comprises a PD-L2 inhibitor.

In some embodiments, the PD-L2 inhibitor comprises an anti-PD-L2 antibody molecule.

In some embodiments, the anti-PD-L2 antibody molecule comprises rHIgM12B7. In one embodiment, the anti-PD-L2 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of rHIgM12B7.

In some embodiments, the immune checkpoint inhibitor polypeptide comprises a Tim-3 inhibitor.

In some embodiments, the Tim3 inhibitor comprises an anti-Tim3 antibody molecule.

In some embodiments, the anti-Tim3 antibody molecule comprises MBG 453. In one embodiment, the anti-Tim3 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of MBG 453.

In some embodiments, the immune checkpoint inhibitor polypeptide comprises a killer IgG-like receptor (KIR) inhibitor.

In some embodiments, the immune checkpoint inhibitor polypeptide is an anti-KIR antibody molecule comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of any KIR antibody molecule amino acid sequence listed in Tabic 26; and/or a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of any KIR antibody molecule amino acid sequence listed in Table 26.

In one embodiment, the anti-KIR antibody molecule comprises a heavy chain variable region comprising the amino acid sequence of any heavy chain variable region listed in Table 26, e.g., SEQ ID NOs: 277 or 279. In one embodiment, the anti-KIR antibody molecule comprises a heavy chain variable region comprising the amino acid sequence having at least one, Lw o or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 26, e.g., SEQ ID NOs: 277 or 279. In one embodiment, the anti-KIR antibody molecule comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 26, e.g., SEQ ID NOs: 277 or 279.

In one embodiment, the anti-KIR antibody molecule comprises a heavy chain comprising the amino acid sequence of any heavy chain listed in Table 26, e.g., SEQ ID NO: 277. In one embodiment, the anti-KIR antibody molecule comprises a heavy chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any heavy chain listed in Table 26, e.g., SEQ ID NO: 277. In one embodiment, the anti-KIR antibody molecule comprises a heavy chain comprising an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy' chain listed in Table 26, e.g., SEQ ID NO: 277.

In one embodiment, the anti-KIR antibody molecule comprises a light chain variable region comprising the amino acid sequence of any light chain variable region listed in Table 26, e.g., SEQ ID NOs: 278 or 279. In one embodiment, the anti-KIR antibody molecule comprises a light chain variable region comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 26, e.g., SEQ ID NOs: 278 or 279. In one embodiment, the anti-KIR antibody molecule comprises a light chain variable region comprising an amino acid sequence having at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 26, e g., SEQ ID NOs: 278 or 279.

In one embodiment, the anti-KIR antibody molecule comprises a light chain comprising the amino acid sequence of any light chain listed in Table 26, e.g., SEQ ID NO: 278. In one embodiment, the anti-KIR antibody molecule comprises a light chain comprising the amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to any light chain listed in Table 26, e.g., SEQ ID NO: 278. In one embodiment, the anti-KIR antibody molecule comprises a light chain comprising an amino acid sequence having at least 95% identity to the amino acid sequence to any light chain listed in Table 26, e.g., SEQ ID NO: 278.

In some embodiments, the KIR inhibitor comprises an anti-KIR antibody molecule.

In some embodiments, the anti-KIR antibody molecule comprises lirilumab. In some embodiments, the anti-KIR antibody is lirilumab (CAS Registry Number: 1000676-41-4). hi one embodiment, the anti-KIR antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of lirilumab.

In some embodiments, the anti-KIR antibody is an antibody that competes for binding with an antibody comprising a complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy' chain complementarity determining region 3 (HC CDR3) of any KIR antibody molecule amino acid sequence listed in Table 26; and/or a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of any KIR antibody molecule amino acid sequence listed in Table 26. In some embodiments, the anti-KIR antibody is an antibody that competes for binding with an antibody comprising a HC or LC of any KIR antibody molecule amino acid sequence listed in Table 26.

Table 26: Exemplary antibody molecules targeting KIR

In some embodiments, the immune checkpoint inhibitor polypeptide comprises a BTLA inhibitor.

In some embodiments, the Tim3 inhibitor comprises an anti-BTLA antibody molecule.

In some embodiments, the anti-BTLA antibody comprises TAB004. An alternative name for cudarolimab is JS004. TAB004 and other antibodies that specifically bind to BTLA are disclosed in US Pat. 8,518,405 and 9,845,362, the content of each is incorporated herein by reference in its entirety. In one embodiment, the anti- BTLA antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of tremelimumab. In some embodiments, the anti- BTLA antibody is an antibody that competes for binding with an anti- BTLA antibody disclosed in US Pat. 8,518,405 or 9,845,362, the content of each which is incorporated herein by reference in its entirety.

(1) a nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor described herein, and

(2) one or more (e.g., 1 , 2, 3, 4, or more) immune checkpoint inhibitor polypeptides (e.g., an antibody molecule or peptide).

In some embodiments, the one or more immune checkpoint inhibitor polypeptides are each independently an antibody molecule. In some embodiments, the one or more antibody molecules are independently chosen from an anti-PD-1, anti-PD-Ll, anti-CTLA-4, anti-LAG3, anti-PD-L2, anti-Tim3, anti -KIR, and anti-BTLA antibody molecule. In some embodiments, the one or more immune checkpoint inhibitor polypeptides comprises 2 antibody molecules. In some embodiments, the two antibody molecules are independently chosen from an anti-PD-1, anti-PD-Ll, anti-CTLA-4, an anti-LAG3 antibody molecule, anti-PD-L2, anti-Tim3, anti-KIR, and an anti-BTLA.

In some embodiments, the 2 antibody molecules comprise an anti-PD-1 antibody molecule (e.g., an anti-PD-1 antibody molecule described herein) and a second, different anti-PD-1 antibody molecule (e.g., an anti-PD-1 antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-PD-Ll antibody molecule (e.g., an anti-PD-Ll antibody molecule described herein) and a second, different anti-PD-Ll antibody molecule (e.g., an anti-PD-Ll antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-CTLA-4 antibody molecule (e.g., an anti- CTLA-4 antibody molecule described herein) and a second, different anti- CTLA-4 antibody molecule (e.g., an anti- CTLA-4 antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-LAG3 antibody molecule (e.g., an anti-LAG3 antibody molecule described herein) and a second, different anti-LAG3 antibody molecule (e.g., an anti-LAG3 antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-PD-L2 antibody molecule (e.g., an anti-PD-L2 antibody molecule described herein) and a second, different anti-PD-L2 antibody molecule (e.g., an anti-PD-L2 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-Tim3 antibody molecule (e.g., an anti-Tim3 antibody molecule described herein) and a second, different anti-Tim3 antibody molecule (e.g., an anti-Tim3 antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-KIR antibody molecule (e.g., an anti-KIR antibody molecule described herein) and a second, different anti-KIR antibody molecule (e.g., an anti-KIR antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-BTLA antibody molecule (e.g., an anti-BTLA antibody molecule described herein) and a second, different anti-BTLA antibody molecule (e.g., an anti-BTLA antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-PD-1 antibody molecule (e.g., an anti-PD-1 antibody molecule described herein) and an anti-PD-Ll antibody molecule (e.g., an anti-PD- L1 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-1 antibody molecule (e.g., an anti-PD-1 antibody molecule described herein) and an anti-CTLA-4 antibody molecule (e.g., an anti- CTLA-4 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-1 antibody molecule (e.g., an anti-PD-1 antibody molecule described herein) and an anti-LAG3 antibody molecule (e.g., an anti-LAG3 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-1 antibody molecule (e.g., an anti-PD-1 antibody molecule described herein) and an anti-PD-L2 antibody molecule (e.g., an anti-PD- L2 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-1 antibody molecule (e.g., an anti-PD-1 antibody molecule described herein) and an anti-Tim3 antibody molecule (e.g., an anti-Tim3 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-1 antibody molecule (e.g., an anti-PD-1 antibody molecule described herein) and an anti-KIR antibody molecule (e.g., an anti-KIR antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-1 antibody molecule (e.g., an anti-PD-1 antibody molecule described herein) and an anti-BTLA antibody molecule (e.g., an anti- BTLA antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-PD-Ll antibody molecule (e.g., an anti-PD-Ll antibody molecule described herein) and an anti-CTLA-4 antibody molecule (e.g., an anti- CTLA-4 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-Ll antibody molecule (e.g., an anti-PD-Ll antibody molecule described herein) and an anti- LAG3 antibody molecule (e g., an anti-LAG3 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-Ll antibody molecule (e.g., an anti-PD-Ll antibody molecule described herein) and an anti-PD-L2 antibody molecule (e.g., an anti-PD-L2 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-Ll antibody molecule (e.g., an anti-PD-Ll antibody molecule described herein) and an anti-Tim3 antibody molecule (e.g., an anti-Tim3 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-Ll antibody molecule (e.g., an anti-PD-Ll antibody molecule described herein) and an anti-KIR antibody molecule (e.g., an anti-KIR antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-Ll antibody molecule (e.g., an anti- PD-Ll antibody molecule described herein) and an anti-BTLA antibody molecule (e.g., an anti-BTLA antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-CTLA-4 antibody molecule (e.g., an anti- CTLA-4 antibody molecule described herein) and an anti-LAG3 antibody molecule (e.g., an anti-LAG3 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-CTLA-4 antibody molecule (e.g., an anti-CTLA-4 antibody molecule described herein) and an anti-PD-L2 antibody molecule (e.g., an anti-PD-L2 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-CTLA-4 antibody molecule (e.g., an anti- CTLA-4 antibody molecule described herein) and an anti-Tim3 antibody molecule (e.g., an anti-Tim3 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti- CTLA-4 antibody molecule (e.g., an anti-CTLA-4 antibody molecule described herein) and an anti-KIR antibody molecule (e.g., an anti-KIR antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-CTLA-4 antibody molecule (e g., an anti-CTLA-4 antibody molecule described herein) and an anti-BTLA antibody molecule (e.g., an anti-BTLA antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-PD-L2 antibody molecule (e.g., an anti-PD-L2 antibody molecule described herein) and an anti-Tim3 antibody molecule (e.g., an anti- Tim3 antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-L2 antibody molecule (e.g., an anti-PD-L2 antibody molecule described herein) and an anti-KIR antibody molecule (e.g., an anti-KIR antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-PD-L2 antibody molecule (e.g., an anti-PD-L2 antibody molecule described herein) and an anti-BTLA antibody molecule (e g., an anti-BTLA antibody molecule described herein).

