Attorney Docket No.370602-7080WO1(00277) LIGAND-DIRECTED GENE EDITING APPROACHES CROSS-REFERENCE TO RELATED APPLICATION The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. 5 Provisional Patent Application No.63/659,753, filed June 13, 2024, which is incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted 10 in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on June 13, 2025, is named “370602- 7080WO1(00277) Sequence Listing.xml” and is 112 kilobytes in size. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND 15 DEVELOPMENT This invention was made with government support under CA286340 awarded by National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE DISCLOSURE 20 Biological information transfer, defined specifically as the residue-by-residue transfer of sequence information between nucleic acids and protein, is a fundamental aspect of molecular biology with extensive experimental and theoretical implications. The elucidation of this phenomenon over half a century ago has directly facilitated remarkable progression in basic science research, with numerous applications in biotechnology and medicine. Early 25 research on the flow of biological information often involved the use of bacteriophage (phage) as a central experimental subject, spurring the genetic and functional characterization of these bacterial viruses. Decades later, phage display technology was developed and refined, whereby phage is genetically manipulated to display unique peptides on the capsid surface for various applications, including protein binding interaction discovery and ligand- 30 directed targeting. Gene editing approaches are an increasingly useful tool in biotechnology and molecular medicine. The global cancer gene therapy market size accounted for USD 2.95 billion in 2023, and it is expected to increase to around USD 18.11 billion by 2033 with a - 1 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) CAGR of 19.9% from 2024 to 2033, according to recent reports. Further, the global gene editing market size is estimated at USD 5.3 billion in 2023 and is anticipated to reach around USD 10.6 billion by 2028, growing at a CAGR of 15.0% from 2024 to 2028, even faster than more traditional gene therapy approaches. 5 A need exists for novel gene editing systems that overcome current limitations, including off‐target effects of more traditional CRISPR technology and which enable the targeting of specific cancer‐causing genes or pathways, allowing for more precise treatment that minimizes damage to healthy cells. The invention of the current disclosure addresses this need. 10 SUMMARY OF THE INVENTION As described herein, the present disclosure relates to methods of gene editing in a target cell, comprising contacting the cell with an engineered phage comprising a fusion protein comprising a ligand-binding polypeptide and a CRISPR/Cas gene editing system. 15 In one aspect, the invention provides an engineered phage comprising: (a) a fusion protein comprising a ligand-binding polypeptide fused to a phage coat protein, and (b) a nucleic acid encoding a CRISPR/Cas gene editing system. In certain embodiments, the nucleic acid encoding the CRISPR/Cas gene editing 20 system comprises one or more guide RNAs specific for one or more endogenous DNA sequences and a nucleic acid encoding a Cas protein. In certain embodiments, at least one guide RNA is operably linked to a first promoter, and the nucleic acid encoding a Cas protein is operably linked to a second promoter. 25 In certain embodiments, the Cas protein is a wildtype Cas protein. In certain embodiments, the Cas protein is an engineered Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of Cas9, SpCas9, SaCas9, CasΦ, Cas12f, NanoCas, Cas12a, Cas13, Cas9-nickase (Cas9n), nuclease dead Cas9 (dCas9), dCas9 fused to the non-specific endonuclease FokI (dCas9- 30 FokI), an enhanced Cas9, a high-fidelity Cas9, eSpCas9, spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non-Sp” Cas9s, and any variant thereof. In certain embodiments, the Cas protein is a fusion protein. In certain embodiments, the Cas protein is fused to a deaminase. In certain embodiments, the Cas protein is fused to a reverse transcriptase. - 2 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) In certain embodiments, the fusion protein comprises a ligand-binding polypeptide that binds specifically to a ligand expressed on the surface of a target cell. In certain embodiments, the ligand-binding polypeptide is RGD4C. In certain embodiments, the ligand-binding polypeptide comprises the amino acid 5 sequence of SEQ ID NO: 72. In certain embodiments, the first promoter is a U6 promoter. In certain embodiments, the second promoter is a CMV promoter. In certain embodiments, the ligand expressed on the surface of the target cell is an αv integrin. 10 In certain embodiments, the fusion protein comprises a phage coat protein selected from the group comprising pIII protein, pVI protein, pVII protein, pVIII protein, rpVIII protein, and pIX protein. In certain embodiments, the engineered phage of the above aspects or embodiments or any aspect or embodiment disclosed herein is an adeno-associated virus/phage (AAVP). 15 In certain embodiments, the AAVP is encoded by a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NOs: 73-75. In another aspect, the current invention provides a method of performing gene editing in a target cell, comprising contacting the cell with an engineered phage comprising: (a) a fusion protein comprising a ligand-binding polypeptide fused to a phage 20 coat protein and (b) a nucleic acid encoding a CRISPR/Cas gene editing system. In certain embodiments, the nucleic acid encoding the CRISPR/Cas gene editing system comprises one or more guide RNAs specific for one or more endogenous DNA sequences and a nucleic acid encoding a Cas protein. In certain embodiments, at least one guide RNA is operably linked to a first 25 promoter, and wherein the nucleic acid encoding a Cas protein is operably linked to a second promoter. In certain embodiments, the Cas protein is a wildtype Cas protein. In certain embodiments, the Cas protein is an engineered Cas protein. In certain embodiments, the Cas protein is selected from the group consisting of 30 Cas9, SpCas9, SaCas9, CasΦ, Cas12f, NanoCas, Cas12a, Cas13, Cas9-nickase (Cas9n), nuclease dead Cas9 (dCas9), dCas9 fused to the non-specific endonuclease FokI (dCas9- FokI), an enhanced Cas9, a high-fidelity Cas9, eSpCas9, spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non-Sp” Cas9s, and any variant thereof. In certain embodiments, the Cas protein is a fusion protein. - 3 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) In certain embodiments, the Cas protein is fused to a deaminase. In certain embodiments, the Cas protein is fused to a reverse transcriptase. In certain embodiments, the fusion protein comprises a ligand-binding polypeptide that binds specifically to a ligand expressed on the surface of a target cell. 5 In certain embodiments, the ligand-binding polypeptide is RGD4C. In certain embodiments, the ligand-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 72. In certain embodiments, the first promoter is a U6 promoter. In certain embodiments, the second promoter is a CMV promoter. 10 In certain embodiments, the ligand expressed on the surface of the target cell is an αv integrin. In certain embodiments, the fusion protein comprises a phage coat protein selected from the group comprising pIII protein, pVI protein, pVII protein, pVIII protein, rpVIII protein, and pIX protein. 15 In certain embodiments, the engineered phage is an adeno-associated virus/phage (AAVP). In certain embodiments, the AAVP is encoded by a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NOs: 73-75. In another aspect, the current invention provides a method for treating, 20 ameliorating, and/or preventing cancer in a subject in need thereof, the method comprising administering to the subject an effect amount of an engineered phage comprising: (a) a fusion protein comprising a ligand-binding polypeptide fused to a phage coat protein and (b) a nucleic acid encoding a CRISPR/Cas gene editing system; wherein the CRISPR/Cas gene editing system comprises one or more guide RNAs 25 specific for one or more cancer-related genes and a nucleic acid encoding a Cas protein; and wherein the ligand-binding polypeptide specifically binds a ligand expressed by a cancer cell. In certain embodiments, the one or more cancer-related genes are selected from the 30 group consisting of GRP78, aminopeptidase N (APN), aminopeptidase A (APA), vascular endothelial growth factor receptor 2 (VEGFR2), polo-like kinase 1 (PLK1), interleukin-11 receptor alpha (IL11RA), phosphoinositide 3-kinase catalytic subunit alpha (PI3KCA), and any combination thereof. In certain embodiments, the one or more guide RNAs are operably linked to a first - 4 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) promoter, and the nucleic acid encoding a Cas protein is operably linked to a second promoter. In certain embodiments, the Cas protein is n wildtype Cas protein. In certain embodiments, the Cas protein is an engineered Cas protein. 5 In certain embodiments, the Cas protein is selected from the group consisting of Cas9, SpCas9, SaCas9, CasΦ, Cas12f, NanoCas, Cas12a, Cas13, Cas9-nickase (Cas9n), nuclease dead Cas9 (dCas9), dCas9 fused to the non-specific endonuclease FokI (dCas9- FokI), an enhanced Cas9, a high-fidelity Cas9, eSpCas9, spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non-Sp” Cas9s, and any variant thereof. 10 In certain embodiments, the Cas protein is a fusion protein. In certain embodiments, the Cas protein is fused to a deaminase. In certain embodiments, the Cas protein is fused to a reverse transcriptase. In certain embodiments, the ligand-binding polypeptide is RGD4C. In certain embodiments, the ligand-binding polypeptide comprises the amino acid 15 sequence of SEQ ID NO: 72. In certain embodiments, the first promoter is a U6 promoter. In certain embodiments, the second promoter is a CMV promoter. In certain embodiments, the ligand expressed by the cancer cell is an αv integrin. In certain embodiments, the phage coat protein is selected from the group 20 comprising pIII protein, pVI protein, pVII protein, pVIII protein, rpVIII protein, and pIX protein. In certain embodiments, the engineered phage is an adeno-associated virus/phage (AAVP). In certain embodiments, wherein the AAVP is encoded by a nucleic acid sequence 25 comprising the nucleic acid sequence of SEQ ID NOs: 73-75. BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of specific embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of 30 illustrating the disclosure, exemplary embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. FIG.1 is a diagram showing example CRISPR/Cas transgene configurations that can be integrated into AAVP. Phage replication and coat protein genes other than pIII are - 5 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) omitted for simplicity. Various CRISPR/Cas transgene configurations which may be inserted into the AAVP genome for expression of components for various CRISPR/Cas- based applications. The most basic configuration includes a gRNA guide sequence insertion site (asterisk) adjacent to a gRNA scaffold together with a Cas nuclease, which may be 5 wildtype (WT) or engineered. Another configuration includes a catalytically deactivated Cas nuclease (dCas), for targeted gene silencing. Another configuration includes a deaminase (fused to the Cas nuclease), for base editing. Another configuration includes a reverse transcriptase (fused to the Cas nuclease), as well as a template sequence insertion site (triangle), for programmable sequence insertion (prime editing). The gRNA (and associated) 10 sequences and Cas (and associated) sequences of the CRISPR/Cas transgenes are transcriptionally controlled by U6 and CMV promoters (or other suitable promoters), respectively, and can be introduced into the AAVP backbone by ligation of compatible ends after enzymatic digestion (designated here as restriction enzyme digestion site or RDS). Multiplexed gRNA guide sequences under U6 promoter control targeting different genes for 15 combined anti-tumor effects can also be incorporated into the AAVP backbone. FIGs.2A-2B are diagrams illustrating Phage Display Technology. (FIG.2A) A filamentous phage display vector can be engineered to display a peptide by insertion of the peptide-encoding sequence into the N-terminal region of the pIII minor coat protein gene. The peptide is displayed such that it is accessible for receptor binding. (FIG.2B) Filamentous 20 phage display vectors can be used to screen for interactions by exposing phage displaying a peptide library to an immobilized target receptor. Unbound phage is removed by washing, bound phage is recovered by bacterial infection, and the coding sequence of the peptide is ascertained by sequencing the pIII gene insert region. FIG.3 illustrates gene editing with CRISPR/Cas. The Cas nuclease is shown in 25 complex with gRNA and genomic target DNA (black) (PDB ID: 4008 is used as an example). The gRNA consists of a scaffold sequence which allows for Cas/gRNA ribonucleoprotein complex formation as well as a programmable guide sequence which directs the Cas nuclease to the DNA target site by sequence complementarity. The Cas nuclease then induces a double-strand break (arrowheads) ~3 bp upstream of the required 30 PAM sequence. Subsequent DNA repair results in the formation of indel mutations in the target DNA (adapted from Ran et al., 2013 and Nishimasu et al., 2014). FIG.4 is a diagram illustrating a schematic representation of the ligand-directed AAVP-Cas particle and genomic organization of the critical components. Following AAVP administration, the targeted AAVP-Cas particle binds to a specific receptor expressed on the - 6 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) cell surface and undergoes receptor-mediated internalization. Once inside the cell, the AAV ITRs, which flank the transgene cassette, mediate its expression. Delivery of a CRISPR/Cas transgene encoding a Cas nuclease and gRNA allows for programmable editing of a desired gene target. 5 FIG.5 illustrates the engineering of the AAVP-Cas plasmid. An AAVP plasmid, consisting of the pIII minor coat protein gene, a tetracycline resistance gene, AAV ITRs, and a pre-existing transgene (dashed line) was digested with the restriction enzyme NotI to obtain the AAVP backbone. The pIII gene contains a targeting peptide sequence, in this case RGD4C. Phage replication and coat protein genes other than pIII are omitted for simplicity. 