WO2025097000A1 - Antibody-based methods for treating polycystic kidney disease - Google Patents

Abstract

The present disclosure provides methods for treating a renal disease in a subject in need thereof, comprising a complex of (i) a 3E10 antibody, and (ii) a therapeutic nucleic acid cargo. Compositions comprising the complex and their use are also provided.

Description

ANTIBODY-BASED METHODS FOR TREATING POLYCYSTIC KIDNEY DISEASE

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with support under RC2 DK120534, awarded by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and R35 CA197574, awarded by the National Cancer Institute (NCI) of the National Institutes of Health. The United States government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63/595,323, filed on November 1, 2023, the disclosure of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] The content of the electronically submitted sequence listing (Name: 2681_154PC01_Sequencelisting_ST26.xml; Size: 129,369 bytes; and Date of Creation: October 31, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

[0004] PKD (polycystic kidney disease) is a common genetic disorder that affects about 500,000 people in the United States alone. Polycystic kidney disease is classified into ADPKD (autosomal dominant polycystic kidney disease) and ARPKD (autosomal recessive polycystic kidney disease). In both types of polycystic kidney disease, many cysts develop in the cortex and medulla of the kidney, leading to kidney dysfunction accompanied by substantial atrophy and fibrosis. As the disease progresses, the kidneys develop hypertrophy, leading to kidney failure requiring dialysis. There is a need to develop targeted therapeutics to treat PKD and other kidney diseases that benefit from targeted therapeutic delivery to the diseased organ and its cells. BRIEF SUMMARY OF THE INVENTION

[0005] In some aspects, the present disclosure provides a method for treating a renal disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a complex comprising: (a) a 3E10 antibody or antigen binding fragment thereof, wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of XYGMH (SEQ ID NO: 20), wherein X = D, N, R, L, or K and corresponds to an amino acid residue at position 31 of the 3E10 heavy chain; and (b) a therapeutic nucleic acid cargo.

[0006] In some aspects, the 3E10 antibody or antigen binding fragment thereof comprises: (a) a heavy chain variable region (VH) CDR1 comprising SEQ ID NO: 15 or SEQ ID NO: 9, a VH CDR2 comprising SEQ ID NO: 10, and a VH CDR3 comprising SEQ ID NO: 11; and (b) a light chain variable region (VL) CDR1 comprising SEQ ID NO: 12, a VL CDR2 comprising SEQ ID NO: 13, and a VL CDR3 comprising SEQ ID NO: 14.

[0007] In some aspects, the 3E10 antibody or antigen binding fragment thereof comprises: (a) a heavy chain variable domain (VH) comprising SEQ ID NO: 16; or SEQ ID NO: 18, and (b) a light chain variable domain (VL) comprising SEQ ID NO: 17 or SEQ ID NO: 19; or (c) a heavy chain variable domain (VH) comprising any one of the sequences set forth in SEQ ID NOs: 21-26, and (d) a light chain variable domain (VL) comprising any one of the sequences set forth in SEQ ID NOs: 27-32.

[0008] In some aspects, the 3E10 antibody or antigen binding fragment thereof comprises: (a) a heavy chain comprising SEQ ID NO: 3 or SEQ ID NO: 1, and (b) a light chain comprising SEQ ID NO: 4 or SEQ ID NO: 2.

[0009] In some aspects of the methods disclosed herein, the 3E10 antibody or antigen binding fragment thereof is humanized. In some aspects, the humanized 3E10 antibody or antigen binding fragment thereof comprises: (a) a heavy chain comprising SEQ ID NO: 5 or SEQ ID NO: 55, and (b) a light chain comprising SEQ ID NO: 6 or SEQ ID NO: 68.

[0010] In some aspects of the methods disclosed herein, the complex is a non-covalent complex. In some aspects of the methods disclosed herein, the complex is a covalent complex.

[0011] In some aspects of the methods disclosed herein, the renal disease comprises a hyperproliferative disease or disorder of the kidney. In some aspects, the renal disease comprises polycystic kidney disease, von Hippel Lindau disease, Tuberous Sclerosis, Bardet-Biedel Syndrome, nephronophthisis, or renal cell carcinoma. In some aspects the renal disease is polycystic kidney disease. In some aspects, the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD). In some aspects, the polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD).

[0012] In some aspects of the methods disclosed herein, the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to epithelial cells of a kidney.

[0013] In some aspects of the methods disclosed herein, the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal cells expressing ENT2.

[0014] In some aspects of the methods disclosed herein, the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal cells expressing Na, K+-ATPase.

[0015] In some aspects of the methods disclosed herein, the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal cells expressing ENT2 and Na, K+-ATPase.

[0016] In some aspects of the methods disclosed herein, the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal cells expressing ENT2 and Na, K+-ATPase, wherein the ENT2 and NA, K+-ATPase colocalize on the plasma membrane.

[0017] In some aspects of the methods disclosed herein, the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal tubular epithelial cells. In some aspects, the renal tubular epithelial cells comprise proximal tubule, Loop of Henle, distal tubule, and/or collecting tubule cells.