In some embodiments, the 2 antibody molecules comprise an anti-Tim3 antibody molecule (e.g., an anti-Tim3 antibody molecule described herein) and an anti-KIR antibody molecule (e.g., an anti-KIR antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti- Tim3 antibody molecule (e.g., an anti-Tim3 antibody molecule described herein) and an anti-BTLA antibody molecule (e.g., an anti-BTLA antibody molecule described herein). In some embodiments, the 2 antibody molecules comprise an anti-KIR antibody molecule (e.g., an anti-KIR antibody molecule described herein) and an anti-BTLA antibody molecule (e.g., an anti- BTLA antibody molecule described herein).

Modulating Gene Expression

The present disclosure is further directed, in part, to a method of modulating, e.g., decreasing, expression of a target gene, e.g., MYC, comprising providing an expression repressor (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repressor nucleic acid) or an expression repression system described herein (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repression system or nucleic acid), and contacting the target gene e.g., MYC, and/or operably linked transcription control element(s) with the expression repressor or the expression repression system. In some embodiments, modulating, e.g., decreasing expression of a target gene, e.g., MY C comprises modulation of transcription of a target gene, e.g., MY C as compared with a reference value, e.g., transcription of a target gene, e.g., MYC in absence of the expression repressor or the expression repression system. In some embodiments, the method of modulating, e g., decreasing, expression of a target gene, e.g., MYC are used ex vivo, e.g., on a cell from a subject, e.g., a mammalian subject, e.g., a human subject. In some embodiments, the method of modulating, e.g., decreasing, expression of a target gene, e.g., MYC are used in vivo, e.g., on a mammalian subject, e.g., a human subject. In some embodiments, the method of modulating, e g., decreasing, expression of a target gene, e.g., MYC are used in vitro, e.g., on a cell or cell line described herein.

The present disclosure is further directed, in part to a method of treating a condition associated with mis-regulation, e.g., over-expression of a target gene, e.g., MYC in a subject, comprising administering to the subject an expression repressor (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repressor nucleic acid) or an expression repression system described herein (or a nucleic acid encoding the same, or pharmaceutical composition comprising said expression repression system or nucleic acid). Conditions associated with overexpression of particular genes are known to those of skill in the art. Such conditions include, but are not limited to, metabolic disorders, cancer (e.g., solid tumors), and hepatitis.

Methods and compositions as provided herein may treat a condition associated with overexpression or mis-regulation of a target gene, e.g., MY C by stably or transiently altering (e.g., decreasing) transcription of a target gene, e.g., MYC. In some embodiments, such a modulation persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or longer or any time therebetween. In some embodiments, such a modulation persists for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, or 7 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., permanently or indefinitely). Optionally, such a modulation persists for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years.

In some embodiments, a method or composition provided herein may decrease expression of a target gene, e.g., MYC in a cell by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100%) relative to expression of the target gene in a cell not contacted by the composition or treated with the method.

In some embodiments, a method provided herein may modulate, e.g., decrease, expression of a target gene, e.g., MYC by disrupting a genomic complex, e.g., an anchor sequence-mediated conjunction, associated with said target gene. A gene that is associated with an anchor sequence-mediated conjunction may be at least partially within a conjunction (that is, situated sequence-wise between a first and second anchor sequences), or it may be external to a conjunction in that it is not situated sequence-wise between a first and second anchor sequences, but is located on the same chromosome and in sufficient proximity to at least a first or a second anchor sequence such that its expression can be modulated by controlling the topology of the anchor sequence-mediated conjunction. Those of ordinary skill in the art will understand that distance in three-dimensional space between two elements (e.g., between the gene and the anchor sequence-mediated conjunction) may, in some embodiments, be more relevant than distance in terms of base pairs. In some embodiments, an external but associated gene is located within 2 Mb, within 1.9 Mb, within 1.8 Mb, within 1.7 Mb, within 1.6 Mb, within 1.5 Mb, within 1.4 Mb, with 1.3 Mb, within 1.3 Mb, within 1.2 Mb, within 1.1 Mb, within 1 Mb, within 900 kb, within 800 kb, within 700 kb, within 500 kb, within 400 kb, within 300 kb, within 200 kb, within 100 kb, within 50 kb, within 20 kb, within 10 kb, or within 5 kb of the first or second anchor sequence.

In some embodiments, modulating expression of a gene, e.g., MYC comprises altering accessibility of a transcriptional control sequence to a gene, e.g., MYC. A transcriptional control sequence, whether internal or external to an anchor sequence-mediated conjunction, can be an enhancing sequence or a silencing (or repressive) sequence.

In some embodiments, such provided technologies may be used to treat a gene mis-regulation disorder e.g., MYC gene mis-regulation disorder e.g., a symptom associated with a MYC gene mis- regulation in a subject, e.g., a patient, in need thereof. In some embodiments, such provided technologies may be used to treat a MYC gene mis-regulation disorder or a symptom associated with a MYC gene mis-regulation disorder in a subject, e.g., a patient, in need thereof. In some embodiments, the disorder is associated with MYC mis-regulation, e.g., MYC overexpression, hi some embodiments, the disorder is associated with AFP mis-regulation, e.g., AFP overexpression. In some embodiments, such provided technologies may be used to methylate the promoter of a target gene, e.g., MYC, to treat a gene mis- regulation disorder e.g., MYC gene mis-regulation disorder, e g., a symptom associated with a MYC gene mis-regulation in a subject, e.g., a patient, in need thereof. In some embodiments, such provided technologies may selectively affect the viability of a cell which aberrantly expresses a polypeptide encoded by a target gene, e.g., MYC.

In some embodiments, such provided technologies may be used to treat a hepatic disorder or a disorder e.g. a symptom associated with a hepatic disorder in a subject, e.g., a patient, in need thereof. In some embodiments, such provided technologies may be used to treat a pulmonary disorder or a disorder e.g. a symptom associated with a hepatic disorder in a subject, e.g., a patient, in need thereof. In some embodiments, such provided technologies may be used to treat a neoplasia disorder e.g., a disorder or, a symptom associated with a neoplasia disorder in a subject, e.g., a patient, in need thereof. In some embodiments, such provided technologies may be used to treat a viral infection related disorder e.g. a disorder or a symptom associated with viral infection related disorder in a subject, e.g., a patient, in need thereof. In some embodiments, such provided technologies may be used to treat an alcohol misuse related disorder e.g. a disorder or a symptom associated with an alcohol misuse related disorder in a subject, e.g., a patient, in need thereof. In some embodiments, such provided technologies may be used to treat a neoplasia disorder associated with a viral infection or alcohol misuse, e.g., a disorder or a symptom associated with a neoplasia disorder that is associated with a viral infection or alcohol misuse in a subject, e.g., a patient, in need thereof.

In some embodiments, the condition treated is neoplasia. In some embodiments, the condition treated is tumorigenesis. In some embodiments, the condition treated is cancer. In some embodiments, the cancer is associated with poor prognosis. In some embodiments, the cancer is associated with MYC mis- regulation, e.g., MYC overexpression. In some embodiments, the cancer is associated with AFP mis- regulation, e.g., AFP overexpression. In some embodiments, the cancer is a breast, a hepatic, a colorectal, a lung, a pancreatic, a gastric, and/or a uterine cancer. In some embodiments, the cancer is associated with an infection, e.g., viral, e.g., bacterial. In some embodiments, the cancer is associated with alcohol abuse. In some embodiments, the cancer is hepatocarcinoma.

In some embodiments, the cancer cells are lung cancer cells, gastric, gastrointestinal, colorectal, pancreatic or hepatic cancer cells. In some embodiments, the cancer is hepatocellular carcinoma (HCC), Fibrolamellar Hepatocellular Carcinoma (FHCC), cholangiocarcinoma, Angiosarcoma, secondary liver cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, small cell lung cancer (SCLC), large cell (undifferentiated) carcinoma, triple negative breast cancer, gastric adenocarcinoma, endometrial carcinoma, or pancreatic carcinoma, hi some embodiments, the cancer is metastatic, advanced (non- resectable, or recurrent solid tumor types known for association with the MY C oncogene (e.g., including, but not limited to, hepatocellular carcinoma (HCC), bladder, lung, pancreatic, ovarian, uterine, endometrial, gastric, esophageal, hepatobiliary, colorectal cancer, and neuroblastoma), which has progressed on, relapsed after, are refractory to, or intolerant of standard of care or no alternative treatment exists.

In some embodiments, the cancer is HCC classified by the commonly used Barcelona Clinic Liver Cancer (BCLC staging system. The BCLC staging system classifies HCC patients into the following categories: very early stage (0), Early Stage (A), Intermediate Stage (B), Advanced Stage (C), and Terminal Stage (D), which guides management decisions. The BCLC classification is based on the extent of the tumor burden, the severity of liver disease (Child-Turcotte -Pugh classification (“C-P Score”)) and patient’s performance status.

Patients with early-stage (BCLC Stage 0 or A) may be managed by surgical resection of the tumor. Transarterial therapies are considered for patients who have intermediate stage (BCLC Stage B) HCC. Systemic therapies are recommended for patients who have advanced disease (BCLC Stage C) or have intermediate stage disease (BCLC Stage B) and have progressed following transarterial therapies. First-line systemic therapies include atezolizumab plus bevacizumab, sorafenib, or lenvatinib. Potential second-line therapies for patients who progress or are intolerant of first-line therapies include sorafenib, regorafenib, lenvatinib, ramucirumab, cabozantinib, and checkpoint inhibitors (pembrolizumab or nivolumab plus ipilimumab). Accordingly, in some embodiments, the subject to be treated with a therapy described herein is a subject that has progressed after receiving, has relapsed after, is refractory to, or is intolerant of, a therapy selected from atezolizumab plus bevacizumab, sorafenib, lenvatinib, sorafenib, regorafenib, lenvatinib, ramucirumab, cabozantinib, and checkpoint inhibitors (pembrolizumab or nivolumab plus ipilimumab). The optimal regimen after progression following first-line therapy is unknown. There are very limited options for patients with Terminal (Stage D) HCC, who receive supportive care only. The BCLC staging system takes into account the following variables:

• Tumor stage;

• Functional status of the liver;

• Physical status; and

• Cancer-related symptoms.