10 In parallel, a plasmid containing a CRISPR/Cas9 transgene with a downstream NotI site was modified by site-directed mutagenesis to replace a non-coding region with an additional upstream NotI site, followed by digestion with the NotI restriction enzyme. The desired fragments from each plasmid (the AAVP backbone and the CRISPR/Cas9 transgene) were isolated by gel electrophoresis. The isolated fragments were then ligated via the NotI sticky 15 ends, present on both ends of each fragment, in order to assemble the RGD4C-AAVP-Cas9 plasmid. The CRISPR/Cas9 transgene contains a dual-BbsI restriction digestion site, allowing for insertion of a desired gRNA-encoding DNA oligo with sticky ends compatible with the dual-BbsI site. In this manner, an EMX1 gRNA guide sequence was inserted into the RGD4C-AAVP-Cas9 plasmid directly upstream of the gRNA scaffold sequence. The Cas9 20 coding sequence and gRNA sequence of the CRISPR transgene are under expressional control of CMV and U6 promoters, respectively. FIGs.6A-6F illustrate AAVP-Cas particle production, quantification, and ligand- directed targeting properties. (FIG.6A) Schematic overview of AAVP-Cas9 particle production. MC1061 F’ E. coli was transformed with AAVP-Cas9 plasmid DNA, conferring 25 tetracycline (Tet) resistance, followed by liquid culture growth under tetracycline selective pressure. Particles were precipitated from the culture by multi-step centrifugation with polyethylene glycol. (FIG.6B) Kanamycin/tetracycline LB agar plates following overnight incubation of kanamycin-resistant K91 E. coli infected with either RGD4C-displaying or non-targeted (NT) AAVP-Cas9 particles (107 dilution). (FIG.6C) Bacterial titer 30 quantification of AAVP-Cas9 particles as determined by serial dilution and infection of K91 E. coli. Data shown as means ± SEM, n = 3 technical replicates. (FIG.6D) Quantification of AAVP-Cas9 particles by qPhage, a quantitative real-time PCR approach which allows for phage genome copy number determination. Data shown as means ± SEM, n = 3 technical replicates. (FIG.6E) Cell-surface binding of AAVP-Cas9 particles in KS1767 cells as - 7 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) determined by the BRASIL assay. Data shown as means ± SEM, n = 3 experimental replicates, **p < 0.005, Welch’s t test. (FIG.6F) Fluorescence microscopy of immunocytochemistry with anti-phage antibody and DAPI for binding and internalization of AAVP-Cas9 particles in KS1767 cells. Scale bar, 20μm. 5 FIGs.7A-7D illustrate the evaluation of AAVP-Cas transgene expression and CRISPR/Cas gene editing in vitro. KS1767 cells were treated with AAVP-Cas9 particles, with a serum-free incubation period of 4 hours, and harvested for various downstream analyses following one week. (FIG.7A) Quantitative real-time PCR for Cas9 mRNA and gRNA expression using Cas9-specific and gRNA-specific primers and probes. Data shown as 10 means ± SEM, n = 3, *p < 0.05, Welch’s t test. (FIG.7B) Flow cytometry with phycoerythrin (PE) fluorophore-conjugated anti-Cas9 antibody for detection of Cas9 protein expression. Dashed box highlights shift in PE signal frequency distribution. Data shown as means ± SEM, n = 2, *p < 0.05, Welch’s t test. (FIG.7C) Indel distribution at the EMX1 gRNA genomic target region from cells treated with RGD4C-AAVP-Cas9-EMX1 versus untreated 15 cells as determined by amplicon NGS. Dotted line indicates the expected DSB site of the EMX1 gRNA. (FIG.7D) Top ten most frequent indel-containing sequencing reads at the expected DSB site (black arrowhead) of the EMX1 gRNA (line), 3 bp upstream of the PAM sequence, from cells treated with RGD4C-AAVP-Cas9-EMX1. FIGs.8A-8D illustrate the design and screening of GRP78 and APN gRNA 20 candidates. (FIG.8A) Schematic representation of the positioning of the five gRNA candidates (lines) within the target exons of the GRP78 and APN genes along with corresponding amino acid residues at the expected DSB sites (indicated by arrowheads). (FIG.8B) Gel electrophoresis of PCR products for each gRNA candidate genomic target site from KS1767 cells treated by Cas9/gRNA ribonucleoprotein co-transfection. Two amplicons 25 (A1 and A2) were produced for each gene to accommodate the positional differences of the gRNA target sites. (FIG.8C) Gene editing efficiency of each gRNA candidate as determined by Sanger sequencing of PCR products followed by bioinformatic analysis with DECODR software. (FIG.8D) Sanger sequencing reads for the gRNA candidates with the highest editing efficiency. Arrowheads indicate the expected DSB sites and underlined bases 30 represent the desired indel mutation site intended to result in a disruptive frameshift of the coding sequence. FIGs.9A-9D illustrate the validation of the lead GRP78 gRNA candidate. (FIG.9A) Phase-contrast microscopy of KS1767 cells treated by Cas9/gRNA ribonucleoprotein co- transfection with the GRP78 gRNA lead candidate and the benchmark EMX1 gRNA serving - 8 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) as a positive control for gene editing. Scale bar, 50μm. (FIG.9B) Proliferation of KS1767 and MCF7 cells treated by Cas9/gRNA ribonucleoprotein co-transfection with the GRP78 and EMX1 gRNAs. Data shown as means ± SD, n = 3. (FIGs.9C-9D) Indel distribution at the GRP78 and EMX1 gRNA genomic target regions from KS1767 cells treated by Cas9/gRNA 5 ribonucleoprotein co-transfection versus untreated cells as determined by amplicon NGS. Dotted line indicates the expected DSB sites of the GRP78 and EMX1 gRNAs. FIG.10 is a map of the RGD4C-AAVP-SpCas9 vector. FIG.11 is a map of the RGD4C-AAVP-SaCas9 vector. FIG.12 is a map of the RGD4C-AAVP-CasPhi vector. 10 DETAILED DESCRIPTION Biological information transfer, defined specifically as the residue-by-residue transfer of sequence information between nucleic acids and protein, is a fundamental aspect of 15 molecular biology with extensive experimental and theoretical implications. The elucidation of this phenomenon over half a century ago has directly facilitated remarkable progression in basic science research, numerous applications in biotechnology and medicine, as well as emerging developments in artificial intelligence. This disclosure describes a novel experimental application of phage-based gene transfer for targeted delivery of a CRISPR/Cas 20 gene editing system. Early research on the flow of biological information often involved the use of bacteriophage (phage) as a central experimental subject, spurring the genetic and functional characterization of these bacterial viruses. Decades later, phage display technology was developed and refined, whereby phage is genetically manipulated to display unique peptides on the capsid surface for various applications, including protein binding interaction 25 discovery and ligand-directed targeting. The latter was utilized here for the targeted delivery of a CRISPR/Cas9 gene editing system. Filamentous phage displaying the tumor-targeting RGD4C peptide was engineered to carry a CRISPR/Cas transgene consisting of a Cas9 nuclease and gRNA sequence. Proof-of-concept evaluation of the engineered vector was performed in vitro, including for cell-surface binding, cellular internalization, post- 30 transduction CRISPR/Cas transgene expression, and post-transduction gene editing, collectively indicating CRISPR/Cas system delivery by targeted gene transfer. In parallel, gRNA screening with Cas9 protein co-transfection resulted in the identification of high editing efficiency gRNA candidates for various cancer target genes, one of which robustly inhibited cell proliferation. - 9 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure 5 pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, selected materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used. It is also to be understood that the terminology used herein is for the 10 purpose of describing particular embodiments only and is not intended to be limiting. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. 15 “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an 20 immune response. This immune response may involve either antibody production, or the activation of specific immunologically competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic deoxyribonucleic acid (DNA). A skilled artisan will understand that any DNA comprised of 25 nucleotide sequences or a partial nucleotide sequence encoding a protein or peptide that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than 30 one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. - 10 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual. “Allogeneic” refers to any material derived from a different animal of the same species. “Xenogeneic” refers to any material derived from an animal of a different species. 5 The term “cleavage” refers to the breakage of covalent bonds, such as in the backbone of a nucleic acid molecule or the hydrolysis of peptide bonds. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible. Double-stranded cleavage can occur as a result of two distinct 10 single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides may be used for targeting cleaved double-stranded DNA. As used herein, the term “conservative sequence modifications” is intended to refer to nucleotide or amino acid modifications that do not change the amino acid sequence or 15 significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence, respectively. Amino acid conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid 20 substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, 25 tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the complementarity-determining regions (CDRs) of an antibody can be replaced with other amino acid residues from the same side chain family and the 30 altered antibody can be tested for the ability to bind antigens using the functional assays described herein. The term “CRISPR/Cas” or “clustered regularly interspaced short palindromic repeats” or “CRISPR” refers to DNA loci containing short repetitions of base sequences followed by short segments of spacer DNA from previous exposures to a virus or plasmid. - 11 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) Bacteria and archaea have evolved adaptive immune defenses termed CRISPR/CRISPR– associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids. In bacteria, the CRISPR system provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage. 5 The “CRISPR/Cas” system or “CRISPR/Cas-mediated gene editing” refers to a class II CRISPR/Cas system that has been modified for genome editing/engineering. It is typically comprised of a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas). “Guide RNA (gRNA)” is used interchangeably herein with “short guide RNA (sgRNA)”. The gRNA is a short synthetic RNA composed of a “scaffold” sequence 10 necessary for Cas-binding and a user-defined ∼20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified. The genomic target of Cas can be changed by changing the targeting sequence present in the gRNA. “Cas” should be interpreted to include any and all types and/or variants of Cas. Examples of Cas variants include but are not limited to Cas9, Streptococcus pyogenes or SpCas9, Staphylococcus 15 aureus or SaCas9, Bacteriophage CasΦ (also known as Cas12j), Acidaminococcus sp. Cas12f (also known as Cas14), NanoCas, Cas12a, Cas13, Cas9-nickase (Cas9n), nuclease dead Cas9 (dCas9) fused to the non-specific endonuclease FokI (dCas9-FokI), “enhanced Cas9”, “high-fidelity Cas9”, eSpCas9, spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non-Sp” Cas9s, and full-nuclease variants. All of the above may be in the20 following forms: wildtype, engineered for increased catalytic activity, catalytically de- activated, fused to a deaminase (for base editing), and/or fused to a reverse transcriptase (for sequence incorporation, i.e. “prime editing”) A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health 25 continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health. The term “downregulation” as used herein refers to the decrease or elimination 30 of gene expression of one or more genes. “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not - 12 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) limited to, anti-tumor activity as determined by any means suitable in the art. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a complementary DNA (cDNA), or a messenger ribonucleic acid (mRNA), to serve as templates for synthesis of other polymers and macromolecules in 5 biological processes having either a defined sequence of nucleotides [i.e., ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA)] or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is10 identical to the mRNA sequence and is usually provided in sequence listings, and the non- coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. As used herein, “endogenous” refers to any material from or produced inside an organism, cell, tissue or system. 15 As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system. The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter. “Expression vector” refers to a vector comprising a recombinant polynucleotide 20 comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., 25 naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and AAV) that incorporate the recombinant polynucleotide. “Homologous” as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit 30 position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two - 13 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between 5 two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a 10 direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical. The term “immunoglobulin” or “Ig,” as used herein is defined as a class of 15 proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the B cell receptor (BCR) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common 20 circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate 25 hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen. The term “immune response” as used herein is defined as a cellular and humoral response to an antigen that occurs when lymphocytes and antigen-presenting cells identify antigenic molecules as foreign and induce the formation of antibodies and/or activate 30 lymphocytes to remove the antigen. The immune response can be mediated by acellular and cellular components. The acellular components include physical barriers and signaling molecules such as cytokines. The cellular response is mediated by both innate immune cells such as macrophages, neutrophils, dendritic cells, and adaptive immune cells such as lymphocytes (T and B). - 14 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) Both cellular and humoral aspects contribute to the production of antibodies, clearance of the antigen, and the development of immunological memory. “Isolated” means altered or removed from the natural state. For example, a nucleic acid 5 or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. 10 The term “knockdown” as used herein refers to a decrease in gene expression of one or more genes. The term “knockout” as used herein refers to the ablation of gene expression of one or more genes. A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses 15 are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. Human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV) are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve 20 significant levels of gene transfer in vivo. The term “adeno-associated virus” or “AAV” as used herein refers to small, nonpathogenic, single-stranded DNA viruses of the genus Dependoparvovirus. Their ability to readily infect both dividing and quiescent human cells and tissues with minimal immune responses and no obvious pathogenicity has led to the use of AAV as vectors for gene therapy 25 and vaccines. The term “phage” or “bacteriophage” as used herein refer to viruses that evolved to infect and replicate within prokaryotic or archaeal cells. Bacteriophages can comprise either RNA or DNA genomes and can have protein capsid structures of varying complexity. In humans, phage therapy has been used as an alternative to antibiotics for the treatment of 30 bacterial infection. Phage particles can also be engineered to infect eukaryotic cells, and as such make attractive vectors for gene therapy in that they can be easily expanded to vast quantities in bacterial cultures and their novel structure means pre-existing immunity in humans is relatively low. - 15 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) The term “limited toxicity” as used herein, refers to the peptides, polynucleotides, cells and/or antibodies of the disclosure manifesting a lack of substantially negative biological effects, anti-tumor effects, or substantially negative physiological symptoms toward a healthy cell, non- tumor cell, non-diseased cell, non-target cell or population of 5 such cells either in vitro or in vivo. By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the disclosure. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids. 10 By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial 15 therapeutic response in a subject, preferably a human. In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases when discussing nucleic acid sequences are used. “A” or “a” refers to adenosine, “C” or “c” refers to cytosine, “G” or “g” refers to guanosine, “T” or “t” refers to thymidine, and “U” or “u” refers to uridine. 20 Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s). 25 The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the 30 promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or - 16 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) infusion techniques. The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general 5 knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a 10 recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means. As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no 15 limitation is placed on the maximum number of amino acids that can comprise a proteins or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by 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 20 proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. 25 The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the 30 promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific - 17 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) manner. A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. 5 An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. A “tissue-specific” promoter is a nucleotide sequence which, when operably linked 10 with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen15 from one species may also bind to that antigen from one or more species. But such cross- species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. 20 In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to 25 proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used therein, may be 30 a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine. Preferably, the subject is human. As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated - 18 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In 5 some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro. A “target site” or “target sequence” refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. “Target site” or “target sequence” can also refer 10 to a protein sequence that defines a portion of a protein to which a binding molecule or polypeptide may specifically bind under conditions sufficient for binding to occur. The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state. 15 The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. In the case of a targeted phage, the exogenous nucleic acid is initiated by a ligand- receptor binding event followed by a receptor-mediated internalization event. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, 20 transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The phrase “under transcriptional control” or “operatively linked” as used herein 25 means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide. A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous 30 vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into - 19 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno- associated viral vectors, retroviral vectors, lentiviral vectors, AAVP, and the like. Ranges: Throughout this disclosure, various aspects of the disclosure can be 5 presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 10 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. 15 The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. 20 Description In one aspect, the current disclosure relates to targeted phage‐based platforms for tissue‐specific gene editing which combines the use of two technologies: (1) hybrid adeno‐ associated virus and phage constructs (AAVP) and (2) CRISPR/Cas gene editing systems. 25 In certain embodiments, the platform makes use of the hybrid AAVP vector for phage display‐based ligand‐directed delivery of a nucleic acid. In certain embodiments, incorporated into AAVP as the deliverable transgene component from (1). The merging of these two technologies allows for customization in terms of both tissue‐specificity and gene editing based on the targeting moiety displayed on the capsid and the CRISPR guide 30 RNA (gRNA) encoded within the CRISPR/Cas transgene, respectively. Both the targeting moiety encoding sequence, and the transgene are embedded within the phage genome of AAVP for particle production in bacterial culture. In certain embodiments, the platform collectively is referred to as AAVP‐Cas, AAVP-CRISPR, or AAVP-CRISPR/Cas. - 20 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) Filamentous Phage Display In certain embodiments, the disclosure includes phage particles displaying polypeptides used to target the particles to certain tissues and act as epitopes for 5 stimulating specific immune responses. These polypeptides can be displayed on the surface of the phage particles by being fused to phage coat proteins in a manner similar to that used in phage display. Phage display is a method using bacteriophage particles as scaffolds to display recombinant libraries of peptides or proteins and provide a vehicle to recover and amplify the peptides or proteins that bind to putative ligand molecules or 10 antigens. In some embodiments of the current invention, polypeptides fused to phage coat proteins are used as antigens to stimulate immune responses and to direct the phage particles to specific tissues. In some embodiments, the coat proteins of the phage particles can comprise either an antigenic polypeptide or a tissue-targeting polypeptide. In some embodiments, the phage particles comprise coat proteins that express both a tissue- 15 targeting polypeptide and an antigenic polypeptide. Phage that presents proteins or peptides as a fusion with a phage coat protein are designed to contain appropriate coding regions of the coat proteins. A variety of bacteriophage and coat proteins may be used. Examples include, without limitation, M13 gene III, gene VI, gene VII, gene VIII, and gene IX; fd minor coat protein pIII (Saggio et 20 al., Gene 152:35, 1995); lambda D protein (Sternberg & Hoess, Proc. Natl. Acad. Sci. USA 92:1609, 1995; Mikawa et al., J. Mol. Biol.262:21, 1996); lambda phage tail protein pV (Maruyama et al., Proc. Natl. Acad. Sci. USA 91:8273, 1994; U.S. Patent No. 5,627,024); fr coat protein (WO 96/11947; DD 292928; DD 286817; DD 300652); X29 tail protein gp9 (Lee, Viol.69:5018, 1995); MS2 coat to protein; T4 small outer capsid 25 protein (Ren et al., Protein Sci.5:1833, 1996), T4 nonessential capsid scaffold protein IPIII (Hong and Black, Virology 194:481, 1993), or T4 lengthened fibritin protein gene (Efimov, Virus Genes 10:173, 1995); PRD-1 gene III; Q33 capsid protein (as long as dimerization is not interfered with); and P22 tail spike protein (Carbonell & Villaverde, Gene 176:225, 1996). Techniques for inserting a foreign coding sequence into a phage 30 gene sequence are well known to one of ordinary skill in the art (see e.g., Sambrook et al., Molecular Cloning: A Laboratory Approach, Cold Spring Harbor Press, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Co., NY, 1995). Compared to other bacteriophage, filamentous phage in general are attractive for - 21 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) use as display scaffolds for polypeptides, with M13 being particularly amenable for a number of reasons: (1) the 3D structure of the virion is known; (2) the processing of the coat protein is well understood; (3) the genome is small enough to allow relatively large payload proteins; (4) the sequence of the genome is known; (5) the virion is physically 5 resistant to shear, heat, cold, urea, guanidinium Cl, low pH, and high salt; (6) it is easily cultured and stored, with no unusual or expensive media requirements for the infected cells; (7) it has a high burst size with each infected cell yielding 100 to 1,000 M13 progeny after infection; and (8) it is easily harvested and concentrated. The filamentous phage include: M13, fl, fd, Ifl, Ike, Xf, Pf1, f88.4 or “Type 88” 10 and Pf3. (Webster (1996) Chapter 1, Biology of the Filamentous Bacteriophage, in Kay et al., eds. (1996) Phage Display of Peptides and Proteins). The entire life cycle of the filamentous phage M13, a common cloning and sequencing vector, is well understood in the art. The genetic structure (the complete sequence, the identity and function of the ten genes, and the order of transcription and location of the promoters) of M13 is well known 15 as is the physical structure of the virion. Because the genome is small (6423 bp), cassette mutagenesis is practical on M13, as is single- stranded oligonucleotide directed mutagenesis. The M13 genome is expandable and M13 does not lyse cells. Because the M13 genome is extruded through the membrane and coated by a large number of identical protein molecules, it can be used 20 as a cloning vector. Thus, payload genes can be engineered into M13, and they can be carried along in a stable manner. The fd pIII minor coat protein is a non-limiting outer surface protein utilized in many phage display systems because it is present in only a few copies and because its location and orientation in the virion are known. For example, only three to five copies of 25 protein pIII are displayed on the ends of each phage particle. The limited number of pIII proteins present make peptides fused to them present at a low valency per particle, which is desirable in situations where a limited number of displayed peptides per phage particle is desired; for example, in the selection of high-affinity interactions. In certain embodiments, tissue-targeting and antigenic polypeptides can be fused to the pIII protein 30 such that they are displayed on the surface of the phage particle. Each fd bacteriophage expresses about 2,700 copies of the pVIII major coat protein which are arranged in stacked helical arrays of five proteins. The f88 vectors (including f88-4; GenBank Accession AF218363) are Type 88 vectors, in which the phage genome bears two genes VIII, encoding two different types of pVIII molecule. - 22 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) One pVIII is recombinant (i.e., bears a foreign DNA insert) and is also known as rpVIII and the other wild type. The recombinant gene VIII is synthetic and differs in nucleotide sequence from the wild-type gene (though it largely encodes the wild-type amino acid sequence). The f88 virion is a mosaic, its coat being composed of both wild-type and 5 recombinant pVIII subunits; the latter typically comprise about 150 of the 3900 subunits. This allows hybrid pVIII proteins with quite large foreign peptides to be displayed on the virion surface, even though the hybrid protein by itself cannot support phage assembly. As a result, peptides expressed in fusion with pVIII or rpVIII proteins are present at a relatively high valency of around 200 copies per phage particle. The increased avidity 10 effect of high valency pVIII or rpVIII display permits selection of low-affinity ligands or is advantageous when relatively large amounts of the fused peptide are needed. In certain embodiments of the current disclosure, tissue-targeting and antigenic polypeptides can be fused with the pVIII or rpVIII protein of the engineered phage particles. In some embodiments, the engineered phage particles can express both pIII and pVIII or rpVIII 15 fusion coat proteins such that antigenic peptides can be targeted to specific tissues in order to stimulate optimal immune responses. Bacteriophage as Vaccines Bacteriophages possess a number of qualities that make them ideal candidates for 20 use as vaccine platforms. Phage particles are highly stable under harsh conditions and can be easily and inexpensively produced at large-scale quantities using well-established manufacturing techniques. Phage particles also possess potent adjuvant capabilities, in that they are readily recognized by the mammalian immune system without being pathogenic due to their inability to infect eukaryotic cells. While the use of phage as medical 25 treatments originally focused on their inherent anti-bacterial function, current uses harness their potent immunogenic potential. In certain embodiments of the current disclosure, phage particles are engineered to express specific antigenic polypeptides in fusion with phage coat proteins. In this way, immune recognition and priming against the phage particles also stimulate immune responses against the fusion polypeptide, thus providing 30 beneficial immune responses against specific epitopes. In some embodiments, these immune responses are directed against epitopes derived from the coronavirus proteins, thus acting as an immunotherapy against coronavirus infection or providing protective immunity against a potential coronavirus infection. In some embodiments, the phage particles further comprise elements of adeno-associated virus (AAV) genome and are - 23 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) AAVP hybrid vectors capable of delivering the viral gene or fragments thereof to target cells that will express and present glycosylated viral antigens to the immune system. Adeno-Associated Virus/Phage (AAVP) 5 AAV are relatively small, non-enveloped viruses with a ~4 Kb genome that is flanked by inverted terminal repeats (ITRs). The genome contains two open reading frames, one of which provides proteins necessary for replication and the other provides components required for construction of the viral capsid. Wild-type AAV is typically found in the presence of adenovirus as the adenoviruses provide helper proteins that are 10 essential for packaging of the AAV genome into virions. Therefore, AAV production piggybacks on co-infection with adenovirus and relies on three key elements: the ITR- flanked genome, the open-reading frames, and adeno-helper genes. Due to their non- pathogenic ability to readily infect human cells, AAV is well-studied as a vector for gene delivery. AAV may be readily obtained and their use as vectors for gene delivery has been 15 described in, for example, Muzyczka, 1992; U.S. Patent No.4,797,368, and PCT Publication WO 91/18088. Construction of AAV vectors is known in the literature. AAVP are hybrid vectors combining elements of AAV and filamentous bacteriophage. Namely, AAVP gene expression is under the control of a eukaryotic transgene cassette flanked by internal terminal repeats (ITRs) of AAV2 and inserted in an 20 intergenomic region of a bacteriophage. In this way, the vector combines the specificity of phage vectors with the genetic characteristics of AAV, yielding a virus that can reproduce specifically and easily in prokaryotic cells, yet is able to efficiently infect and transduce mammalian cells with the expression profile similar to AAV. Hence, the AAVP vector possesses favorable characteristics of mammalian and prokaryotic viruses and does 25 not suffer from the disadvantages that those individual vectors normally carry. The advantages of phage or AAVP particles as antigen carrier vaccines are listed: (1) they are highly stable under harsh environmental conditions and their large- scale production is extremely cost-effective if compared to traditional methods used for vaccine production; (2) several studies have demonstrated that phage-based vaccines do 30 not induce detectable toxic side effects and because phage and AAVP do not replicate inside eukaryotic cells, their use is generally considered safe when compared to other classic viral-based vaccination strategies; (3) unlike conventional peptide-based vaccines that may often become inactivated due to minimal temperature excursions (~1oC), phage or AAVP vaccines have no cumbersome and expensive requirements for - 24 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) keeping a stringent so-called “cold-chain” during field applications, particularly in the developing world. In certain embodiments of the current invention, the engineered phage particles of the disclosure further comprise genomic elements of AAV and are AAVP hybrid vectors. 