[0018] In some aspects of the methods disclosed herein, the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to cystic epithelial cells of a kidney.

[0019] In some aspects of the methods disclosed herein, the therapeutic nucleic acid cargo comprises a nucleotide sequence encoding polycystin-1 (PCI) protein, or a portion thereof. In some aspects, the therapeutic nucleic acid cargo comprises polycystin-1 (PCI) C- terminal tail (CTT)-encoding nucleotide sequence. [0020] In some aspects of the methods disclosed herein, the therapeutic nucleic acid cargo comprises the sequence set forth in SEQ ID NO: 8. In some aspects, the sequence of the therapeutic nucleic acid cargo is

ATGGTGATCTTGCGCTGGCGGTATCACGCCCTTCGCGGAGAACTGTACAGACC GGCTTGGGAGCCTCAGGACTACGAGATGGTGGAACTGTTTCTGCGGCGGCTC AGACTTTGGATGGGGCTCTCCAAGGTCAAGGAGTTCAGGCACAAGGTCCGCT TCGAGGGGATGGAACCGTTGCCATCCCGGTCGAGCCGGGGATCTAAAGTGTC GCCCGATGTGCCACCCCCTTCCGCGGGCTCCGACGCCTCCCACCCGAGCACCA GCAGCTCCCAGCTCGACGGCCTGTCGGTGTCCCTGGGCCGGCTGGGTACCCGC TGCGAACCTGAACCCTCGAGACTGCAAGCCGTGTTCGAGGCCCTGCTGACTC AGTTCGACCGCCTGAACCAAGCAACCGAGGACGTGTACCAGCTGGAACAGCA GCTGCATTCGCTGCAAGGAAGGAGATCCTCCCGGGCGCCGGCTGGCTCATCA AGAGGTCCGAGCCCCGGACTGCGCCCCGCCCTCCCTTCCCGCCTCGCCCGGGC CTCCCGGGGAGTGGATCTCGCAACCGGACCGAGCCGAACCCCTCTGAGGGCC AAGAACAAGGTCCACCCCTCCTCAACT (SEQ ID NO: 8).

[0021] In some aspects, the therapeutic nucleic acid encodes an amino acid polypeptide comprising the sequence set forth in SEQ ID NO: 7.

[0022] In some aspects, the therapeutic nucleotide encodes an amino acid polypeptide comprising a PDK1 protein sequence as set forth in any one of SEQ ID NOs: 34-36, or a portion thereof.

[0023] In some aspects of the methods disclosed herein, administration of the complex to the subject suppresses cystic phenotype, thereby treating the subject. In some aspects, administration of the complex to the subject preserves and/or improves renal function, thereby treating the subject. In some aspects, administration of the non-covalent complex to the subject preserves and/or improves renal function, thereby treating the subject.

[0024] In some aspects, the present disclosure provides a method for treating a renal disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a non-covalent complex comprising a 3E10 antibody or antigen binding fragment thereof comprising SEQ ID NO: 1, and a light chain comprising SEQ ID NO: 2, and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8. [0025] In some aspects, the present disclosure provides a method for treating a renal disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a non-covalent complex comprising a 3E10 antibody or antigen binding fragment thereof comprising SEQ ID NO: 3, and a light chain comprising SEQ ID NO: 4, and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8.

[0026] In some aspects, the present disclosure provides a method for treating a renal disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a non-covalent complex comprising a 3E10 antibody or antigen binding fragment thereof comprising SEQ ID NO: 5, and a light chain comprising SEQ ID NO: 6, and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8. In some aspects of the methods disclosed herein, the therapeutic nucleic acid encodes a protein comprising SEQ ID NO: 7.

[0027] In some aspects, the present disclosure provides a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo for use in treating a renal disease in a subject in need thereof, wherein the 3E10 antibody or antigen binding fragment thereof comprises: a heavy chain comprising SEQ ID NO: 1, and a light chain comprising SEQ ID NO: 2; a heavy chain comprising SEQ ID NO: 3, and a light chain comprising SEQ ID NO: 4; or a heavy chain comprising SEQ ID NO: 5, and a light chain comprising SEQ ID NO: 6, and wherein the therapeutic nucleic acid cargo comprising the sequence set forth in SEQ ID NO: 8. In some aspects, the complex is a non-covalent complex. In some aspects, the complex is a covalent complex.

[0028] In some aspects, the present disclosure provides a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of XYGMH (SEQ ID NO: 20), wherein X = D, N, R, L, or K and corresponds to an amino acid residue at position 31 of the 3E10 heavy chain.

[0029] In some aspects, the present disclosure provides a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) CDR1 comprising SEQ ID NO: 15 or SEQ ID NO: 9, a VH CDR2 comprising SEQ ID NO: 10, and a VH CDR3 comprising SEQ ID NO: 11; and a light chain variable region (VL) CDR1 comprising SEQ ID NO: 12, a VL CDR2 comprising SEQ ID NO: 13, and a VL CDR3 comprising SEQ ID NO: 14.