Five stages (0 and A through D) are identified based on the variables mentioned above.

The C-P Score is used to estimate the risk of mortality in patients with cirrhosis and consists of the following factors: Total bilimb in level, albumin, Prothrombin Time and INR, degree of ascites and the grade of hepatic encephalopathy. In some embodiments, the C-P Score is performed at patient screening, on Cycle Day 1 predose, every 6 weeks for the first year, every 3 months until disease progression, at tire end of treatment/early termination visit, and as clinically indicated. In some embodiments, the C-P Score is performed frequently in accordance with clinical practice if the participant develops hepatic adverse events of special interest until the AE resolves. Hepatic encephalopathy graded according to West Haven criteria for semi-quantitative grading of Mental Status: Grade 1: Trivial lack of awareness; euphoria or anxiety; shortened attention span; impaired performance of addition or subtraction; Grade 2: Lethargy or apathy; minimal disorientation for time or place; subtle personality change; inappropriate behavior; Grade 3: Somnolence to semi-stupor, but responsive to verbal stimuli. Confusion; Gross disorientation; Grade 4: Coma (unresponsive to verbal or noxious stimuli).

In some embodiments the condition treated is a hepatic disease. In some embodiments the condition treated is associated with MYC mis-regulation, e.g., MYC overexpression. In some embodiments the condition treated is a chronic disease. In some embodiments the condition treated is a chronic liver disease. In some embodiments the condition treated is a viral infection. In some embodiments, the condition treated is an alcohol misuse associated disorder.

In some embodiments the condition treated is a pulmonary disease. In some embodiments the condition treated is associated with MYC mis-regulation, e.g., MYC overexpression. In some embodiments the condition treated is a chronic disease. In some embodiments the condition treated is a chronic pulmonary disease. In some embodiments, such provided technologies may be used to treat or reduce lung cancer growth, metastasis, drug resistance, and/or cancer stem cell (CSC) maintenance. In some embodiments, the condition treated is a carcinoma, e.g., non-small cell lung cancer (NSCLC). In some embodiments, the chronic pulmonary disease is associated with tobacco misuse.

In some embodiments, the cancer hepatocarcinoma subtype SI (HCC SI), hepatocarcinoma subtype S2 (HCC S2), or hepatocarcinoma subtype S3 (HCC S2). In some embodiments, the HCC subtype is associated with MYC overexpression. In some embodiments, the cancer is HCC SI or HCC S2. In some embodiments, the cancer subtype is associated with aggressive tumor and poor clinical outcome.

In some embodiments, the disclosure provides a treatment regimen that may be devised for the subject on the basis of the HCC subtype in the subject, e.g., a personalized approach to tailor the aggressiveness of treatment based on HCC subtype on a subject. In some embodiments, the disclosure provides a method of treatment using the expression repressors or expression repressor systems disclosed herein, the method comprising, identifying the HCC subtype in a patient and determine a dosage and administration schedule of said expression repressors and/or expression repressor systems based on the HCC subtype identification.

Methods are described herein to deliver agents, or a composition as disclosed herein to a subject for treatment of a disorder such that the subject suffers minimal side effects or systemic toxicity in comparison to chemotherapy treatment. In some embodiments, the subject does not experience any significant side effects typically associated with chemotherapy, when treated with the agents and/or compositions described herein. In some embodiments, the subject does not experience a significant side effect including but not limited to alopecia, nausea, vomiting, poor appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, oral ulcers, itchy skin, peeling, nerve and muscle damage, auditory changes, weight loss, diarrhea, immunosuppression, bruising, heart damage, bleeding, liver damage, kidney damage, edema, mouth and throat sores, infertility, fibrosis, epilation, moist desquamation, mucosal dryness, vertigo and encephalopathy when treated with the agents and/or compositions described herein. In some embodiments, the subject does not show a significant loss of body weight when treated with the agents and/or compositions described herein.

The agents and compositions described herein can be administered to a subject, e.g., a mammal, e.g., in vivo, to treat or prevent a variety of disorders as described herein. This includes disorders involving cells characterized by altered expression patterns of MYC.

Epigenetic Modification

The present disclosure is further directed, in part, to a method of epigenetically modifying a target gene, a transcription control element operably linked to a target gene, or an anchor sequence (e.g., an anchor sequence proximal to a target gene or associated with an anchor sequence-mediated conjunction operably linked to a target gene), the method comprising providing an expression repressor (or nucleic acid encoding the same ) or an expression repression system (e.g., expression repressor(s)), or nucleic acid encoding the same or pharmaceutical composition comprising said an expression repressor (or nucleic acid encoding the same ) or expression repression system or nucleic acid; and contacting the target gene or a transcription control element operably linked to the target gene with the expression repressor or the expression repression system, thereby epigenetically modifying the target gene, e.g., MYC or a transcription control element operably linked to the target gene, e.g., MYC.

In some embodiments, a method of epigenetically modifying a target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC comprises increasing or decreasing DNA methylation of the target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC. In some embodiments, a method of epigenetically modifying a target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MY C comprises increasing or decreasing histone methylation of a histone associated with the target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC. In some embodiments, a method of epigenetically modifying a target gene, e.g., MY C or a transcription control element operably linked to a target gene, e.g., MYC comprises decreasing histone acetylation of a histone associated with the target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC. In some embodiments, a method of epigenetically modifying a target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC comprises increasing or decreasing histone sumoylation of a histone associated with the target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC. In some embodiments, a method of epigenetically modifying a target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC comprises increasing or decreasing histone phosphorylation of a histone associated with the target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC.

In some embodiments, a method of epigenetically modifying a target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC may decrease the level of the epigenetic modification by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to 100%) relative to the level of the epigenetic modification at that site in a cell not contacted by the composition or treated with the method. In some embodiments, a method of epigenetically modifying a target gene, e.g., MYC or a transcription control element operably linked to a target gene, e.g., MYC may increase the level of the epigenetic modification by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% (and optionally up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000%) relative to the level of the epigenetic modification at that site in a cell not contacted by the composition or treated with the method. In some embodiments, epigenetic modification of a target gene, e.g., MY C or a transcription control element operably linked to a target gene, e.g., MY C may modify the level of expression of the target gene, e.g., MYC, e.g., as described herein.

In some embodiments, an epigenetic modification produced by a method described herein persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or longer or any time therebetween. In some embodiments, such a modulation persists for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, or 7 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely). Optionally, such a modulation persists for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years.

In some embodiments, an expression repressor, or an expression repression system for use in a method of epigenetically modifying a target gene, e.g., MY C or a transcription control element operably linked to a target gene, e.g., MY C comprises an expression repressor comprising an effector moiety that is or comprises an epigenetic modifying moiety. For example, a effector moiety may be or comprise an epigenetic modifying moiety with DNA methyltransferase activity, and an endogenous or naturally occurring target sequence (e.g. a target gene, e.g., MYC or transcription control element) may be altered to increase its methylation (e.g., decreasing interaction of a transcription factor with a portion of target gene, e.g., MY C or transcription control element, decreasing binding of a nucleating protein to an anchor sequence, and/or disrupting or preventing an anchor sequence-mediated conjunction), or may be altered to decrease its methylation (e.g., increasing interaction of a transcription factor with a portion of a target gene, e.g., MYC or transcription control element, increasing binding of a nucleating protein to an anchor sequence, and/or promoting or increasing strength of an anchor sequence-mediated conjunction).

Kits

The present disclosure further directed, in part, to a kit comprising an expression repressor or an expression repression system, e.g., expression repressor(s), described herein, and an immune checkpoint inhibitor polypeptide (e.g., an antibody molecule or peptide). In some embodiments, a kit comprises an expression repressor or an expression repression system (e.g., the expression repressor(s) of the expression repression system), an immune checkpoint inhibitor polypeptide (e.g., an antibody molecule or peptide), and instructions for the use of said an expression repressor or expression repression system and the immune checkpoint inhibitor polypeptide (e.g., an antibody molecule or peptide). In some embodiments, a kit comprises a nucleic acid encoding the expression repressor or a nucleic acid encoding the expression repression system or a component thereof (e.g., the expression repressor(s) of the expression repression system), an immune checkpoint inhibitor polypeptide (e.g., an antibody molecule or peptide), and instructions for the use of said expression repressor (and/or said nucleic acid) and/or said expression repression system (and/or said nucleic acid) and the immune checkpoint inhibitor polypeptide (e.g., an antibody molecule or peptide). In some embodiments, a kit comprises a cell comprising a nucleic acid encoding the expression repressor or a nucleic acid encoding the expression repression system or a component thereof (e.g., the expression repressor(s) of the expression repression system) and instructions for the use of said cell, nucleic acid, and/or said expression repressor or expression repression system.

In some aspects, the kit comprises a) a container comprising a composition comprising a system comprising two expression repressors, comprising a first expression repressor comprising a first targeting moiety and optionally a first effector moiety, wherein the first expression repressor binds to a transcription regulatory element (e.g., a promoter or transcription start site (TSS)) operably linked to target gene, e.g., MYC or to a sequence proximal to the transcription regulatory element and an expression repressor comprising a second targeting moiety and optionally a second effector moiety, wherein the second expression repressor binds to a genomic locus located in a super enhancer region of a target gene, e.g., MYC.

In some embodiments the kit further comprises b) a set of instructions comprising at least one method for treating a disease or modulating, e.g., decreasing the expression of target gene, e.g., MYC within a cell with said composition. In some embodiments, the kits can optionally include a delivery vehicle for said composition (e.g., a lipid nanoparticle). The reagents may be provided suspended in the excipient and/or delivery vehicle or may be provided as a separate component which can be later combined with the excipient and/or delivery vehicle. In some embodiments, the kits may optionally contain additional therapeutics to be co-administered with the compositions to affect the desired target gene expression, e.g., MYC gene expression modulation. While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated. Such media include but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

In some embodiments, a kit comprises a unit dosage of an expression repressor an expression repression system, e.g., expression repressor(s), described herein, or a unit dosage of a nucleic acid, e.g., a vector, encoding an expression repression system, e.g., expression repressers), described herein.