5 In certain embodiments, the AAVP of the disclosure comprise fusion coat proteins comprising tissue-targeting polypeptide that direct the AAVP to cells expressing specific target ligands. In certain embodiments, the AAVP of the disclosure are gene delivery vectors that express exogenous proteins in target cells. For instance, in certain embodiments, the exogenous protein is a viral protein that is expressed in tissue-resident 10 antigen-presenting cells, thereby stimulating an adaptive immune response against the exogenous protein. In certain embodiments, the viral protein is an S protein from a coronavirus, and the AAVP of the disclosure acts as a vaccine or immunotherapy. In some preferred embodiments, the S protein is derived from SARS-CoV-2. In some preferred embodiments, the S protein is derived from MHV. 15 Tissue-Targeting Ligands The cells of the body express unique surface proteins or molecules which account for the extensive morphological and functional diversity of the tissues which they comprise. These unique molecules or groups of molecules can be targeted by specific 20 ligands to deliver agents such as drug or imaging molecules to specific tissues in both in vitro and in vivo experimental models, as well as directly in human patients. These tissue- targeting ligands can be specific for normal tissue, as well as diseases or disorders including but not limited to cancer, viral infections, bacterial infections, or otherwise normal cells involved in disease states. 25 Tissue-targeting polypeptides can take a number of forms, including but not limited to antibodies or antigen-binding fragments thereof, and ligands of receptors expressed by the target cells or fragments thereof. Recent studies have identified that peptides of about 7-15 amino acids in length can bind to cell surface ligands with relatively high affinity and specificity. Given their relatively short length, these ligand- 30 binding polypeptides can be easily attached to molecules or proteins by chemical conjugation or expressed as fusion proteins by genetic engineering. CRISPR/Cas9 The CRISPR/Cas9 system is a facile and efficient system for inducing targeted - 25 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) genetic alterations. Target recognition by the Cas9 protein requires a ‘seed’ sequence within the guide RNA (gRNA) and a conserved di-nucleotide containing protospacer adjacent motif (PAM) sequence adjacent to the gRNA-binding region. The CRISPR/Cas9 system can thereby be engineered to cleave virtually any DNA sequence 5 by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR/Cas9 system can simultaneously target multiple genomic loci by co- expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes. The Cas9 protein and guide RNA form a complex that identifies and cleaves 10 target sequences. Cas9 is comprised of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC. The RecI domain binds the guide RNA, while the Bridge helix binds to target DNA. The HNH and RuvC domains are nuclease domains. Guide RNA is engineered to have a 5’ end that is complementary to the target DNA sequence. Upon binding of the guide RNA to the Cas9 protein, a conformational change 15 occurs activating the protein. Once activated, Cas9 searches for target DNA by binding to sequences that match its protospacer adjacent motif (PAM) sequence. A PAM is a two to six nucleotide base sequence within one nucleotide downstream of the region complementary to the guide RNA. In one non-limiting example, the PAM sequence is 5’-NGG-3’. When the Cas9 protein finds its target sequence with the appropriate PAM, 20 it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. Then the RuvC and HNH nuclease domains cut the target DNA after the third nucleotide base upstream of the PAM. One non-limiting example of a CRISPR/Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Publication No. US20140068797. CRISPRi 25 induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or 30 transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes. CRISPR/Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the - 26 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) target gene. In certain embodiments, the CRISPR/Cas system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, 5 Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleases known in the art, and any combination thereof. In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter, such as 10 one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline). However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector. 15 In certain embodiments, guide RNA(s) and Cas9 can be delivered to a cell as a ribonucleoprotein (RNP) complex. RNPs are comprised of purified Cas9 protein complexed with gRNA and are well known in the art to be efficiently delivered to multiple types of cells, including but not limited to stem cells and immune cells (Addgene, Cambridge, MA, Mirus Bio LLC, Madison, WI). 20 The guide RNA is specific for a genomic region of interest and targets that region for Cas endonuclease-induced double strand breaks. The target sequence of the guide RNA sequence may be within a locus of a gene or within a non-coding region of the genome. In certain embodiments, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 25 37, 38, 39, 40 or more nucleotides in length. Guide RNA (gRNA), also referred to as “short guide RNA” or “sgRNA”, provides both targeting specificity and scaffolding/binding ability for the Cas9 nuclease. The gRNA can be a synthetic RNA composed of a targeting sequence and scaffold sequence derived from endogenous bacterial crRNA and tracrRNA. gRNA is 30 used to target Cas9 to a specific genomic locus in genome engineering experiments. Guide RNAs can be designed using standard tools well known in the art. In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between a target sequence and a guide sequence promotes the formation - 27 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In certain embodiments, a target sequence is located in the 5 nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or nucleus. Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near 10 (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As with the target sequence, it is believed that complete complementarity is not needed, provided this is sufficient to be functional. In certain embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of 15 the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, 20 with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of 25 the coding sequence of a second element, and oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or 30 more in at least one intron, or all in a single intron). In certain embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and - 28 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, 5 transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In certain embodiments, a tagged CRISPR enzyme is used to identify the location of a target 10 sequence. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian and non-mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems 15 include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell (Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy 1:13-26). 20 In certain embodiments, the CRISPR/Cas is derived from a Class II CRISPR/Cas system. In other embodiments, the CRISPR/Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species. The term “Cas9” should be interpreted to include any and all types and/or variants of Cas9. Examples of Cas9 variants include but are not 25 limited to Cas9-nickase (Cas9n), nuclease dead Cas9 (dCas9) fused to the non-specific endonuclease FokI (dCas9-FokI), “enhanced Cas9”, “high-fidelity Cas9”, eSpCas9, spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non-Sp” Cas9s, and full nuclease variants. In general, Cas proteins comprise at least one RNA recognition and/or RNA 30 binding domain. RNA recognition and/or RNA binding domains interact with the guiding RNA. Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. The Cas proteins can be modified to increase nucleic acid binding affinity and/or specificity, alter an - 29 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) enzymatic activity, and/or change another property of the protein. In certain embodiments, the Cas-like protein of the fusion protein can be derived from a wild type Cas9 protein or fragment thereof. In other embodiments, the Cas can be derived from modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can 5 be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, and so forth) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein. In general, a Cas9 protein comprises at least two nuclease (i.e., DNase) domains. For example, a Cas9 protein can comprise a 10 RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains work together to cut single strands to make a double-stranded break in DNA. (Jinek, et al., 2012, Science, 337:816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC- like or an HNH-like nuclease domain). For example, the Cas9-derived protein can be 15 modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent). In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein is able to introduce a nick into a double-stranded nucleic acid (such protein is termed a “nickase”), but not cleave the double-stranded DNA. In any of the above-described embodiments, any or 20 all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and/or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art. In one non-limiting embodiment, a vector drives the expression of the CRISPR 25 system. The art is replete with suitable vectors that are useful in the present invention. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The vectors of the present invention may also be used for 30 nucleic acid standard gene delivery protocols. Methods for gene delivery are known in the art (U.S. Patent Nos.5,399,346, 5,580,859 & 5,589,466, incorporated by reference herein in their entireties). Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4th - 30 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitable 5 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 (e.g., WO 01/96584; WO 01/29058; and U.S. Patent No.6,326,193). Methods of Use 10 In certain embodiments, the current invention provides a method of gene editing in a target cell, comprising contacting the cell with an engineered phage comprising a fusion protein comprising a ligand-binding polypeptide fused to a phage coat protein and a nucleic acid encoding a CRISPR/Cas gene editing system. In certain embodiments, the nucleic acid encoding the CRISPR/Cas gene editing system comprises one or more guide 15 RNAs specific for one or more endogenous DNA sequences and a nucleic acid encoding a Cas protein. In certain embodiments, the guide RNA is operably linked to a first promoter, and the nucleic acid encoding a Cas protein is operably linked to a second promoter. In certain embodiments, the first promoter is a U6 promoter, and the second promoter is a CMV promoter. 20 In certain embodiments, the Cas protein is a wildtype Cas protein. In certain embodiments, the Cas protein is an engineered Cas protein. It is contemplated that any Cas protein or variant thereof known in the art can be used with the CRISPR/Cas systems of the present invention, and that a skilled artisan would be able to select a specific Cas protein appropriate for use. Non-limiting examples of Cas proteins include Cas9, SpCas9, 25 SaCas9, CasΦ, Cas12f, NanoCas, Cas12a, Cas13, Cas9-nickase (Cas9n), nuclease dead Cas9 (dCas9), dCas9 fused to the non-specific endonuclease FokI (dCas9-FokI), an enhanced Cas9, a high-fidelity Cas9, eSpCas9, spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non-Sp” Cas9s, and any variant thereof. In certain embodiments, the Cas protein is a fusion protein, in which the Cas 30 protein is fused with another protein. In certain embodiments, the Cas protein is fused to a deaminase. In certain embodiments, the Cas protein is fused to a reverse transcriptase. In certain embodiments, the fusion protein which comprises a phage coat protein and a ligan-binding polypeptide comprises a ligand-binding polypeptide that binds specifically to a ligand expressed on the surface of the target cell. In certain embodiments, - 31 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) the ligand-binding polypeptide is RGD4C, which comprises the acid sequence of SEQ ID NO: 72. In certain embodiments, the ligand expressed on the surface of the target cell is an αv integrin. In certain embodiments, the fusion protein comprises a phage coat protein selected from the group comprising pIII protein, pVI protein, pVII protein, pVIII protein, 5 rpVIII protein and pIX protein. In certain embodiments, the therapeutic engineered phage is an adeno-associated virus/phage (AAVP). In certain embodiments, the current invention provides a method for treating, ameliorating, and/or preventing cancer in a subject in need thereof, comprising 10 administering to the subject an effect amount of an engineered phage comprising a fusion protein comprising a ligand-binding polypeptide fused to a phage coat protein and a nucleic acid encoding a CRISPR/Cas gene editing system, wherein the CRISPR/Cas gene editing system comprises one or more guide RNAs specific for one or more cancer- related genes and a nucleic acid encoding a Cas protein, and wherein the ligand-binding 15 polypeptide specifically binds a ligand expressed by a cancer cell. In certain embodiments, the one or more cancer-related genes are selected from the group consisting of GRP78, aminopeptidase N (APN), aminopeptidase A (APA), vascular endothelial growth factor receptor 2 (VEGFR2), polo-like kinase 1 (PLK1), interleukin-11 receptor alpha (IL11RA), phosphoinositide 3-kinase catalytic subunit 20 alpha (PI3KCA), and any combination thereof. In certain embodiments, the one or more guide RNAs are operably linked to a first promoter, and the nucleic acid encoding a Cas protein is operably linked to a second promoter. In certain embodiments, the first promoter is a U6 promoter, and the second promoter is a CMV promoter. In certain embodiments, the Cas protein is a wildtype Cas 25 protein. In certain embodiments, the Cas protein is an engineered Cas protein. It is contemplated that any Cas protein or variant thereof known in the art can be used with the CRISPR/Cas systems of the present invention, and that a skilled artisan would be able to select a specific Cas protein appropriate for use. Non-limiting examples of Cas proteins include Cas9, SpCas9, SaCas9, CasΦ, Cas12f, NanoCas, Cas12a, Cas13, Cas9-nickase 30 (Cas9n), nuclease dead Cas9 (dCas9), dCas9 fused to the non-specific endonuclease FokI (dCas9-FokI), an enhanced Cas9, a high-fidelity Cas9, eSpCas9, spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non-Sp” Cas9s, and any variant thereof. In certain embodiments, the Cas protein is a fusion protein comprising a Cas protein fused with another protein. In certain embodiments, the Cas protein is fused to a deaminase. In - 32 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) certain embodiments, the Cas protein is fused to a reverse transcriptase. In certain embodiments, the fusion protein which comprises a phage coat protein and a ligan-binding polypeptide comprises a ligand-binding polypeptide that binds specifically to a ligand expressed on the surface of the target cell. In certain embodiments, 5 the ligand-binding polypeptide is RGD4C, which comprises the acid sequence of SEQ ID NO: 72. In certain embodiments, the ligand expressed on the surface of the target cell is an αv integrin. In certain embodiments, the fusion protein comprises a phage coat protein selected from the group comprising pIII protein, pVI protein, pVII protein, pVIII protein, rpVIII protein, and pIX protein. 