The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of tire disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. EXAMPLES

Example 1. ZF9-MQ1 ZF3-KRAB downregulates PD-L1 expression in human HCC cell lines

The objective of this study is to determine the effect of ZF9-MQ1 ZF3-KRAB treatment on surface protein and mRNA PD-L1 expression of Interferon-y (IFN-y) stimulated Human HCC cell lines (Hep 3B and SK-HEP-1). MYC is known to regulate immunosuppression by increasing the expression of the immune checkpoint gene products CD47 and PD-L1 in multiple cancers by binding directly to their promoters, thereby preventing highly proliferative cancer cells from eliciting an immune response. MYC- driven cancer cells co- opt this mechanism to bypass immune detection. Inhibition of MYC therefore represents a mechanism for restoration of host immune response against tumors, as well as an approach to potentiating the effect of immune checkpoint inhibitors.

To assess this, all test articles were formulated using SSOP lipids for cellular delivery. Lipid nanoparticles (LNP) were formulated using the NanoAssemblr® Spark™ according to the steps outlined in the spark formulations protocol. Hep 3B cells and SK-HEP-1 cells were maintained in EMEM media with 10% FBS. They were passaged 1:3 when they reached 80% confluency. An 80% confluent flask was used to create a single cell suspension of Hep 3B or SK-HEP-1 cells, which were then plated in 12 well plates at 100,000 cells per well in growth media. Cells were treated in duplicate for surface PD-L1 expression analysis and mRNA analysis of PD-L1. Cells were exposed to treatment continuously for 48 hours with ZF9-MQ1 ZF3-KRAB or short non-coding mRNA at 1 iig/ml. A subset of cells was also left untreated as a negative control. Following 24 hours of incubation period with ZF9-MQ1 ZF3-KRAB or non-coding mRNA treatment, cells were stimulated with human IFN-y at 50 ng/ml. Following 24-hour stimulation with IFN-y (total 48 hours incubation with ZF9-MQ1 ZF3-KRAB or short non-coding mRNA), cells in one plate were harvested for flow cytometry analysis of surface PD-L1 expression (antiHuman PD-L1 antibody (BV711)) while the second plate was lysed with RLT Plus Lysis buffer for mRNA extraction using RNeasy Plus 96 Kit. The lysed sample was bound to an RNA column, washed with buffers, and eluted off the column with RNAse free water. Total RNA was then converted to cDNA with RT Lunascript. The cDNA was then analyzed through ACT qPCR with a MYC (target) or PD-L1 (target) and GAPDH (reference) probe.

Surface PD-L1 protein expression (flow cytometry) and PD-L1 and MYC mRNA expression levels (RT-PCR) were assessed 48 hrs after ZF9-MQ1 ZF3-KRAB (DS, HA-tagged; MR-30723) or negative control mRNA (short non-coding mRNA; MR-30626) treatment delivered via an SSOP LNP. After 24-hours treatment with ZF9-MQ1_ZF3-KRAB or negative control mRNA the cells were stimulated with 50 ng/ml of IFN-y for 24 hours. Treatment with ZF9-MQ1_ZF3-KRAB down -regulated the surface protein expression as well as the mRNA expression levels of PD-L1 that was induced by IFN-y treatment in Hep 3B and SK-HEP-1 cells (Figs. 2A-2D). Treatment with ZF9-MQ 1_ZF3-KRAB down-regulated the mRNA expression levels of MYC independent of IFN-y treatment of HCC cell lines. This result suggests reducing PD-L1 ligand expression will allow the host immune cells to selectively kill tumors cells and prevent immunosuppression within the tumor microenvironment. These data suggest PD-L1 inhibition through ZF9-MQ1 ZF3-KRAB in combination with anti-PD-1 treatment should further add to the anti-tumor effect in HCC.

Example 2. 48 h treatment of ZF9-MQ1 ZF54-KRAB reduces MYC mRNA level and cell surface PD-L1 expression in human non small cell lung cancer cell lines

In this example, MYC mRNA levels and cell surface PD-L1 expression levels were accessed in cells from the human lung cancer cell lines H2009 or H460 after treatment with PBS (untreated), or 1 pg/ml of scrambled RNA (control) or MR-32054 (bicistronic mRNA that encodes ZF9-MQ1 ZF54- KRAB) formulated in LNP containing disulfanediylbis(ethane-2,l-diyl)bis(piperidine-l,4- diyl)bis(ethane-2,l-diyl)bis(oxy)bis(2-oxoethane-2,l-diyl)bis(4,l -phenylene) dioleate (SS-OP) for 24 h followed by treatment with vehicle (no IFNy) or 10 ng/ml IFNy (IFNg) for an additional 24 h. qRT-PCRto assess MYC mRNA levels: whole cell RNA was processed to make complementary DNA (cDNA) (using a poly-A primer) that was then used for reverse transcription polymerase chain reaction (RT-PCR) analysis using Taqman probes (Thermo Fisher) specific for human MYC mRNA transcripts. GAPDH mRNA transcript levels were used for normalization across groups.

Flow cytometry to assess PD-L1 surface expression levels: cells were stained anti-PD-Ll antibody molecule or isotype control conjugated with BV711 (Biolegend) with LIVE/DEAD™ Fixable Aqua (Invitrogen). Flow cytometry (Aurora cytek immunocytometer) was used to capture PD-L1 expression in live cells. The median intensity of surface PD-L1 level was analyzed using FlowJo software (BD).

These data show that 48 h treatment of MR-32054 reduces MY C mRNA level and cell surface PD-L1 expression with or without IFNy treatment during the last 24h of experiment in H2009 cells (Figs. 3A and 3B) (MYC: -97% downregulation with or without IFNy, PD-L1: 73% or 48% downregulation with or without IFNy compared to untreated in that condition, respectively). These data also show that 48 h treatment of MR-32054 reduces MYC mRNA levels and cell surface PD-L1 expression with or without IFNy treatment during the last 24h of experiment in H460 cells (Figs. 4A and 4B) (MYC: 95% or 87% downregulation with or without IFNy, respectively, PD-L1 : 59% or 12% downregulation with or without IFNy compared to untreated in the same condition, respectively. Example 3. ZF17-MQ1 decreases MYC protein and mRNA production while increasing MYC promoter methylation in a mouse HCC cell line

This study demonstrates the use of ZF17-MQ1 in a model of hepatocellular carcinoma. In this example, the mouse hepatocellular carcinoma cell line HEPA1-6 was used to evaluate expression repressors targeted to the MYC IGD in mouse. Expression repressors were developed as surrogates to ZF9-MQ1 ZF3-KRAB that target the mouse genome and evaluated for ability to downregulate MYC mRNA and protein levels and reduce mouse HCC cell viability.

A panel of mouse surrogate expression repressors were generated and screened in HEPA1-6 cells by seeding 10,000 cells per well in duplicate plates for mRNA or viability analysis. The 96-well plates were treated in triplicate with the candidate expression repressors at 0.6 or 1.2 pg/mL. Cells were incubated 72 hrs. Following the incubation period, one 96-well plate was lysed with Cell-Titer Gio and luminescence was quantified using the Gio Max where high luminescence values indicates high cell viability and low values indicate low cell viability. The second 96-well plate was lysed with RLT Plus Lysis buffer for mRNA extraction using RNeasy Plus 96 Kit. The lysed sample was bound to an RNA column, washed with buffers, and eluted off. mRNA was then converted to cDNA with RT Lunascript. cDNA was then analyzed through A ACT qPCR with a MY C (target) and GAPDH (reference) probe. This screen indicated that ZF 17-MQ 1 was a possible expression repressor for targeting MY C in the mouse IGD. RNA and Viability analysis was repeated for ZF17-MQ1 at 96 hrs and MYC protein levels were analyzed by transfecting 100,000 cells in a 12 well plate with ZF17-MQ1 or control GFP mRNA. Cells were incubated for 24 or 48 hrs. Cells were then lysed in RIPA buffer and protein levels quantified using the Pierce BCA protein assay (23225). Equal amounts of protein were loaded for each sample and separated by size using the Invitrogen NuPAGE mini gel system. Protein was then transferred to PVDF membrane using the Invitrogen IBlot2 gel transfer device. Membranes were probed overnight with anti- MYC antibody (ABCAM, ab32072). Anti-ACTIN antibody (Cell Signaling, 8H10D10) was used as a loading control. Signal was then visualized and quantified using the LICOR imaging system using fluorescent secondary antibodies to the MYC and ACTIN antibody species.

Quantitative methylation PCR (qMSP) was used to determine on-target methylation of tire MY C promoter by ZF17-MQ1. Cells were treated with ZF17-MQ1 for 24 or 48 hrs and DNA was prepared using Qiagen DNeasy kit. DNA was then bisulfite converted using the ZYMO gold conversion kit. Converted DNA was then amplified using PCR primers specific to methylated MY C promoter DNA. This signal was quantified using the AACT relative to an ACTIN house keeping control and a positive 100% methylated control. This study found significant increase in methylation signal following treatment with ZF17-MQ1. Treatment with ZF17-MQ1 resulted in a significant decrease in MYC mRNA levels in HEPA1-6 cells (Fig. 5A). ZF17-MQ1 treatment also triggered an increase in methylation of the MYC promoter in these cells (Fig. 5B).

This study found that ZF17-MQ1 targets the mouse MYC IGD. ZF17-MQ1 treatment in mouse HCC cells HEPA1-6 show significant downregulation of MYC mRNA, cell viability and MYC protein levels through an increase in MY C promoter methylation.

Example 4. ZF17-MQ1 reduces growth of subcutaneously-grafted HCC tumor cells in a mouse syngeneic model

In this example, disease was induced in female C57/BL6 by the implantation of HEPA1-6 tumor cells into the left flank. Treatment was initiated when mean tumor volume reached approximately 200 mm³. Mice were divided into treatment groups (7-8 mice each) so that mean tumor volume in each group was approximately equal. Mice were injected intravenously with PBS, ZF17-MQ1 at 3 mg/kg or the positive control standard of care drug sorafenib at 50 mg/kg (every day). ZF17-MQ1 was dosed every 5 days for 4 doses then given a 2 week drug holiday and provided two further doses. All animals were weighed daily and assessed visually. Tumor size was measured 3 times per week.

The results show that ZF 17 -MQ 1 significantly reduces animal tumor burden and, following a drug holiday, re-dosing the animals resulted in full tumor depletion. These data show that expression repressors can effectively reduce tumor burden in HCC tumor grafts done in immune-competent animals.