10 In certain embodiments, the therapeutic engineered phage is an adeno-associated virus/phage (AAVP). Pharmaceutical Compositions Pharmaceutical compositions of the present disclosure may comprise the 15 engineered phage particles as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate-buffered saline (PBS) and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating 20 agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure are preferably formulated for a number of administration routes including oral, inhalation, nasal, nebulization, intravenous injection, intramuscular injection, subcutaneous injection, and/or transdermal injection. Pharmaceutical compositions of the present disclosure may be administered in a 25 manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient’s disease, and the type and functional nature of the patient’s immune response to the phage particles, although appropriate dosages may be determined by clinical trials. 30 The engineered phage particles of the disclosure can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Phage particle compositions may be administered multiple times at dosages within these ranges. Administration of the phage particles of the - 33 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) disclosure may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art. It can generally be stated that a pharmaceutical composition comprising the engineered phage particles described herein may be administered at a dosage of at least 5 about 107, about 108, about 109, about 1010, about 1011, about 1012, or about 1013 transducing units (TU) or phage particles / kg, including all integer values within those ranges. Dosage size can be adjusted according to the weight, age, and stage of the disease of the subject being treated. Phage particles may also be administered multiple times at these dosages. The phage particles can be administered by using infusion techniques that 10 are commonly known in the art of immunotherapy or vaccinology. The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. The administration of the phage compositions of the disclosure may be carried out 15 in any convenient manner known to those of skill in the art. The phage of the present disclosure may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans- arterially, subcutaneously, intranasally, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously, or 20 intraperitoneally. In other instances, the phage of the disclosure are injected directly into a site of inflammation in the subject, a local disease site in the subject, a LN, an organ, a tumor, and the like. It should be understood that the method and compositions that would be useful in the present disclosure are not limited to the particular formulations set forth in the 25 examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the phage particles, expansion and culture methods, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. The practice of the present disclosure employs, unless otherwise indicated, 30 conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” - 34 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and 5 Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. 10 EXPERIMENTAL EXAMPLES The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the disclosure is not limited to these Examples, but rather encompasses all variations that are evident as a result 15 of the teachings provided herein. The materials and methods employed in these experiments are now described. Genetic engineering of AAVP-CRISPR/Cas constructs. The AAVP backbone, including the phage genome, tetracycline resistance gene, and AAV ITRs, was originally developed from the fUSE5 phage display vector (Hajitou, A., Trepel, M., Lilley, C. E., 20 Soghomonyan, S., Alauddin, M. M., Marini, F. C., 3rd, ... Arap, W. (2006). A hybrid vector for ligand-directed tumor targeting and molecular imaging. Cell, 125(2), 385-398. https://doi.org/10.1016/j.cell.2006.02.042). The version of the AAVP construct used as starting material in the current work contained the RGD4C peptide sequence inserted into the pIII minor coat protein gene. A CRISPR/Cas9 plasmid (Addgene #42230) was modified by25 site-directed mutagenesis for NotI restriction digestion site insertion using the Q5 Site- Directed Mutagenesis Kit (New England BioLabs (NEB) #E0554), with custom primers designed using the NEBaseChanger tool. Plasmid DNA of the AAVP construct and the modified CRISPR/Cas9 plasmid were digested with NotI-HF restriction enzyme (NEB #R3189). Restriction digestion products were run on 1% agarose gels and desired fragments 30 (based on size, 9,194 bp for the AAVP backbone fragment and 5,811 bp for the CRISPR/Cas9 transgene fragment) were extracted from the gels and purified using the QIAquick PCR & Gel Cleanup Kit (Qiagen #28506). The purified fragments were ligated with T4 DNA Ligase (NEB #M0202) using a 3:1 molar ratio of AAVP backbone fragment to CRISPR/Cas9 transgene fragment. The non-targeted AAVP-Cas9 construct, which consists - 35 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) of an insertless wildtype pIII minor coat protein gene, was generated in the same manner. Insertion of the EMX1 gRNA sequence into the AAVP-Cas9 constructs was accomplished in three steps. First, complementary single-stranded DNA oligos containing the guide sequence were annealed to obtain a double-stranded DNA oligo with 5’ overhangs 5 complementary to the dual BbsI restriction digestion site within the CRISPR/Cas9 transgene. Second, AAVP-Cas9 plasmid DNA was digested with BbsI-HF restriction enzyme (NEB #R3539). Third, the double-stranded DNA oligo and AAVP-Cas9 digestion product were ligated with T4 DNA Ligase (NEB #M0202) using a 5:1 molar ratio of DNA oligo to digestion product. Preparation of the various plasmids described above involved chemical 10 transformation of DH5α E. coli (NEB # C2987) and isolation using the QIAGEN Plasmid Maxi Kit (Qiagen #12162). Plasmids were selected for and verified by colony PCR using 20μg/mL tetracycline LB agar plates and primers for DNA regions of interest which were validated by Sanger sequencing outsourced to GENEWIZ (Azenta Life Sciences). AAV ITR elements were sequenced using a specialized ITR Sanger sequencing service provided by 15 GENEWIZ. For all of the above, primer/oligo sequences, specific reaction mixtures, and thermal cycler reaction conditions are listed in Tables 1-4. Bioinformatic sequence analysis, annotation, and modification was performed using SnapGene software version 6.2.1. AAVP-Cas9 particle production and quantification. AAVP-Cas9 particles were produced by electroporation transformation of MC1061 F- E. coli (Lucigen #60514-2) with 20 plasmid DNA of the RGD4C-targeted and non-targeted AAVP-Cas9 constructs. For each construct, transformed bacteria were grown in 500 mL LB containing 20 μg/mL tetracycline on a shaker set to 250 rpm at 37°C overnight (16 hours). Phage particles were precipitated by multistep centrifugation with PEG/NaCl as previously described (Hajitou, A., Trepel, M., Lilley, C. E., Soghomonyan, S., Alauddin, M. M., Marini, F. C., 3rd, Arap, W. (2006). A25 hybrid vector for ligand-directed tumor targeting and molecular imaging. Cell, 125(2), 385- 398.). Briefly, the bacterial liquid culture was centrifuged at 10,000 g for 30 minutes at 4°C allowing for removal of the bacterial pellet and collection of the particle-containing supernatant. PEG/NaCl (15% volume) was added to the supernatant followed by incubation on ice for 2 hours and subsequent centrifugation 10,000 g at 4°C for 30 minutes. The 30 resultant pellet, containing precipitated particles, was resuspended in 10mL of PBS and incubated on a shaker set to 250 rpm at 37°C for 30 minutes. PEG/NaCl (15% volume) was added to the solution, followed by incubation on ice for 30 minutes and subsequent centrifugation 10,000 g at 4°C for 30 minutes. The resultant pellet was resuspended in 1 mL to 2 mL of PBS, centrifuged at 13,000 g at room temperature for 10 minutes, and the - 36 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) supernatant passed through a 0.2 μm filter to remove bacterial debris to recover the final AAVP-Cas9 particle stock. AAVP-Cas9 particles were quantified by bacterial titer as well as qPhage (Dias-Neto et al., 2009). For bacterial titer, kanamycin-resistant K91 E. coli was grown in 10mL TB 5 containing 100μg/mL kanamycin until OD600 = 1.8 was reached. The bacteria were infected with 107, 108, and 109 serial dilutions of AAVP-Cas9 stock by incubation at room temperature for 30 minutes, followed by technical triplicate aliquoting of each dilution on kanamycin-tetracycline LB agar plates and incubated at 37°C overnight (16 hours). The following day colonies were counted and the titer, in transducing units (TU) per μL, was 10 calculated. For quantification by qPhage, 103, 104, 105, and 109 serial dilutions of AAVP-Cas9 particles were prepared. Dilutions of corresponding plasmid DNA (used as a standard) were prepared to contain plasmid copies ranging from 108 copies/μL to 103 copies/μL. Technical triplicates of each particle and plasmid dilution were added to qPhage PCR master mix in 15 designated wells of a MicroAmp Fast Optical 96-Well Reaction Plate with Barcode (Applied Biosystems #4346906) and the PCR reaction was run using a QuantStudio 5 Real- Time PCR System (Applied Biosystems #A28574) with the QuantStudio software set to perform the Relative Standard Curve program. Quantity values for each of the 103, 104, 105, and 109 AAVP-Cas9 particle dilutions were automatically calculated by the QuantStudio 20 software relative to the standard curve generated from the corresponding AAVP-Cas9 plasmid DNA dilutions. Genome copies (GC) per μL were determined by taking the mean of dilution-adjusted quantity values for each dilution. Primer sequences, specific reaction mixtures, and thermal cycler reaction conditions are listed in Tables 1-4. Cell Culture. Kaposi sarcoma KS1767 cells were cultured in Roswell Park Memorial 25 Institute (RPMI) 1640 media containing L-glutamine (Gibco #11875119) and additionally supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (referred to hereafter as complete media). Cells were maintained in T75 flasks at an incubation temperature of 37°C and 5% CO2 air concentration in a humidified incubator. Cells were regularly passaged with 0.05% Trypsin-EDTA (Gibco #25300120) when cells reached 80- 30 90% confluency for up to a total of ten passages. Cell-surface binding and internalization assays. Cell-surface binding of AAVP-Cas9 particles was performed by the BRASIL assay (Giordano et al., 2001). In brief, 109 TU of AAVP-Cas9 particles were incubated with KS1767 cells in 200 μL of RPMI media containing 1% BSA on ice for 2 hours. This aqueous phase was transferred on top of a 200 - 37 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) μL organic phase, composed of a 9:1 v/v mixture of dibutyl phthalate (Sigma-Aldrich #524980) and cyclohexane (Sigma-Aldrich #34855), in an Eppendorf tube. Centrifugation at 10,000 g at room temperature. Tubes were centrifuged at 10,000 g at room temperature and snap frozen in liquid nitrogen. The bottom of the tube containing the cell pellet was cut off 5 using sterile scissors and transferred to a new tube in order to prevent cross-contamination of bound and unbound phage particles in the separated phases. Frozen cell pellets were immersed in 100 μL K91 E. coli for 30 minutes at room temperature in order to recover cell- bound AAVP-Cas9 particles by bacterial infection. Cell/bacteria mixtures were aliquoted on kanamycin-tetracycline LB agar plates and incubated at 37°C overnight (16 hours). Plates 10 were retrieved the following day and colonies were counted to determine TU values. Immunocytochemistry for cellular internalization of AAVP-Cas9 particles was performed as follows. An 8-well chamber slide with KS1767 cells at a density of 1.5 x 105 cells/well in complete media followed by overnight incubation at 37°C and 5% CO2. The next day, cells were blocked with RPMI media containing 30% FBS for 1 hour and 15 subsequently incubated with 109 TU of AAVP-Cas9 particles in RPMI media containing 2% FBS for 2 hours. Cells were then washed 5 times with PBS, washed 5 times with glycine buffer (50 mM glycine, 150mM NaCl), washed 3 times with PBS, fixed with 4% paraformaldehyde/PBS (Electron Microscopy Solutions #15714), washed 3 times with PBS, permeabilized with 0.2% Triton X-100 solution (Sigma-Aldrich #93443), washed 5 times 20 with PBS, and blocked with 1% BSA/PBS at room temperature for 2 hours. Next, cells were incubated with 1:500 anti-phage rabbit polyclonal primary antibody (Sigma-Aldrich #B7786) in 1% BSA/PBS at room temperature for 2 hours, washed 5 times with 1% BSA/PBS, labelled with 1:300 anti-rabbit CY3-conjugated donkey polyclonal secondary antibody (Jackson ImmunoResearch #711-165-152) in 1% BSA/PBS at room temperature 25 for 1 hour in the dark, washed 5 times with 1% BSA/PBS, fixed with 4% paraformaldehyde, and washed 2 times with PBS. Cells were then stained with Vectashield Mounting Medium with DAPI (Vector Laboratories #H-1800) at room temperature for 2 hours in the dark. Images of stained cells were acquired by confocal fluorescence microscopy using an Olympus IX83 inverted microscope. 30 Transduction of cells with AAVP-Cas9 particles. KS1767 cells were seeded in a poly-D-lysine coated 24-well plate (Thermo Scientific #152025) at a density of 2.5x104 cells/well. The following day, cells were treated with 2.0x106 TU/cell of AAVP-Cas9 particles in 200 μL of serum-free RPMI media per well. Cells were incubated on a rotator platform set to 30 rpm at 37°C and 5% CO2 for 4 hours, after which an additional 300 μL of - 38 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) complete media was added per well and the plate returned to stationary incubation at 37°C and 5% CO2. Media was replaced with 500 μL complete media per well the following day and every two days afterwards. One-week post-treatment, cells were harvested either by trypsinization for quantitative real-time PCR or with acutase (Millipore #SCR005) for flow 5 cytometry. For gene editing analysis, this protocol was modified by using 5.0x106 TU/cell of AAVP-Cas9 particles and treating cells twice, with the second treatment two days after the first. Gene expression assays. Quantitative real-time PCR was performed as follows. RNA from harvested cells using the PureLink RNA Mini Kit (Invitrogen #12183025) following 10 the manufacturer’s protocol. Extracted RNA was then used as a template for cDNA library preparation using the ImProm-II Reverse Transcription System (Promega #A3800) following the manufacturer’s protocol. Primers and TaqMan probes specific to the Cas9 coding sequence, and the non-variable gRNA scaffold sequence were used to individually quantify Cas9 mRNA and gRNA levels. Primers and TaqMan probe for the human 18S 15 ribosomal housekeeping gene (Thermo Scientific Hs99999901_s1) were used for gene expression normalization. All probes contained a 6-Carboxyfluorescein (6-FAM) fluorescent dye on the 5’ end as well as a minor groove binder (MGB) and a non-fluorescent quencher (NFQ) on the 3’ end (Applied Biosystems #4331182). Technical triplicates of cDNA library from each treatment condition were added to TaqMan PCR master mix in 20 designated wells of a MicroAmp Fast Optical 96-Well Reaction Plate with Barcode (Applied Biosystems #4346906) and the PCR reaction was run using a QuantStudio 5 Real- Time PCR System (Applied Biosystems #A28574) with the QuantStudio software set to perform the Comparative CT (ΔΔCT) program. CT values were automatically calculated by the QuantStudio software and relative gene expression was manually calculated using the 25 2−ΔΔCт method. For undetectable CT values of untreated control samples, values equal to the greatest detectable untreated control CT values were imputed for calculation. Primer and probe sequences, specific reaction mixtures, and thermal cycler reaction conditions are listed in Tables 2-4. Flow cytometry was performed as follows. Harvested cells were centrifuged at 1200 30 rpm for 1 minute at 4°C to obtain a cell pellet, and the same centrifugation was performed between each washing step. Cells were washed once with PBS, fixed with 1% paraformaldehyde/PBS, washed 2 times with 0.1% BSA/PBS, permeabilized by incubation with methanol on ice for 30 minutes, washed 3 times with 0.1% BSA/PBS, labelled with 1:50 anti-Cas9 PE-conjugated mouse monoclonal antibody (Cell Signaling Technology - 39 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) #35193) in 0.1% BSA/PBS on ice for 2 hours in the dark, and washed 3 times with PBS. Cells were analyzed using a BD Accuri C6 Plus Flow Cytometer configured for 488 nm excitation and FL2 (585 nm ± 20 nm) detection wavelengths and data were processed using FloJo software (version 10.10.0). 5 Design and screening of gRNA candidates. Genomic DNA sequences were analyzed for potential gRNA target sites using the Benchling CRISPR Guide RNA Design Tool (www.benchling.com/crispr). Sequence analysis and annotation were performed using SnapGene software version 6.2.1. Custom gRNA oligos for the five top-scoring gRNA candidates for each gene (as listed in Table 1) were ordered through the Invitrogen 10 TrueGuide Synthetic gRNA service, which provides oligos containing guide sequences appended to the non-variable scaffold sequence. KS1767 cells in a 24-well plate, seeded at a density of 2.5x104 cells/well in 500 µL complete media on the previous day, were co- transfected with gRNA oligos and Cas9 protein (Invitrogen #A36496) using Lipofectamine CRISPRMAX Transfection Reagent (Invitrogen #CMAX00003) following the 15 manufacturer’s protocol. The amounts of gRNA and Cas9 protein used per well were 0.24 µg and 1.25 µg, respectively. Cells were incubated at 37°C and 5% CO2 and were harvested by trypsinization three days post-treatment. Gene editing analysis for each gRNA candidate was performed via Sanger sequencing, as described below. Next-generation and Sanger sequencing. DNA was extracted from harvested cells 20 using the DNeasy Blood & Tissue Kit (Qiagen #69506) following the manufacturer’s protocol. Next-generation sequencing (NGS) was performed by PCR amplification of genomic target sites using NEBNext Ultra II Q5 Master Mix (NEB #M0544) with primers designed to produce amplicons 400-500 bp in size. PCR products were purified using AMPure XP magnetic beads (Beckman Coulter #A63881) following the manufacturer’s 25 protocol. Purified PCR products were submitted to GENEWIZ (Azenta Life Sciences) for Amplicon-EZ, an amplicon NGS service which involves use of an Illumina MiSeq instrument for paired-end sequencing with Illumina partial adapters for read length of 2 x 250 bp and depth of ~50,000 reads, providing raw data in FASTQ format. Raw data was analyzed using CRISPResso2 gene editing analysis software (Clement et al., 2019), with 30 minimum sequence homology for filtering set to 90%. Sanger sequencing of extracted DNA was performed by PCR amplification of genomic target sites using GoTaq DNA polymerase (Promega #M300A) and submission of unpurified PCR products to GENEWIZ (Azenta Life Sciences) for sequencing. Sanger sequencing reads were analyzed using ‘Deconvolution of Complex DNA Repair’ (DECODR) gene editing analysis software (Bloh et al., 2021), - 40 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) Cell proliferation assay. A 96-well RTCA E-plate (Agilent #5232368001) was seeded with either KS1767 cells or MCF7 breast cancer cells (ATCC #HTB-22) at a density of 2.5x103 cells/well in 100 µL complete media and incubated overnight at 37°C and 5% 5 CO2. The following day, cells were co-transfected with gRNA oligos and Cas9 protein (Invitrogen #A36496) using Lipofectamine CRISPRMAX Transfection Reagent (Invitrogen #CMAX00003) following the manufacturer’s protocol. The amounts of gRNA and Cas9 protein used per well were 50 ng and 250 ng, respectively. The 96-well E-plate was then mounted on an Agilent xCELLigence RTCA SP instrument inside an incubator set to 37°C 10 and 5% CO2. The instrument program was set to perform a cell index reading once every hour. Instrument analysis was left to proceed until readings for untreated control cells plateaued (approximately 140 hours for KS1767 cells and 165 hours or MCF7 cells), indicating maximal confluency or inhibition of growth due to media nutrient depletion. Cell Index is a measure of electronic cell-sensor impedance (between proliferating cells and the 15 gold microelectrodes of the RTCA E-plate) and directly correlates with the number of viable cells. Statistical Analysis. All data were analyzed using GraphPad Prism software version 10.1.0. Statistical details specific to each experiment are reported in the respective figure legends. Number of samples (n) represent experimental replicates (not technical replicates), 20 unless otherwise stated. Statistical measurements include mean, mean ± SEM, mean ± SD, and mean ± range. Differences between groups were tested for statistical significance with Welch's t test (assuming unequal variances), with statistical significance set to p < 0.05. Asterisk notations represent statistically significant p values as * p < 0.05 and ** p < 0.005. 25 Table 1: List of gRNA sequences used in this disclosure.
- 41 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
Table 2: List of DNA sequences used for the experimental examples.
- 42 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 43 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
Table 3: Reaction mixtures.
- 44 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 45 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
Table 4: Thermal cycler and incubation conditions.
- 46 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
Table 5: Exemplary AAVP vectors
- 47 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 48 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 49 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 50 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 51 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 52 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 53 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 54 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 55 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 56 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 57 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 58 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 59 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
- 60 - 55699615.3
Attorney Docket No.370602-7080WO1(00277)
Example 1: Methods of biological information transfer Genetic information was initially presumed to be stored within protein, largely attributed to its variability in chemical composition compared to other cellular 5 macromolecules, as was experimentally observable at the time. It was, therefore, highly surprising when the discovery was made in the mid-1900s that nucleic acid is in fact the genetic material of life. Subsequent experimentation revealed the intricate relationship between protein and nucleic acid, more specifically deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Of note, determination of the structure of DNA, identification of 10 RNA as an intermediate messenger, elucidation of the genetic code and its triplet codon nature, among various other works, were of paramount importance. These multiple lines of research painted an illuminating picture of biological information transfer, eloquently summarized by Francis Crick through the ‘Sequence Hypothesis’ and the ‘Central Dogma of Molecular Biology’. It is worth noting that what is transferred is neither matter nor energy, 15 making biological information transfer a truly unique phenomenon of nature. While the Sequence Hypothesis and the more commonly cited Central Dogma are fundamental aspects of molecular biology, they are often misconstrued. The Sequence Hypothesis states that “the specificity of a piece of nucleic acid is expressed solely by the sequence of its bases, and that this sequence is a (simple) code for the amino acid sequence 20 of a particular protein”. In contrast, “the central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred from protein to either protein or nucleic acid” – or, in shorter form – “once (sequential) information has passed into protein it cannot get out again”. The Sequence Hypothesis is, therefore, a ‘positive’ statement describing how information transfer 25 may occur while the Central Dogma is a ‘negative’ statement that imposes a restriction on this information transfer. This distinction is imperative as these two principles lay at the very - 61 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) core of the biological sciences, upon which numerous theoretical and experimental works are grounded. Moreover, the Sequence Hypothesis and Central Dogma have withstood the test of time in terms of scientific validity, despite various proposed challenges. Biological information transfer also deterministically extends to the level of protein structure, whereby 5 the folding of a protein is governed by its amino acid sequence – or, as stated more specifically in Anfinsen’s Thermodynamic Hypothesis, “the native conformation is determined by the totality of interatomic interactions and hence by the amino acid sequence”. Phage engineering for ligand-directed gene transfer 10 Bacteriophage (phage) was the central experimental subject of several of the original groundbreaking studies concerning biological information transfer. These include the aforementioned identification of DNA as genetic material, discovery of the intermediary role of RNA, and elucidation of the genetic code. Phage is among the most extensively studied biological entities, having provided fundamental insights as noted above, as well as through 15 utilization for various applications, including food preservation, antimicrobial therapeutic approaches, and phage display. The latter application is a fundamental aspect of the current work and was recently recognized with a Nobel Prize in Chemistry. Phage display involves the genetic modification of a coat protein to ‘display’ a foreign moiety, typically a peptide, on the virion surface. Filamentous phages M13 and fd, 20 which are non-lytic and have a single-stranded DNA genome, have been the most commonly used strains for phage display applications. For purposes of interaction discovery, a peptide library is utilized, from which the sequences of specific peptides that bind to receptors of interest can be enriched and identified. The most common method by which this is accomplished involves insertion of peptide library-encoding DNA oligos (typically consisting 25 of millions of unique sequences) into the amino terminus region of the pIII minor coat protein gene of filamentous phage ‘fusion’ vectors (FIG.2A). These fusion vectors are propagated in Escherichia coli (E. coli) to produce phage particles displaying the inserted peptides (one peptide per phage particle). The phage particles are then exposed to a receptor of interest that is immobilized onto a plastic surface, in the most basic form of the application (FIG.2B). 30 Unbound phage is removed by washing and receptor-bound phage is recovered by bacterial infection, thereby isolating specific ligand-receptor interactions in an unbiased manner. Multiple rounds of this selection process may be performed for maximal enrichment, and peptide ligand sequences are subsequently determined by sequencing of the insert-containing pIII gene of the recovered phage. - 62 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) The in vivo application of phage display further advanced this technology and its capabilities for the identification of tissue-specific peptide ligands (Pasqualini, R., & Ruoslahti, E. (1996). Organ targeting in vivo using phage display peptide libraries. Nature, 380(6572), 364-366). Screening by in vivo phage display involves systemic administration 5 (via intravenous injection) of phage displaying a peptide library, harvesting tissue of interest following circulation, recovery of phage from the tissue by bacterial infection, repeated rounds of administration/recovery for maximal enrichment, and sequencing of the enriched peptide coding sequence. Bioinformatic analysis and in vitro binding validation are then performed to determine the receptor of the recovered peptide ligand. In vivo phage display 10 led to the discovery of the molecular heterogeneity of the vasculature of different tissues, and that this heterogeneity can be exploited for ligand-directed targeting. Numerous tissue- specific peptide ligands have since been identified by in vivo phage display for both normal and diseased tissues. Particularly relevant to the current work is the cyclic CDCRGDCFC (SEQ ID NO: 72) (abbreviated RGD4C) peptide, which preferentially homes to tumor tissue 15 via αv integrins over-expressed on angiogenic vasculature and tumor cells (Arap, W., Pasqualini, R., & Ruoslahti, E. (1998). Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science, 279(5349), 377-380; Koivunen, E., Wang, B., & Ruoslahti, E. (1995). Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins. Biotechnology (N Y), 13(3), 265-270; 20 Pasqualini, R., Koivunen, E., & Ruoslahti, E. (1997). Alpha v integrins as receptors for tumor targeting by circulating ligands. Nat Biotechnol, 15(6), 542-546). Further building upon in vivo phage display technology, a phage-based vector capable of ligand-directed gene transfer was developed. In brief, a filamentous phage display vector was engineered to display a targeting peptide and carry a transgene flanked by the inverted 25 terminal repeat (ITR) genetic elements of adeno-associated virus (AAV), collectively referred to as ‘AAVP’ (Hajitou, A., Rangel, R., Trepel, M., Soghomonyan, S., Gelovani, J. G., Alauddin, M. M., Arap, W. (2007). Design and construction of targeted AAVP vectors for mammalian cell transduction. Nat Protoc, 2(3), 523-531.; Hajitou, A., Trepel, M., Lilley, C. E., Soghomonyan, S., Alauddin, M. M., Marini, F. C., 3rd, Arap, W. (2006). A hybrid vector 30 for ligand-directed tumor targeting and molecular imaging. Cell, 125(2), 385-398.). Modification of the displayed peptide allows for targeting the vector to a specific tissue in a ligand-directed manner, while the ITRs serve to mediate transgene expression following cellular internalization. Work with AAVP has been primarily focused on tumor-specific delivery of therapeutic and diagnostic (imaging) transgenes using RGD4C and other tumor- - 63 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) targeting peptides, with effective outcomes in mouse models of various cancer types. Mechanistically, AAVP exploits biological information transfer in two different ways. First, the phage display aspect of the vector makes use of a unique sequence-structure relationship, with the targeting peptide being structurally dependent on genetic manipulation of the pIII 5 coat protein. Second, the gene transfer aspect of the vector is entirely dependent on storage of a desired protein product in the form of expressible sequence information within the vector genome. In both cases, biological information transfer from the DNA level to the protein level is required for vector functionality. 