Example 5. The combination of expression repressors plus anti-PD-1 antibody molecules or anti- PD-L1 antibody molecules shows benefit and tolerability in reducing tumor burden of subcutaneously-grafted HCC tumor cells in mice

In this example, the ability of ZF17-MQ1 to reduce in vivo Hepal-6 tumor burden in combination with immune checkpoint blockers anti-PD-1 antibody molecules and anti-PD-Ll antibody molecules was tested.

In this example, disease was induced in ninety-two (92) female C57B/6mice by the implantation of Hepa 1-6 tumor cells into the left flank. Treatment was initiated when mean tumor volume reaches approximately 100-150 mm³. Mice were be divided into treatment groups so that mean tumor volume in each group in approximately equal. Mice were injected TV, IP or PO with test article. All animals were weighed daily and assessed visually. Tumors size was measured on Mondays, Wednesdays and Fridays or 3 times per week. In this study, negative control GFP mRNA and test article ZF17-MQ1 mRNA (MR- 30296) were evaluated for their effect on tumor growth using the Hepal .6 Hepatocellular Carcinoma subcutaneous graft model in B6 mice by dosing at 1 mg/kg Q5D. Test agents were evaluated alone and in combination with anti-PD-1 or PD-L1 immune checkpoint inhibitor antibody molecules (bioXcell clones RMP1-14 and 10F.9G2, respectively, dosed once per week at 10 mg/kg).

Treatment with ZF17-MQ1 at 1 mg/kg significantly reduced tumor growth relative to PBS controls and negative control GFP mRNA (p<0.0001). Treatment with anti-PD-1 therapy at 10 mg/kg significantly reduced tumor burden relative to PBS (p=0.0002) and GFP controls (p<0.0001). Treatment with anti-PD-Ll therapy at 10 mg/kg significantly reduced tumor burden relative to PBS (p=0.0001) and GFP controls (p<0.0001). The combination ofZF17-MQl with anti-PD-1 or anti-PD-Ll therapy significantly reduced tumor burden relative to PBS (p=0.0001) and GFP controls (p<0.0001) (Fig. 6A). Of note, the combination of ZF17-MQ1 with anti-PD-1 reduced tumor burden compared to treatment with either agent alone, and the combination of ZF17-MQ1 and anti-PD-Ll reduced tumor burden relative to treatment with either agent alone. All combinations were well tolerated by animal body weights (Fig. 6B).

These results show that combination therapies that include an epigenetic regulator of MY C and an immune checkpoint inhibitor display superior efficacy at curbing growth of liver-derived tumors in comparison to therapies in which either class of agent is provided individually.

Example 6. The combination of expression repressors plus anti-PD-1 antibody molecules or anti- PD-Ll antibody molecules shows benefit and tolerability in reducing tumor burden of subcutaneously-grafted colon adenocarcinoma cells in mice

In this example, the ability of ZF17-MQ1 in combination with immune checkpoint inhibitor antibody molecules anti-PD-1 and anti-PD-Ll to reduce in vivo tumor burden resulting from subcutaneous graft of MC-38 cells, a murine colon adenocarcinoma cell line, was tested in mice.

In this example, disease was induced in eighty-two (82) female C57B/6mice by the implantation of MC-38 tumor cells into the left flank. Treatment was initiated when mean tumor volume reached approximately 100-150 mm³. Mice were divided into treatment groups so that mean tumor volume in each group was approximately equal. Mice were injected IV, IP or PO with test article. All animals were weighed daily and assessed visually. Tumor size was measured on Mondays, Wednesdays and Fridays or 3 times per week, hr this study, negative control GFP mRNA and test article ZF17-MQ1 mRNA (MR- 30296) were evaluated for their effect on tumor growth using the MC38 subcutaneous graft model in B6 mice by dosing at 3 mg/kg Q5D. Test agents were evaluated alone and in combination with anti-PD-1 or PD-L1 therapeutics (dosed twice per week at 10 mg/kg).

Treatment with ZF17-MQ1 at 3 mg/kg significantly reduced tumor growth relative to PBS controls (p=0.0003). Treatment with anti-PD-1 therapy at 10 mg/kg significantly reduced tumor burden relative to PBS (p<0.0001) and GFP controls (p=0.0348). Treatment with anti-PD-Ll therapy at 10 mg/kg significantly reduced tumor burden relative to PBS (p<0.0001) and GFP controls (p=0.0126). Of note, the combination of ZF17-MQ1 with anti-PD-1 therapy significantly reduced tumor burden vs. ZF17-MQ1 alone (p=0.0050) and anti-PD-1 alone (p=0.0028) (Fig. 7A). All combinations were well tolerated by animal body weights (Fig. 7B).

These results indicate that the combination therapies that include an epigenetic regulator of MYC and an immune checkpoint inhibitor display superior efficacy at curbing growth of colon-derived tumors in comparison to therapies in which either class of agent is provided individually. Taken together with the results from the previous example, these results suggest that such combination therapies may be effective for halting growth of a wide variety of tumor types.

Example 7. Combination of ZF17-MQ1 and checkpoint inhibitor antibodies in mice bearing subcutaneously-grafted Lewis-lung carcinoma cells inhibits tumor growth to a greater extent than monotherapy

This example demonstrates the use of expression repressors in combination with immune checkpoint inhibitor antibody molecules in an animal model for lung cancer.

C56B16/J mice bearing subcutaneous Lewis-lung carcinoma (LLC1) tumors were treated with PBS (vehicle), ZF17-MQ1 (MR-30296, formulated in MC3), GFP mRNA (formulated in MC3), anti-PD- 1 (RMP1-14) antibody molecule, anti-PD-Ll antibody molecule (10F.9G2), a combination of ZF17-MQ1 + anti-PD-1, or a combination of ZF17-MQ1 + anti-PD-Ll, and tumor growth was measured (Fig. 8 and 9). PBS and LNPs were administered intravenously, and antibodies were administered intraperitoneally. Mouse body weights were also measured (Fig. 10).

Dose/regimen: ZF17-MQ1, GFP LNP, and PBS were dosed at 3 mg/kg, Q5D for 6 administrations. Antibodies were administered at 10 mg/kg at Q3D for 3 weeks. Body weights were also measured.

Tumor measurements: The length and width of tumors were measured twice weekly. Tumor volumes were calculated as width² x length/2. Body weights were measured daily.

These results show that ZF17-MQ1 treatment inhibits tumor growth in the LLC1 subcutaneous tumor model. Hie average tumor volumes at day 22 when PBS-treated mice were coming off study were 2005 mm³, 1289 mm³, 2077 mm³, 1025 mm³, 1315 mm³, 843 mm³, 724 mm³ in PBS, ZF17-MQ1, GFP mRNA, anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, ZF17-MQ1 + anti-PD-1, and ZF17- MQ1 + anti-PD-Ll groups, respectively. Although average tumor volumes in ZF17-MQ1 treated mice were indistinguishable from either anti-PD-1 or anti-PD-Ll -treated mice, ZF17-MQ1 combined with either anti-PD-1 or anti-PD-Ll were significantly different from either ZF17-MQ1, anti-PD-1, or anti-PD- LI treatments indicating that combination therapy exhibits activity to a greater extent than monotherapy in this model (Figs. 8-10).

These results demonstrate that in comparison to the monotherapies, ZF17-MQ1 in combination with anti-PD-1 antibody molecule or anti-PD-Ll antibody molecule was more effective in inhibiting tumor growth in the in vivo LLC1 subcutaneous tumor model.

Example 8. Combination of ZF17-MQ1 and checkpoint blocking antibodies in mice bearing subcutaneously-grafted colon carcinoma cells inhibits tumor growth to a greater extent than monotherapy

This example demonstrates the use of expression repressors in combination with immune checkpoint inhibitor antibody molecules in a mouse model for colon cancer.

C56B16/J mice bearing subcutaneous CT26 tumors were treated with PBS (vehicle), ZF17-MQ1 (MR-30296 formulated in MC3), GFP mRNA (formulated in MC3), anti-PD-1 (RMP1-14) antibody molecule, anti-PD-Ll antibody molecule (10F.9G2), a combination of ZF17-MQ1 + anti-PD-1, or a combination of ZF17-MQ1 + anti-PD-Ll, and tumor growth was measured (Fig. 11 and 12). PBS and LNPs were administered intravenously, and antibodies administered intraperitoneally. Mouse body weights were also measured (Fig. 13).

Dose/regimen: ZF17-MQ1, GFP LNP, and PBS were dosed at 3 mg/kg, Q5D for 6 administrations. Antibodies were administered at lOmg/kg at Q3D for 3 weeks. Body weights were also measured.

Tumor measurements: The length and w idth of tumors were measured twice weekly. Tumor volumes were calculated as width² x length/2. Body weights were measured daily.

These results show that ZF17-MQ1 treatment inhibits tumor growth in the CT26 subcutaneous tumor model. The average tumor volumes when PBS-treated mice were coming off of the study were 2169 mm³, 1503 mm³, 2093 mm³, 1375 mm³, 1203 mm³, 907 mm³, 832 mm³ in PBS, ZF17-MQ1, GFP mRNA, anti-PD-1 antibody molecule, anti-PD-Ll antibody molecule, ZF17-MQ1 + anti-PD-1 antibody molecules, and ZF17-MQ1 + anti-PD-Ll antibody molecules groups, respectively. Although average tumor volumes in ZF17-MQ1 treated mice were indistinguishable from either anti-PD-1 antibody molecules or anti-PD-Ll antibody molecules treated mice, ZF17-MQ1 combined with either anti-PD-1 antibody molecules or anti-PD-Ll antibody molecules were significantly different from either ZF17- MQ1, anti-PD-1 antibody molecules, or anti-PD-Ll antibody molecules treatments indicating that combination therapy exhibits activity to a greater extent than monotherapy in this model (Figs. 9-11). These results demonstrate that in comparison to the monotherapies, ZF17-MQ1 in combination with anti-PD-1 antibody molecule or anti-PD-Ll antibody molecule was more effective in inhibiting tumor growth in the in vivo CT26 SQ tumor model.

Example 9. MYC Overexpression is Associated with Decreased Survival Probability with Pembrolizumab treatment in NSCLC

Analysis of human patient NSCLC tumor data from Tempus labs. Biopsies from tumor samples were analyzed for MYC mRNA levels and compared to data reporting on time to progression. Figure 14 shows a decrease in survival probability of patients with over-expressed MY C compared to underexpressed. These results suggest that patients with higher MYC mRNA levels have poorer survival on Pembrolizumab (trending different).