10 Programmable gene editing with CRISPR-Cas systems Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) sequences and CRISPR-associated (Cas) proteins function in prokaryotic immunity against foreign nucleic acids. This is accomplished three stages: (i) adaptation, recognition and incorporation of foreign nucleic acid sequences as spacers between repeats in the CRISPR locus; (ii) 15 expression, transcription and processing of the incorporated sequences into CRISPR RNAs (crRNAs); and (iii) interference, crRNA-guided recruitment of Cas effector nucleases against foreign nucleic acid targets by sequence homology. CRISPR/Cas systems have been repurposed for programmable gene editing, generally involving a Cas nuclease and a crRNA- equivalent guide RNA (gRNA) (FIG.3). The programmable aspect refers to designating the 20 sequence of the gRNA such that it directs the Cas nuclease to a DNA target of choice. Once appropriately positioned, the Cas nuclease cleaves the target DNA at a specific distance relative to a protospacer-adjacent motif (PAM) sequence. The result may be gene disruption by insertion/deletion (indel) mutations, or sequence modification in the presence of an appropriately designed template. This capability to edit genetic sequence, specifically by the 25 RNA-guided incorporation of novel sequence information, situates CRISPR/Cas gene editing systems as mechanistically unique mediators of biological information transfer. In light of the technological advancement of programmable gene editing with CRISPR/Cas, this work was recognized with the 2020 Nobel Prize in Chemistry. Since its development, CRISPR/Cas gene editing has garnered widespread use in 30 biological and medical research, particularly for gene therapy applications. While the high specificity of CRISPR/Cas gene editing is greatly advantageous, in vivo delivery of the required CRISPR/Cas components to specific tissue compartments is a major obstacle. Current delivery methods, including direct injection, mammalian viral vectors, and nanoparticles are invasive or have limited specificity. Targeting of mammalian viral vectors - 64 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) and nanoparticles for CRISPR/Cas gene editing is an ongoing area of research and holds considerable potential. While such delivery vehicles offer improved capability in reaching tissues with limited accessibility, such as beyond the blood-brain barrier, they inherently remain subject to non-specific cellular internalization. For disorders of an inherited or 5 congenital nature where all or most cells of the body contain the pathogenic mutation(s) and need to be repaired, this is a desired outcome. However, for disorders where somatic mutations arise only in a specific tissue, such as in various cancers, avoidance of delivery to healthy tissue is desired in order to avoid off-target effects. In either case, addressing the obstacle of tissue-specific CRISPR-Cas delivery has proven to be highly complex. 10 In certain aspects, the examples disclosed herein demonstrate the development of a phage-based vector for ligand-directed delivery of a CRISPR/Cas gene editing system. The experimental paradigm epitomizes how biological information transfer may be employed for practical, and potentially therapeutic, applications. CRISPR/Cas gene editing systems and their unique mode of biological information 15 transfer hold therapeutic promise for a multitude of diseases, including in cancer. However, in vivo delivery of CRISPR/Cas gene editing systems has been a major challenge, both in terms of reaching target tissues as well as sparing healthy tissue from undesired gene editing activity. The hybrid AAVP vector, which combines the ligand-directed targeting attributes of phage display and gene expression enhancement from AAV genetic elements, was 20 hypothesized to be a prospective solution. A previous study demonstrated that phage can be used to deliver a CRISPR/Cas transgene to human cells in vitro, although results were preliminary and further evaluation is required. Herein, AAVP vectors were engineered to comprise a transgene encoding a Cas nuclease and gRNA for the delivery of these components via the RGD4C tumor-targeting 25 peptide in an in vitro setting (FIG.4). In parallel, potentially therapeutic gRNA candidates were screened for editing of genes involved in cancer, specifically the genes encoding glucose-regulated protein 78 (GRP78) and aminopeptidase N (APN). Example 2: Leveraging biological information transfer for phage-based CRISPR/Cas 30 delivery The engineering of a proof-of-concept AAVP-Cas9 construct involved a multi-step strategy consisting of various molecular cloning techniques (FIG.5). The plasmid encoding the AAVP components displaying the RGD4C targeting peptide on the pIII minor coat protein was digested with the NotI restriction enzyme to obtain the AAVP backbone, a DNA - 65 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) fragment ~9.2 kb in length. A CRISPR transgene containing the sequence for Cas9, and the scaffold of the gRNA derived from Streptococcus pyogenes (S. pyogenes) was previously developed. A plasmid containing this transgene (Addgene #42230) was modified by site- directed mutagenesis to contain an additional NotI restriction digestion site for excision of the 5 transgene (~5.8 kb) and compatibility with the AAVP backbone. Following digestion with NotI, the desired DNA fragments of the AAVP backbone and the CRISPR transgene were independently isolated by gel electrophoresis separation and extraction. The two DNA fragments were then ligated, resulting in a plasmid encoding the RGD4C-AAVP-Cas9 construct (~15 kb). The CRISPR transgene contains a dual BbsI restriction digestion site 10 directly upstream of the gRNA scaffold sequence for insertion a desired guide sequence in the form of a double-stranded DNA oligo with BbsI-compatible overhangs. For the proof-of- concept purposes of this work, a guide sequence targeting the empty spiracles homeobox 1 (EMX1) gene, typically used as a benchmark for CRISPR gene editing (Nishimasu et al., 2014; Ran et al., 2013; Tycko et al., 2016), was synthesized and inserted (Tables 1 and 2).15 Collectively, this construct contains the various elements required for ligand-directed phage- based delivery of a CRISPR/Cas gene editing system, as illustrated in FIG.4. Additional AAVP-Cas constructs are contemplated in FIG.1. AAVP-Cas9 particles were produced from both the engineered RGD4C-AAVP-Cas9 plasmid as well as from a similarly generated non-targeted negative control carrying the 20 CRISPR transgene but lacking the RGD4C peptide (NT-AAVP-Cas9). Particle production (FIG.6A) involved transformation of MC1061 F’ E. coli with the AAVP-Cas9 plasmids, large-scale growth of transformed bacteria in liquid media, and particle purification by polyethylene glycol (PEG) precipitation and subsequent filtration. Particles were quantified using two methods: bacterial titration and qPhage, a quantitative real-time PCR approach for25 phage quantification (Dias-Neto et al., 2009). Bacterial titers indicated both RGD4C- displaying and non-targeted AAVP-Cas9 particles were viable, i.e. capable bacterial infection (FIG.6B), and were present in typical quantitative variations with RGD4C-displaying particles yielding a slightly lower titer than non-targeted particles (FIG.6C). Quantification of particle genome copies by qPhage were consistent with bacterial titers (FIG.6D), based on 30 the expected relative fold difference between transducing unit and genome copy values. Particles were also used as a direct template for PCR amplification using primers spanning the CRISPR/Cas transgene, ITRs, and targeting peptide region of the pIII coat protein gene in order to verify the genetic integrity of these elements by Sanger sequencing. Assessments of the in vitro functionality AAVP-Cas9 construct were performed using - 66 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) Kaposi sarcoma KS1767 cells, which have previously been used for proof-of-concept work with AAVP displaying the RGD4C peptide (Hajitou, A., Trepel, M., Lilley, C. E., Soghomonyan, S., Alauddin, M. M., Marini, F. C., 3rd, ... Arap, W. (2006). A hybrid vector for ligand-directed tumor targeting and molecular imaging. Cell, 125(2), 385-398.). Cell- 5 surface binding of AAVP-Cas9 particles was analyzed by the ‘Biopanning and Rapid Analysis of Selective Interactive Ligands’ (BRASIL) assay. As anticipated, BRASIL indicated binding of RGD4C-AAVP-Cas9 but not the non-targeted vector (FIG.6E). To assess cellular internalization, immunocytochemistry was performed using KS1767 cells incubated with AAVP-Cas9 particles and anti-phage antibody. Fluorescence microscopy 10 revealed internalization of the targeted particles but not the non-targeted particles (FIG.6F). Previously established protocols for the AAVP vector outline in vitro transduction and place maximal transgene expression at one-week post-treatment (Hajitou, A., Rangel, R., Trepel, M., Soghomonyan, S., Gelovani, J. G., Alauddin, M. M., Arap, W. (2007). Design and construction of targeted AAVP vectors for mammalian cell transduction. Nat 15 Protoc, 2(3), 523-531.; Hajitou, A., Trepel, M., Lilley, C. E., Soghomonyan, S., Alauddin, M. M., Marini, F. C., 3rd, Arap, W. (2006). A hybrid vector for ligand-directed tumor targeting and molecular imaging. Cell, 125(2), 385-398.). KS1767 cells were treated with AAVP-Cas9 particles and harvested for various analyses following one week. While binding and internalization of the AAVP vector occur potently, post-internalization 20 transgene expression in vitro has been documented to be substantially limited with efficiencies ranging from 2-10%, primarily attributed to endosomal-lysosomal sequestration and degradation of the vector. Therefore, high-sensitivity assays were used to evaluate AAVP-Cas9 functionality. Quantitative real-time PCR (qPCR) showed simultaneous expression of both Cas9 mRNA and gRNA, which are prerequisites for the Cas9/gRNA 25 ribonucleoprotein complex, in cells treated with RGD4C-AAVP-Cas9 (FIG.7A). Flow cytometry using anti-Cas9 antibody allowed for a qualitative and quantitative assessment of Cas9 protein levels, indicating the presence of Cas9 protein although at low levels (FIG. 7B), as anticipated with the limited post-internalization expression efficiency of the AAVP vector. Next-generation sequencing (NGS) of the genomic region targeted by the EMX1 30 gRNA harbored by the RGD4C-AAVP-Cas9 vector revealed precise gene editing with an accumulation of indels at the expected double-strand break site (FIGs.7C and 7D). Collectively, these results substantiate the producibility of viable RGD4C-AAVP- Cas9 particles, demonstrate the targeted binding and internalization properties of the vector, elucidate post-internalization transgene expression at the levels of RNA and protein, as well - 67 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) as demonstrate gene editing at a benchmark target site in an in vitro setting. While post- internalization transgene expression is a limitation in vitro, various efforts for enhancing transduction efficiency of the AAVP vector are underway and AAVP has been an efficacious vector in vivo in numerous prior studies (Staquicini, F. I., Hajitou, A., Driessen, W. H., 5 Proneth, B., Cardo-Vila, M., Staquicini, D. I., Pasqualini, R. (2021). Targeting a cell surface vitamin D receptor on tumor-associated macrophages in triple-negative breast cancer. Elife, 10.; Staquicini, F. I., Smith, T. L., Tang, F. H. F., Gelovani, J. G., Giordano, R. J., Libutti, S. K., Pasqualini, R. (2020). Targeted AAVP-based therapy in a mouse model of human glioblastoma: a comparison of cytotoxic versus suicide gene delivery strategies. Cancer Gene 10 Ther, 27(5), 301-310). In addition to performative evaluation, these data illustrate the various aspects of biological information transfer which are mechanistically inherent to AAVP-Cas, including the sequence-structure relationship of the targeting peptide, transfer of the multi- component CRISPR/Cas transgene, and CRISPR/Cas-mediated gene editing. 15 Example 3: CRISPR/Cas gene editing strategies for cancer In parallel to engineering the AAVP-Cas vector, with anticipation of future in vivo application, various cancer-relevant targets for CRISPR/Cas-mediated gene editing were considered. It has been previously demonstrated that CRISPR/Cas transgenes can accommodate multiple gRNAs while maintaining high levels of editing efficiency. This 20 ‘multiplex’ targeting capability is therapeutically favorable, particularly in cancer where there is mutational and expressional dysregulation of multiple genes. While such dysregulation differs between cancer types, and even intratumorally, some genes may be more broadly suitable as targets for gene editing. The target genes studied here were glucose-regulated protein 78 (GRP78), also known as HSPA5, and aminopeptidase N (APN), also known as 25 CD13. GRP78 is over-expressed in a variety of different cancers, promoting both proliferation and angiogenesis. APN is also over-expressed in various cancers and plays a key role in angiogenesis. Thus, gRNA candidates for disruptive gene editing, i.e. impairing gene product functionality, of both GRP78 and APN were designed and screened. gRNA scaffolds can be designed to target other relevant genes, such as aminopeptidase A (APA), Vascular 30 endothelial growth factor receptor 2 (VEGFR2), polo-like kinase 1 (PLK1), interleukin-11 receptor alpha (IL11RA), and phosphoinositide 3-kinase catalytic subunit alpha (PI3KCA), all with anti-angiogenic or anti-proliferative effect. The gRNA scaffolds can be multiplexed to target several genes simultaneously for greater efficacy (FIG.1). Genomic DNA sequences were obtained from the NCBI Gene database (Brown et al., - 68 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) 2015) and exon sequences were analyzed for potential gRNA target sites using the Benchling CRISPR Guide RNA Design Tool, which provides and scores gRNA candidate sequences based on a predictive algorithm for maximal editing efficiency (Doench et al., 2016). Given the goal of disruptive gene editing, target sites proximal to N-terminal domains were 5 prioritized, as frameshifts caused by indels further upstream are more likely to be detrimental. Five top-scoring gRNA candidates from these regions were selected for screening (FIG.8A, Table 1). DNA from KS1767 cells co-transfected with Cas9 protein and the gRNA candidates, synthesized by Invitrogen, was analyzed for gene editing by Sanger sequencing of target region PCR products (FIGs.8B-8D). Gene editing efficiencies, as determined by 10 ‘Deconvolution of Complex DNA Repair’ (DECODR) software analysis of Sanger sequencing reads, of the gRNA candidates ranged from ~30-80% (FIG.8C). During screening, it was observed that cells treated with the GRP78 gRNA candidate found to have the highest gene editing efficiency were less confluent compared to control conditions (FIG.9A). To examine whether this gRNA inhibited cell proliferation, a 15 pathogenic function of GRP78 in cancer (Lee, 2014), KS1767 and MCF7 breast cancer cells were co-transfected and monitored by real-time cell analysis (RTCA) using an xCELLigence instrument (Agilent Technologies). Co-transfection was performed using Cas9 protein in combination with either the GRP78 gRNA or the EMX1 gRNA, which serves as a positive control for gene editing but does not inhibit proliferation. Proliferation following treatment 20 with the GRP78 gRNA was robustly inhibited in both KS1767 and MCF7 cell lines (FIG. 9B). NGS was also performed using DNA from cells treated with the lead GRP78 and EMX1 gRNAs, revealing accumulation of indels at the expected double-strand break sites and confirming gene editing (FIGs.9C and 9D). Together, these data elucidate the gene editing efficiencies of GRP78 and APN gRNA candidates and highlight a GRP78 gRNA with anti- 25 proliferative therapeutic potential. Example 4: Selected Discussion The discovery and characterization of the transfer of sequence information between biological molecules is arguably one of the most impactful scientific breakthroughs of the 30 past century. This flow of information, characterized specifically as the “residue-by-residue transfer of sequential information”, navigates between nucleic acids to protein and extends deterministically to biological structure. Considering its fundamental nature and long- standing conceptual consistency, biological information transfer may even be deemed to rise to the level of scientific law. While Francis Crick is most frequently credited in this subject - 69 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) area, given his postulation of the Sequence Hypothesis and the Central Dogma, it is important to recognize that many other scientists and their research were of monumental importance. As exemplified by phage display, synthetic gene transfer, and CRISPR gene editing, applications involving biological information transfer have been and continue to be transformative in 5 biology and medicine. The work described in this disclosure pertains to a phage-based construct engineered for ligand-directed delivery of a CRISPR/Cas gene editing system. Various molecular cloning techniques were used for the incorporation of a transgene encoding a Cas9 nuclease and gRNA into a version of the AAVP vector displaying the tumor-targeting RGD4C peptide. 