Example 10. Bicistronic ZF9-MQ1 ZF3-KRAB dose monotherapy

Participants will be considered evaluable for a dose limiting toxicity (DLT) if they received at least 1 dose of bicistronic ZF9-MQ1 ZF3-KRAB and completed the 28-day DLT observation period (unless the participant does not complete the DLT period because he or she experiences a Grade 5 toxicity prior to the end of the DLT period, in which case they will remain evaluable for DLT).

After providing informed consent, participants will undergo screening assessments (up to 28 days prior to enrollment).

Once enrolled in the study, Bicistronic ZF9-MQ1 ZF3-KRAB will be administered via intravenous infusion once every 2 weeks (Day 1 and Day 15 of each 28-day cycle). Bicistronic ZF9- MQ1 ZF3-KRAB will be an LNP aqueous emulsion comprising a unit dose of 5 mg/5 ml (1 mg/mL concentration) for intravenous administration. For the first dose level in Part 1, premedication will be administered prior to each dose of bicistronic ZF9-MQ 1 ZF3-KRAB at least 60 minutes prior to the start of infusion including, but not limited to, oral acetaminophen (e.g., 500 mg), intravenous Hl blocker (e.g., diphenhydramine, e.g., 50 mg, or equivalent), and intravenous corticosteroids (e.g., dexamethasone, e.g., 10 mg, or equivalent). For dose level 2 and beyond, the same premedication is required, with the exception of intravenous corticosteroids (e.g., dexamethasone, e.g., 10 mg, or equivalent), which may be added at the Investigator’s discretion after discussion with the Sponsor Medical Monitor.

The first dose of bicistronic ZF9-MQ1_ZF3-KRAB will be administered in the hospital setting during dose escalation. A sentinel dosing strategy will be used during dose escalation. Dose escalation will begin at 0.02 mg/kg and is planned to proceed to the provisional dose levels of 0.05 mg/kg, 0.08 mg/kg, 0.125 mg/kg, and 0.15 mg/kg (approximately 5 dose levels total). After the second cycle is completed, individual participants may be considered for treatment at a dose of bicistronic ZF9-MQ1 ZF3-KRAB higher than the dose to which they were assigned. The participant must not have experienced any Common Terminology Criteria for Adverse Events (CTCAE) Grade >2 adverse event at the initial dose level assigned for at least 2 cycles of therapy in order to be treated at a higher dose. Intermediate dose levels within the proposed range may be used based upon pharmacokinetics, pharmacodynamics, clinical observations, and/or safety observations. Any intermediate dose levels will be lower than the next planned dose escalation level. The MTD is the dose level at which < 1/6 participants experience a DLT, or the highest dose level tested if DLTs are not observed at any of the dose levels.

Tumor imaging will be performed by computed tomography (CT) scan with contrast or magnetic resonance imaging (MRI) at baseline and every 6 weeks (± 1 week) for the first year, then every 3 months (± 3 weeks) until disease progression. In addition, if disease progression is suspected due to clinical or symptomatic deterioration, tumor imaging will be performed to confirm disease progression. Responses will be assessed according to modified Response Evaluation Criteria in Solid Tumors (mRECIST) for HCC and RECIST 1.1 for non-HCC solid tumor participants based on Investigator assessment.

Bicistronic ZF9-MQ1 ZF3-KRAB will be administered until disease progression, unacceptable toxicity (including DLT), withdrawal of consent, Investigator decision to withdraw the participant, death, start of subsequent treatment, or up to 2 years, whichever comes first. A participant may continue study treatment after confirmation of disease progression as long as the participant is deriving clinical benefit, as judged by the Investigator.

Participants who achieve a complete response (CR) by 2 tumor imaging assessments conducted at least 4 weeks apart may discontinue study treatment at the Investigator’s discretion after confirmation of the CR.

Disease status for participants who discontinue study drugs due to reasons other than disease progression will continue to be monitored every 12 weeks until start of new anticancer therapy, disease progression, withdrawal of consent, death or end of study, whichever comes first. Participants will be followed for OS until death, withdrawal of consent, up to 2 years, or end of the study, whichever comes first.

Safety assessments will include the incidence, severity of adverse events (including SAEs), laboratory abnormalities, electrocardiogram (ECG) changes, cytokines, C-reactive protein (CRP), and complement. Plasma samples will be collected to assess PK. Blood samples and tumor biopsy samples (if available) may be collected.

Adverse events will be graded according to the National Cancer Institute (NCI) CTCAE version 5.0. Progression of the underlying disease will not be considered an AE unless considered related to tire study drug by the Investigator. Adverse events will be collected from the time of signing informed consent until 30 days after last administration of study drug or initiation of new anticancer therapy, whichever occurs first. After stopping study drug, adverse events that are >Grade 1 will be followed until resolution of the AE to < Grade 1 or baseline or until the start of new anticancer therapy, whichever occurs first.

Example 11. Combination of bicistronic ZF9-MQ1 ZF3-KRAB and a checkpoint inhibitor for treatment of a subject

Participants will receive bicistronic ZF9-MQ1_ZF3-KRAB via intravenous infusion every 2 weeks (Day 1 and Day 15 of each 28 days/4-week cycle). The checkpoint inhibitor will be taken starting on Cycle 1 Day 1 . On the same days bicistronic ZF9-MQ1 ZF3-KRAB is administered, the checkpoint inhibitor is administered at least 1 hour and preferably less than 3 hours after completion of the bicistronic ZF9-MQ1 ZF3-KRAB infusion.

In some embodiments, the checkpoint inhibitor comprises pembrolizumab. Pembrolizumab will be administered as an IV infusion of 400 mg every 6 weeks over 30 minutes (- 5/+ 10 minutes). On days when bicistronic ZF9-MQ1 ZF3-KRAB and pembrolizumab are both administered, pembrolizumab infusion will be administered at least 1 hour and optionally less than 3 hours after completion of the bicistronic ZF9-MQ1 ZF3-KRAB infusion.

In some embodiments, the checkpoint inhibitor comprises atezolizumab. In certain embodiments, atezolizumab may be administered as an IV infusion of 840 mg every 2 weeks over about 30 minutes or about 60 minutes. In some embodiments, atezolizumab may be administered as an IV infusion of 1200 mg every 3 weeks over about 30 minutes or about 60 minutes. In some embodiments, atezolizumab may be administered as an IV infusion of 1650 mg every 4 weeks over about 30 minutes or about 60 minutes. On days when bicistronic ZF9-MQ1 ZF3-KRAB and atezolizumab are both administered, atezolizumab infusion will be administered at least 1 hour and optionally less than 3 hours after completion of the bicistronic ZF9-MQ1 ZF3-KRAB infusion.

Similar to Example 10, for the first dose level in Part 2, premedication will be administered prior to each dose of bicistronic ZF9-MQ1 ZF3-KRAB at least 60 minutes prior to tire start of infusion including, but not limited to, oral acetaminophen (e.g., 500 mg), intravenous Hl blocker (e.g., diphenhydramine, e.g., 50 mg, or equivalent), and intravenous corticosteroids (e.g., dexamethasone, e.g., 10 mg, or equivalent). For dose level 2 and beyond, the same premedication is required, with the exception of intravenous corticosteroids (e.g., dexamethasone, e.g., 10 mg, or equivalent), which may be added at the Investigator’s discretion after discussion with the Sponsor Medical Monitor. During the safety run-in, the first dose of bicistronic ZF9-MQ1 ZF3-KRAB will be administered in the hospital and participants will be observed for at least 24 hours after their initial infusion and then discharged home if clinically stable at the Investigator’s discretion. Subsequent doses (post-initial dose) for a given participant during the safety run-in and all doses during dose expansion may be administered as an outpatient in an infusion center, and the participant may be discharged home if clinically stable during and after the infusion. Enrollment and treatment of all 3 participants in the safety run-in may occur simultaneously.

Tumor imaging will be performed by CT scan with contrast or MRI every 6 weeks (±1 week) for the first year, then every 3 months (± 3 weeks) until disease progression. In addition, if disease progression is suspected due to clinical or symptomatic deterioration, tumor imaging will be performed to confirm disease progression. Administration of study drugs will continue until disease progression, unacceptable toxicity (including DLT), withdrawal of consent, Investigator decision to withdraw participant, death, start of subsequent treatment, or up to 2 years, whichever comes first. Participants who achieve a CR by 2 tumor imaging assessments conducted at least 4 weeks apart may discontinue study treatment at the Investigator’s discretion after confirmation of the CR. A participant may continue study treatment after confirmation of disease progression as long as the participant is deriving clinical benefit, as judged by the Investigator.

Disease status for participants who discontinue study drags due to reasons other than disease progression will continue to be monitored every 12 weeks (± 3 weeks) until start of new anticancer therapy, disease progression, withdrawal of consent, death or end of study, whichever comes first. Participants will be followed for OS until death, withdrawal of consent, up to 2 years, or end of the study, whichever comes first.

Adverse events will be collected from the time of signing informed consent until 30 days after the last administration of study drags or initiation of new anti cancer therapy, whichever comes first.

Example 12. Combination of ZF17-MQ1 and checkpoint blocking antibodies in Hepal-6 syngeneic mouse model of hepatocellular carcinoma (HCC) to assess infiltrating immune cells in the tumor microenvironment using flow cytometry.

This example demonstrates the use of expression repressors (ZF17-MQ1) in combination with an immune checkpoint inhibitor antibody (anti-PD-1 antibody molecule) in a syngeneic mouse model of HCC (Hepal-6) to assess changes in the infiltrating immune cell populations.

C57B/6 mice bearing subcutaneous Hepal-6 tumors were treated with PBS (vehicle control), ZF17-MQ1 (MR-30296 formulated in MC3), GFP mRNA (formulated in MC3), anti-PD-1 (clone RMP1- 14) antibody molecule, or a combination of ZF17-MQ1 + anti-PD-1 antibody molecule. PBS, GFP mRNA, or ZF17-MQ1 were administered intravenously, and the anti-PD-1 antibody molecule was administered intraperitoneally. GFP-mRNA and ZF17-MQ1 mRNA were administered at 2 mg/kg, Q5D for 4 doses. Anti-PD-1 antibody molecule was administered at 10 mg/kg, QW for 3 doses. Tumor growth was measured three times per week (Fig. 15A). Mouse body weights were also measured daily (Fig. 15B). These results show that ZF17-MQ1 alone, or in combination with an anti-PD-1 antibody molecule, inhibit tumor growth in the Hepal-6 subcutaneous tumor model when compared to PBS or GFP mRNA treatment.

On day 16 after initiation of treatment the mice were sacrificed and tumors were collected and dissociated to measure the tumor immune cell profiling using flow cytometry (Figs. 16A-16H). Treatment with ZF17-MQ1, the anti-PD-1 antibody molecule, or the combination of ZF17-MQ1 + anti-PD-1 antibody molecule, increased CD8 T-cells overall when compared to PBS treated control (Fig. 16D). In addition, the anti-PD-1 antibody molecule or the combination of ZF17-MQ1 + anti-PD-1 antibody molecule, increased overall T cell count (Fig. 16B). ZF17-MQ1 significantly reduced the percentage of inhibitory regulatory T cells (Tregs) infiltrating the tumor as compared to PBS controls (Fig. 16E). In addition, the combination of ZF17-MQ1 with anti-PD-1 antibody molecule significantly reduced the induction of Tregs observed with anti-PD-1 antibody molecule alone (Fig. 16E). This increase in inhibitory Tregs leads to incomplete effectiveness of, and potential resistance to, the anti-PD-1 antibody molecule. These data show ZF17-MQ1 was able to significantly increase the ratio of CD8+ T-cells to Tregs or the ratio of activated CD8+ T-cells to Tregs (Figs. 16G and 16H). These results demonstrate ZF17-MQ1 alone, or in combination with anti-PD-1 antibody molecule, was able to engage cytotoxic T- cells while repressing inhibitory Tregs to more effectively enlist the adaptive immune system to inhibit Hepal-6 tumor growth.

Example 13. Immune cell depletion in combination with ZF17-MQ1 and checkpoint blocking antibodies in Hepal-6 syngeneic mouse model of hepatocellular carcinoma (HCC) to assess contributions of adaptive and innate immunity to tumor growth inhibition.

This example demonstrates the use of expression repressors (ZF17-MQ1) in combination with an immune checkpoint inhibitor antibody molecule (anti-PD-1) in a Hepal-6 syngeneic mouse model of HCC. To assess the contribution of immune cells on tumor growth inhibition of ZF17-MQ 1 alone, or in combination the anti-PD-1 antibody molecule, the syngeneic mouse models were T-cell or NK (natural killer) cell depleted with anti-CD4 antibody, anti-CD8 antibody, or natural killer 1.1 antibody.

Eighty (80) female C57BL/6 mice were inoculated subcutaneously into the left flank with IxlO⁷ Hepal-6 cells. Treatment was initiated when the tumors reached a mean volume of 156.7 mm³ (standard deviation ± 49.6 mm³ range 48.1-372.3 mm³). C57B/6 mice bearing subcutaneous Hepal-6 tumors were left untreated (control) or pre-treated with anti-CD4 (clone GK1.5), anti-CD8a (clone 2.43), or anti- NK1.1 (clone PK136) antibodies twice (400 pg Q5Dx2). Following pre-treatment, the mice (untreated and pre-treated) were then split into 10 subgroups of 8 mice each. The study was performed as follows: Untreated (control) mice were administered with ZF17-MQ1 (MR-30296 formulated in MC3) (Group 1), GFP mRNA (formulated in MC3) (Group 2), anti-PD-1 (clone RMP1-14) antibody molecule (Group 3), or a combination of ZF17-MQ1 + anti-PD-1 antibody molecule (Group 4); anti-CD8a pre-treated groups were administered with ZF17-MQ1 (Group 5) or ZF17-MQ1 + anti-PD-1 antibody molecule (Group 6); anti-CD4 pre-treated groups were administered with ZF17-MQ1 (Group 7) or ZF17-MQ1 + anti-PD-1 antibody molecule (Group 8); and anti-NKl. l pre-treated groups were administered with ZF17-MQ1 (Group 9) or ZF17-MQ1 + anti-PD-1 antibody molecule (Group 10). GFP mRNA and ZF17-MQ1 mRNA were administered intravenously at 2 mg/kg Q5D for 4 doses. Anti-PD-1 antibody molecule was administered intraperitoneally at 10 mg/kg Q5D for 4 doses. Tumor growth was measured three times per week.

These results showed that ZF17-MQl administered in combination with an anti-PD-1 antibody molecule was able to eliminate the tumors in all eight mice more effectively and quicker than either treatment with ZF17-MQ1 or anti-PD-1 antibody molecule alone. Notably , no regrowth of eliminated tumors was seen in any of the mice with eliminated tumors at study termination. Treatment with ZF17- MQ 1 after pre-treatment with anti-CD4 or anti-CD 8a was not as effective as ZF 17 -MQ 1 alone or ZF 17- MQ1 + anti-NKl .l (Fig. 17A). The data from a single outlier mouse is not shown for the ZF17-MQ1 group, indicated by a “star” within Fig. 17A. Similarly, pre-treated subgroups administered ZF17-MQ1 in combination with anti-PD-1 antibody molecule demonstrated greater efficacy in the anti-NKl .1 pretreatment group than the anti-CD4 or anti-CD8a group (Fig. 17B). These data show that efficacy of ZF17-MQ1, either alone or in combination with an anti-PD-1 antibody molecule, may be partially driven through adaptive, but not innate, immunity. These data agree with the tumor infiltrating lymphocyte data in Example 12.

Example 14. Assessment of immune memory of ZF17-MQ1 using Hepal-6 mouse syngeneic model of hepatocellular carcinoma (HCC).

This example demonstrates the use of expression repressors (ZF17-MQ1) alone or in combination with immune checkpoint inhibitor antibody (anti-PD-1) in a syngeneic mouse model of HCC, Hepal-6 generated immune memory to Hepal-6.

C57B/6 mice bearing subcutaneous Hepal-6 tumors were divided into 4 groups (20 mice each for treatment groups and 10 mice for PBS control group). Treatment groups received ZF17-MQ1 (MR- 30296 formulated in MC3; 3 mg/kg IV Q5Dx6), anti-PD-1 antibody molecule (clone RMP1-14; 10 mg/kg IP BIWx4), or a combination ofZF17-MQl plus anti-PD-1 antibody molecule. Groups administered anti-PD-1 antibody molecule alone, and combination treatments (ZF17-MQ1 plus anti-PD-1 antibody molecule) eliminated 20/20 tumors while ZF17-MQ1 eliminated 14/20 tumors after approximately 30 days of treatment (Fig. 18A). The treatment regimens were well tolerated over the course of the study, as seen by change in percent body weight (BW) over baseline (Fig. 18B).

To further assess immune memory of ZF17-MQ1, previously treated mice (e.g., ZF17-MQ1, anti- PD-1 antibody molecule, and combination of ZF17-MQ1 and anti-PD-1 antibody molecule) were (re)challenged with Hepal-6 cells and mouse lung cancer LL/2 cells (control). At about 70 days after last dose of any treatment regimen, age-matched mice naive to Hepal-6 cells, and previously treated mice with no visible Hepal-6 tumors, were (re)challenged with mouse cancer cells: left hind flank with mouse liver cancer Hepal-6 cells (Fig. 19A) and right hind flank with mouse lung cancer LL/2 cells (Fig. 19B). The naive mice grew both Hepal-6 and LL/2 tumors on both flanks. The previously treated mice in all groups grew LL/2 tumors but Hepal-6 tumors failed to be established on left flank. These results demonstrate that ZF17-MQ1 alone or in combination with anti-PDl were able to establish immune memory to previous exposure to Hepal-6 cells.

EQUIVALENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Some aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS We claim:

1. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject:

(1) a nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor, wherein the expression repressor comprises:

(a) a targeting moiety that binds to a MY C locus, and

2. The method of claim 1, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-PD-1 antibody molecule, an anti-PD-Ll antibody molecule, an anti-CTLA4 antibody molecule, an anti-LAG3 antibody molecule, an anti-PD-L2 antibody molecule, an anti-Tim3 antibody molecule, and anti-KIR antibody molecule, or an anti-BTLA antibody molecule.

3. The method of claim 1, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-PD-1 antibody molecule.

4. The method of claim 3, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 18.

5. The method of claim 3 or 4, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region listed in Table 18, e.g., SEQ ID Nos.: 208, 211, 214, or 217; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 18, e.g., SEQ ID Nos.: 208, 211, 214, or 217; or lii) an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 18, e.g., SEQ ID Nos.: 208, 211, 214, or 217.

6. The method of any one of claims 3-5, wherein the immune checkpoint inhibitor antibody molecule comprises a light chain variable region comprising: i) the amino acid sequence of any light chain variable region listed in Table 18, e.g., SEQ ID Nos.: 209, 212, 215, or 218; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 18, e.g., SEQ ID Nos.: 209, 212, 215, or 218; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 18, e.g., SEQ ID Nos.: 209, 212, 215, or 218.

7. The method of any one of claims 3-6, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of cemiplimab according to Kabat or Chothia.

8. The method of any one of claims 3-6, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of dostarlimab according to Kabat or Chothia.

9. The method of any one of claims 3-6, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of mvolumab according to Kabat or Chothia.

10. The method of any one of claims 3-6, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of pembrolizumab according to Kabat or Chothia.

11. The method of any one of claims 3-6, wherein the anti-PD-1 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 19 or 20.

12. Tire method of claim 3, wherein tire immune checkpoint inhibitor polypeptide is an antibody molecule comprising a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of MIH4, according to Kabat or Chothia.

13. The method of claim 1, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-PD-Ll antibody molecule.

14. The method of claim 13, wherein the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 21.

15. The method of claim 13 or 14, wherein the anti-PD-Ll antibody molecule comprises a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region listed in Table 21, e.g., SEQ ID Nos.: 238, 241, or 244; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 21, e.g., SEQ ID Nos.: 238, 241, or 244; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 21, e.g., SEQ ID Nos.: 238, 241, or 244.

16. The method of any one of claims 13-15, wherein the anti-PD-Ll antibody molecule comprises a light chain variable region comprising: i) the amino acid sequence of any light chain variable region listed in Table 21, e.g., SEQ ID Nos.: 239, 242, or 245; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 21, e.g., SEQ ID Nos.: 239, 242, or 245; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 21, e.g., SEQ ID Nos.: 239, 242, or 245.

17. The method of claim 13, where the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of mAb 10F.9G2, according to Kabat or Chothia.