10 The resultant AAVP-Cas9 particles were demonstrated to bind to and internalize in KS1767 cells in a targeted manner. Treatment of KS1767 cells with AAVP-Cas9 particles resulted in simultaneous expression of Cas9 mRNA and gRNA as well as Cas9 protein. Gene editing was also detected in KS1767 cells treated with AAVP-Cas9 particles carrying a benchmark gRNA targeting the EMX1 gene. An anticipated limitation was low in vitro transduction 15 efficiency, which ongoing research efforts seek to address through various strategies, including modification the AAVP vector for the display of endosomal escape peptides as well as gold nanoparticle-mediated transduction of the vector. Engineering and proof-of-concept testing of AAVP-Cas9, as was performed here in an in vitro setting, lays the groundwork for advancing the vector to in vivo experimentation. 20 The manner in which AAVP-Cas leverages biological information transfer is worth addressing, as it allows for a comprehensive delineation of the mechanistic nature of the vector and facilitates the conceptualization of potential therapeutic applications. The three ‘points’ of biological information transfer here include: (i) the sequence-structure relationship of the targeting peptide; (ii) transfer of the multi-component CRISPR/Cas transgene; and (iii) 25 CRISPR/Cas-mediated gene editing. First, display of the targeting peptide, which must be structurally compatible for interaction with its target receptor, is governed by the nucleotide sequence inserted within the pIII gene. While the targeting peptide used in the current work was RGD4C, this sequence may be modified for targeting theoretically any desired tissue, for example lung (Staquicini, D. I., Cardo-Vila, M., Rotolo, J. A., Staquicini, F. I., Tang, F. H. 30 F., Smith, T. L., Arap, W. (2023). Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands. Proc Natl Acad Sci U S A, 120(34), e2220269120.) or adipose. Second, Cas/gRNA ribonucleoprotein expression is dependent upon the transfer of sequence information from the CRISPR/Cas transgene encoding these elements through the transcriptional and translational machinery of the cell. Additionally, - 70 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) modification of the gRNA sequence allows for targeting different genes. Third, the CRISPR/Cas gene editing system delivered by the vector functions to perform gene editing in a gRNA-directed manner. While not specifically explored in the current work, the provision of a nucleotide template containing a desired sequence, either in the form of DNA or RNA, 5 would allow for residue-by-residue transfer of sequence information into the host genome for correcting specific mutations. In relation to this, various gRNA sequences were analyzed for the disruptive editing of GRP78 and APN, both of which are potential therapeutic targets in cancer. For both genes, gRNA candidates with high editing efficiencies were identified. It was also demonstrated that 10 the lead GRP78 gRNA candidate inhibited proliferation in KS1767 Kaposi sarcoma cand MCF7 breast cancer cells in vitro. Given the promotion of proliferation by GRP78 in cancer and the pro-angiogenic function of APN, the GRP78 and APN gRNA candidates were intended for in vivo applications of the RGD4C tumor-targeted AAVP-Cas vector. However, these gRNA candidates are not restricted for use with this vector and may be useful for other 15 therapeutic CRISPR/Cas gene editing strategies. Enumerated Embodiments The following enumerated embodiments are provided, the numbering of which is not 20 to be construed as designating levels of importance. Embodiment 1 provides an engineered phage comprising: (a) a fusion protein comprising a ligand-binding polypeptide fused to a phage coat protein, and (b) a nucleic acid encoding a CRISPR/Cas gene editing system. 25 Embodiment 2 provides the engineered phage of embodiment 1, wherein the nucleic acid encoding the CRISPR/Cas gene editing system comprises one or more guide RNAs specific for one or more endogenous DNA sequences and a nucleic acid encoding a Cas protein. Embodiment 3 provides the engineered phage of embodiment 2, wherein at least one 30 guide RNA is operably linked to a first promoter, and the nucleic acid encoding a Cas protein is operably linked to a second promoter. Embodiment 4 provides the engineered phage of embodiment 2, wherein the Cas protein is a wildtype Cas protein. Embodiment 5 provides the engineered phage of embodiment 2, wherein the Cas - 71 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) protein is an engineered Cas protein. Embodiment 6 provides the engineered phage of embodiment 2, wherein the Cas protein is selected from the group consisting of Cas9, SpCas9, SaCas9, CasΦ, Cas12f, NanoCas, Cas12a, Cas13, Cas9-nickase (Cas9n), nuclease dead Cas9 (dCas9), dCas9 fused 5 to the non-specific endonuclease FokI (dCas9-FokI), an enhanced Cas9, a high-fidelity Cas9, eSpCas9, spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non- Sp” Cas9s, and any variant thereof. Embodiment 7 provides the engineered phage of embodiment 2, wherein the Cas protein is a fusion protein. 10 Embodiment 8 provides the engineered phage of embodiment 7, wherein the Cas protein is fused to a deaminase. Embodiment 9 provides the engineered phage of embodiment 7, wherein the Cas protein is fused to a reverse transcriptase. Embodiment 10 provides the engineered phage of embodiment 1, wherein the fusion 15 protein comprises a ligand-binding polypeptide that binds specifically to a ligand expressed on the surface of a target cell. Embodiment 11 provides the engineered phage of embodiment 10, wherein the ligand-binding polypeptide is RGD4C. Embodiment 12 provides the engineered phage of embodiment 10, wherein the 20 ligand-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 72. Embodiment 13 provides the engineered phage of embodiment 3, wherein the first promoter is a U6 promoter. Embodiment 14 provides the engineered phage of embodiment 3, wherein the second promoter is a CMV promoter. 25 Embodiment 15 provides the engineered phage of embodiment 10, wherein the ligand expressed on the surface of the target cell is an αv integrin. Embodiment 16 provides the engineered phage of embodiment 1, wherein the fusion protein comprises a phage coat protein selected from the group comprising pIII protein, pVI protein, pVII protein, pVIII protein, rpVIII protein, and pIX protein. 30 Embodiment 17 provides the engineered phage of embodiment 1, which is an adeno- associated virus/phage (AAVP). Embodiment 18 provides the engineered phage of embodiment 17, wherein the AAVP is encoded by a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NOs: 73-75. - 72 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) Embodiment 19 provides a method of performing gene editing in a target cell, comprising contacting the cell with an engineered phage comprising: (a) a fusion protein comprising a ligand-binding polypeptide fused to a phage coat protein and (b) a nucleic acid encoding a CRISPR/Cas gene editing system. 5 Embodiment 20 provides the method of embodiment 19, wherein the nucleic acid encoding the CRISPR/Cas gene editing system comprises one or more guide RNAs specific for one or more endogenous DNA sequences and a nucleic acid encoding a Cas protein. Embodiment 21 provides the method of embodiment 20, wherein at least one guide RNA is operably linked to a first promoter, and wherein the nucleic acid encoding a Cas 10 protein is operably linked to a second promoter. Embodiment 22 provides the method of embodiment 20, wherein the Cas protein is a wildtype Cas protein. Embodiment 23 provides the method of embodiment 20, wherein the Cas protein is an engineered Cas protein. 15 Embodiment 24 provides the method of embodiment 20, wherein the Cas protein is selected from the group consisting of Cas9, SpCas9, SaCas9, CasΦ, Cas12f, NanoCas, Cas12a, Cas13, Cas9-nickase (Cas9n), nuclease dead Cas9 (dCas9), dCas9 fused to the non- specific endonuclease FokI (dCas9-FokI), an enhanced Cas9, a high-fidelity Cas9, eSpCas9, spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non-Sp” Cas9s, and 20 any variant thereof. Embodiment 25 provides the method of embodiment 20, wherein the Cas protein is a fusion protein. Embodiment 26 provides the method of embodiment 25, wherein the Cas protein is fused to a deaminase. 25 Embodiment 27 provides the method of embodiment 25, wherein the Cas protein is fused to a reverse transcriptase. Embodiment 28 provides the method of embodiment 19, wherein the fusion protein comprises a ligand-binding polypeptide that binds specifically to a ligand expressed on the surface of a target cell. 30 Embodiment 29 provides the method of embodiment 28, wherein the ligand-binding polypeptide is RGD4C. Embodiment 30 provides the method of embodiment 28, wherein the ligand-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 72. Embodiment 31 provides the method of embodiment 21, wherein the first promoter - 73 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) is a U6 promoter. Embodiment 32 provides the method of embodiment 21, wherein the second promoter is a CMV promoter. Embodiment 33 provides the method of embodiment 28, wherein the ligand 5 expressed on the surface of the target cell is an αv integrin. Embodiment 34 provides the method of embodiment 19, wherein the fusion protein comprises a phage coat protein selected from the group comprising pIII protein, rpVIII protein, pVI protein, pVII protein, pVIII protein, and pIX protein. Embodiment 35 provides the method of embodiment 1, wherein the engineered 10 phage is an adeno-associated virus/phage (AAVP). Embodiment 36 provides the method of embodiment 35, wherein the AAVP is encoded by a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NOs: 73-75. Embodiment 37 provides a method for treating, ameliorating, and/or preventing 15 cancer in a subject in need thereof, the method comprising administering to the subject an effect amount of an engineered phage comprising: (a) a fusion protein comprising a ligand- binding polypeptide fused to a phage coat protein and (b) a nucleic acid encoding a CRISPR/Cas gene editing system; wherein the CRISPR/Cas gene editing system comprises one or more guide RNAs 20 specific for one or more cancer-related genes and a nucleic acid encoding a Cas protein; and wherein the ligand-binding polypeptide specifically binds a ligand expressed by a cancer cell. Embodiment 38 provides the method of embodiment 37, wherein the one or more cancer-related genes are selected from the group consisting of GRP78, aminopeptidase N 25 (APN), aminopeptidase A (APA), vascular endothelial growth factor receptor 2 (VEGFR2), polo-like kinase 1 (PLK1), interleukin-11 receptor alpha (IL11RA), phosphoinositide 3- kinase catalytic subunit alpha (PI3KCA), and any combination thereof. Embodiment 39 provides the method of embodiment 37, wherein the one or more guide RNAs are operably linked to a first promoter, and the nucleic acid encoding a Cas 30 protein is operably linked to a second promoter. Embodiment 40 provides the method of embodiment 37, wherein the Cas protein is n wildtype Cas protein. Embodiment 41 provides the method of embodiment 37, wherein the Cas protein is an engineered Cas protein. - 74 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) Embodiment 42 provides the method of embodiment 37, wherein the Cas protein is selected from the group consisting of Cas9, SpCas9, SaCas9, CasΦ, Cas12f, NanoCas, Cas12a, Cas13, Cas9-nickase (Cas9n), nuclease dead Cas9 (dCas9), dCas9 fused to the non- specific endonuclease FokI (dCas9-FokI), an enhanced Cas9, a high-fidelity Cas9, eSpCas9, 5 spCas9-HF1, HypaCas9, S.pyogenes VQR, EQR and VRER mutants, “non-Sp” Cas9s, and any variant thereof. Embodiment 43 provides the method of embodiment 37, wherein the Cas protein is a fusion protein. Embodiment 44 provides the method of embodiment 43, wherein the Cas protein is 10 fused to a deaminase. Embodiment 45 provides the method of embodiment 43, wherein the Cas protein is fused to a reverse transcriptase. Embodiment 46 provides the method of embodiment 37, wherein the ligand-binding polypeptide is RGD4C. 15 Embodiment 47 provides the method of embodiment 37, wherein the ligand-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 72. Embodiment 48 provides the method of embodiment 39, wherein the first promoter is a U6 promoter. Embodiment 49 provides the method of embodiment 39, wherein the second 20 promoter is a CMV promoter. Embodiment 50 provides the method of embodiment 37, wherein the ligand expressed by the cancer cell is an αv integrin. Embodiment 51 provides the method of embodiment 37, wherein the phage coat protein is selected from the group comprising pIII protein, pVI protein, pVII protein, pVIII 25 protein, rpVIII protein, and pIX protein. Embodiment 52 provides the method of embodiment 37, wherein the engineered phage is an adeno-associated virus/phage (AAVP). Embodiment 53 provides the method of embodiment 52, wherein the AAVP is encoded by a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NOs: 30 73-75. Other Embodiments The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or - 75 - 55699615.3
Attorney Docket No.370602-7080WO1(00277) subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The disclosures of each and every patent, patent application, and publication cited 5 herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations. - 76 - 55699615.3