18. Hie method of claim 13, wherein the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of atezolizumab according to Kabat or Chothia, e.g., CDRs of a sequence of Table 22.

19. The method of claim 13 or 18, wherein the anti-PD-Ll antibody molecule comprising a HC CDR1 comprising an amino acid sequence GFTFSDSWIH (SEQ ID NO: 259), a HC CDR2 comprising an amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 260), and a HC CDR3 comprising an amino acid sequence RHWPGGFDY (SEQ ID NO: 261); and/or a LC CDR1 comprising an amino acid sequence RASQDVSTAVA (SEQ ID NO: 262), a LC CDR2 comprising an amino acid sequence SASFLYS (SEQ ID NO: 263), and a LC CDR3 comprising an amino acid sequence QQYLYHPAT (SEQ ID NO: 264).

20. The method of claim 13, wherein the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of avelumab according to Kabat or Chothia.

21. The method of claim 13, wherein the anti-PD-Ll antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of durvalumab according to Kabat or Chothia.

22. The method of claim 1, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-CTLA-4 antibody molecule.

23. The method of claim 22, wherein the anti-CTLA-4 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 23.

24. The method of claim 22 or 23, wherein the anti-CTLA-4 antibody molecule comprises a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region listed in Table 23, e.g., SEQ ID No: 247; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 23, e.g., SEQ ID No: 247; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 23, e.g., SEQ ID No: 247.

25. Tire method of any one of claims 22-24, wherein the anti-CTLA-4 antibody molecule comprises a light chain variable region comprising: i) the amino acid sequence of any light chain variable region listed in Table 23, e.g., SEQ ID No: 248; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 23, e.g., SEQ ID No: 248; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 23, e.g., SEQ ID No: 248.

26. The method of claim 23, wherein the anti-CTLA-4 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of ipilimumab, according to Kabat or Chothia.

27. The method of claim 1, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-LAG3 antibody molecule.

28. The method of claim 27, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of a sequence of Table 24.

29. The method of claim 27 or 28, wherein the anti-LAG3 antibody molecule comprises a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region listed in Table 24, e.g., SEQ ID No: 250 or 280; li) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any heavy chain variable region provided in Table 24, e.g., SEQ ID No: 250 or 280; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any heavy chain variable region provided in Table 24, e.g., SEQ ID No: 250 or 280.

30. The method of any one of claims 27-29, wherein the anti-LAG3 antibody molecule comprises a light chain variable region comprising: i) the amino acid sequence of any light chain variable region listed in Table 24, e.g., SEQ ID No: 251 or 281; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of any light chain variable region provided in Table 24, e.g., SEQ ID No: 251 or 281; or iii) an amino acid sequence with at least 95% identity to the amino acid sequence of any light chain variable region provided in Table 24, e.g., SEQ ID No: 251 or 281.

31. The method of claim 27, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of relatlimab, according to Kabat or Chothia.

32. The method of claim 27, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of relatlimab, according to table 25.

33. The method of claim 27, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of favezelimab, according to Kabat or Chothia.

34. The method of claim 28, wherein the anti-LAG3 antibody molecule comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 of favezelimab, according to table 25.

35. The method of claim 1, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-PD-L2 antibody molecule.

36. The method of claim 1, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-Tim3 antibody molecule.

37. The method of claim 1, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti- killer IgG-like receptor (KIR) antibody molecule.

38. The method of claim 1, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising an anti-BTLA antibody molecule.

39. The method of any of the preceding claims, wherein the immune checkpoint inhibitor antibody molecule is administered by injection (e.g., subcutaneously or intravenously).

40. Tire method of any of tire preceding claims, wherein the immune checkpoint inhibitor antibody molecule is administered at a dose of about 1 mg/kg to 30 mg/kg or about 100 mg to about 2000 mg, e.g., administered every 1, 2, 3, 4, 5, 6 or 7 weeks.

41. The method of any of claims 1, 2, 13-18, 38, or 39, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising atezolizumab and is administered (e.g., at 840 mg) as an intravenous infusion (e.g., over 30 minutes or over 60 minutes) every 2 weeks.

42. The method of any of claims 1, 2, 13-19, 38, or 39, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising atezolizumab and is administered (e.g., at 1200 mg) as an intravenous infusion (e.g., over 30 minutes or over 60 minutes) every 3 weeks.

43. The method of any of claims 1, 2, 13-19, 38, or 39, wherein the immune checkpoint inhibitor polypeptide is an antibody molecule comprising atezolizumab and is administered (e.g., at 1680 mg) as an intravenous infusion (e.g., over 30 minutes or over 60 minutes) every 4 weeks.

44. The method of any of the preceding claims, wherein after administration, tumor volume decreases in the subject.

45. The method of any of the preceding claims, wherein after administration, MY C mRNA levels decrease in the subject.

46. The method of any of the preceding claims, wherein after administration, surface PD-L1 protein levels decrease in the subject.

47. The method of any of the preceding claims, wherein the targeting moiety binds to a MYC promoter.

48. The method of any of the preceding claims, wherein the targeting moiety binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 83, 2, 3, 75- 86, 97-107, 109, 110, 190-192, or 199-202.

49. The method of any of the preceding claims, wherein the cancer is a hepatocellular carcinoma (HCC), Fibrolamellar Hepatocellular Carcinoma (FHCC), Cholangiocarcinoma, Angiosarcoma, or secondary liver cancer.

50. The method of any of the preceding claims, wherein the nucleic acid comprises an RNA, e.g., an mRNA.

51. The method of any of the preceding claims, wherein: the targeting moiety binds a genomic locus comprising at least 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 83, and the expression repressor comprises the first effector moiety, wherein the effector moiety comprises a DNA methyltransferase.

52. The method of claim 51, wherein the targeting moiety comprises a zinc finger domain.

53. The method of claim 51 or 52, wherein the targeting moiety comprises an amino acid sequence according to SEQ ID NO: 13 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

54. The method of any of claims 51-53, wherein the effector moiety comprises MQ1 or a functional variant or fragment thereof.

55. The method of any of claims 51-54, wherein the effector moiety comprises a sequence of SEQ ID NO: 19 or 87, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

56. The method of any of claims 51-55, wherein the effector moiety comprises a sequence of SEQ ID NO: 129, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

57. The method of any of claims 51-55, wherein the RNA comprises a nucleotide sequence encoding the targeting moiety, wherein the nucleotide sequence encoding the targeting moiety comprises a sequence according to SEQ ID NO: 131 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

58. The method of any of claims 51-57, wherein the RNA comprises a nucleotide sequence encoding the effector moiety, wherein the nucleotide sequence encoding the effector moiety comprises a sequence according to SEQ ID NO: 132, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

59. The method of any of claims 51-58, wherein the RNA comprises a nucleotide sequence according to SEQ ID NO: 130, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

60. The method of any of claims 51-59, wherein the RNA further encodes a second expression repressor, wherein the second expression repressor comprises: a second targeting moiety that binds a second genomic locus, and a second effector moiety.

61. The method of claim 61, wherein the second targeting moiety binds a second genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of SEQ ID NO: 77.

62. The method of claim 60 or 61, wherein the second targeting moiety comprises a zinc finger domain.

63. The method of any of claims 60-62, wherein the second targeting moiety comprises an amino acid sequence according to SEQ ID NO: 7, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

64. The method of any of claims 60-63, wherein the second effector moiety comprises KRAB or a functional variant or fragment thereof.

65. Tire method of any of claims 60-64, wherein the second effector moiety comprises an amino acid sequence according to SEQ ID NO: 18, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

66. The method of any of claims 60-65, wherein the second expression repressor comprises an amino acid sequence according to SEQ ID NO: 24 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.

67. The method of any of claims 60-66, wherein the RNA comprises a nucleotide sequence according to SEQ ID NO: 113.

68. The method of any of the preceding claims, wherein the nucleic acid is formulated in lipid nanoparticles (LNPs), wherein optionally the nucleic acid is encapsulated inside of the LNPs.

69. The method of any of the preceding claims, wherein the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor is administered as an IV infusion, e.g., over 80-120 minutes, e.g., every 2 weeks.

70. The method of any of the preceding claims, wherein the nucleic acid is administered at a dose of about 0.001 mg/kg to 1 .5 mg/kg or about 0.002 mg/kg to 1 .5 mg/kg, e.g., administered every 1, 2, 3, 4, 5, 6 or 7 weeks.

71. The method of any of the preceding claims, wherein the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor and the checkpoint inhibitor polypeptide are administered on different days.

72. The method of any of the preceding claims, wherein the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor and the checkpoint inhibitor polypeptide are administered on the same day.

73. The method of claim 72, wherein the of the nucleic acid (e.g., RNA, e.g., mRNA) encoding the expression repressor is administered prior to administration of the checkpoint inhibitor polypeptide.

74. The method of claim 72 or 73, wherein the checkpoint inhibitor polypeptide is administered between 1-24 hours after completion of administration of the nucleic acid (e.g., RNA, e.g., mRNA) encoding an expression repressor.

75. The method of any of the preceding claims, which results in a reduction of tumor-infiltrating Treg cells (e.g., a reduction by about 10%, 20%, 30%, 40%, or 50%) compared to administration of the immune checkpoint inhibitor polypeptide alone.

76. The method of any of the preceding claims, which results in no increase in tumor-infiltrating Treg cells relative to levels before the administration.

77. The method of any of the preceding claims, which results in an increase in tumor-infiltrating T cells relative to levels before the administration.

78. The method of any of the preceding claims, which results in an increase in tumor-infiltrating CD8+ T cells relative to levels before the administration.

79. The method of any of the preceding claims, which results in an increase in activated tumorinfiltrating CD8+ T cells relative to levels before the administration.

80. The method of any of the preceding claims, which results in an increase in the ratio of tumorinfiltrating CD8+ T cells to tumor-infiltrating Treg cells relative to the ratio before the administration.

81. The method of any of the preceding claims, which results in an increase in the ratio of activated tumor-infiltrating CD8+ T cells to tumor-infiltrating Treg cells relative to the ratio before the administration.

82. The method of any of the preceding claims, which results in a complete remission, e.g., for at least 10, 20, 30, 40, 50, 60, or 70 days.

83. The method of claim 82, wherein the subject does not receive one of both of the nucleic acid encoding the expression repressor or immune checkpoint inhibitor polypeptide during the period of complete remission.