[0030] In some aspects, the present disclosure provides a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen-binding fragment thereof comprises: a heavy chain variable domain (VH) comprising SEQ ID NO: 16; or SEQ ID NO: 18, and a light chain variable domain (VL) comprising SEQ ID NO: 17 or SEQ ID NO: 19; or a heavy chain variable domain (VH) comprising any one of the sequences set forth in SEQ ID NOs: 21-26, and a light chain variable domain (VL) comprising any one of the sequences set forth in SEQ ID NOs: 27-32.

[0031] In some aspects, the present disclosure provides a composition comprising a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 1, and a light chain comprising SEQ ID NO: 2.

[0032] In some aspects, the present disclosure provides a composition comprising a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 3, and a light chain comprising SEQ ID NO: 4.

[0033] In some aspects, the present disclosure provides a composition comprising a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising the sequence set forth in SEQ ID NO: 8, wherein the 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 5, and a light chain comprising SEQ ID NO: 6. In some aspects, the therapeutic nucleic acid encodes an amino acid polypeptide comprising the sequence set forth in SEQ ID NO: 7.

[0034] In some aspects of the compositions described herein, the complex is a non-covalent complex. In some aspects of the compositions described herein, the complex is a covalent complex. [0035] In some aspects, the present disclosure provides a pharmaceutical composition comprising the composition disclosed herein, and a pharmaceutically acceptable excipient. [0036] In some aspects, the present disclosure provides a pharmaceutical kit comprising the pharmaceutical composition disclosed herein, and instructions for use thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0037] FIG. 1 shows In Vivo Imaging System (IVIS) images of KPC tumor, kidney, heart, liver, and skeletal muscle tissues of C57B1/6 mice bearing flank KPC tumors 24 hours postinjection. Brighter (colored) foci show GFP signal intensity.

[0038] FIG. 2 shows immunofluorescence (IF) images of wild-type C57B1/6 (WT) and Pkdl^fl/fl; Pax8^rtTA;TetO-Cre (Pkdl- G) mouse kidneys. ENT2 is shown in in the first row. Basolateral plasma membrane marker Na, K⁺-ATPase is shown as the brightest intensity white. Arrows indicate colocalization of ENT2 and Na, K+-ATPase. Proximal tubules (PT) are shown.

[0039] FIG. 3 shows IVIS images of WT and Pkdl- O mouse kidneys 24 hours postinjection. Brighter foci show IR700 signal intensity.

[0040] FIG. 4 shows IVIS images of Pkdl-KQ mouse kidneys 24 hours post-injection. Brighter foci show GFP signal intensity.

[0041] FIG. 5 shows IF images of Pkdl-KQ mouse kidneys 24 hours post-injection. Cell nuclei DAPI staining and GFP are shown as low and medium bright intensity. Na, K⁺- ATPase is shown as the brightest intensity.

[0042] FIG. 6 shows IF images of Pkdl- Q mouse kidneys 24-hours after the final injection. The first column shows NNT. The middle column shows PCI CTT (detected by Rat anti-HA). The third column shows the images merged. Cell nuclei are shown (DAPI) throughout.

[0043] FIG. 7 shows IF images of Pkdl- Q mouse kidneys 24-hours after the final injection, and untreated control. The top row shows NNT. The bottom row shows PCI CTT (detected by Rat anti-HA).

[0044] FIG. 8 shows Western Blot (WB) images of whole protein lysates of Pkdl-KO mouse kidneys 24-hours after the final injection. PCI CTT was detected using an antibody to the HA epitope tag. [0045] FIG. 9 shows WB images of protein lysates of Pkdl- O mouse kidneys 24-hours after the final injection after immunoprecipitation with anti -HA magnetic beads. PCI CTT was detected using an antibody to the HA epitope tag.

DETAILED DESCRIPTION

[0046] As described herein, there is a need for targeted delivery of therapeutics to treat PKD and other kidney diseases. It is challenging to deliver therapeutic nucleic acids to cystic epithelial cells of the kidney because, for example, the lumens of the cyst are generally disconnected from the lumens of the tubules, potentially limiting direct access from the glomerular filtrate to the apical surfaces of cyst cells. In addition, fibrotic tissue often surrounding cysts further complicates delivery of agents from the systemic circulation to the basolateral surfaces of the cyst cells. The 3E10 antibody, can transport a variety of biologically important molecules into target cells, by penetrating the plasma and nuclear membranes of a cell via the nucleoside transporter ENT2. Accordingly, it has been shown that 3E10 can successfully deliver therapeutic proteins and nucleic acids in vitro and in vivo. Importantly, the 3E10 antibody-mediated delivery has not shown any cellular toxicity in vitro or in vivo. In contrast, other delivery approaches and antibodies that penetrate living cells are frequently toxic or injurious and can trigger some of the pathologic manifestations of the autoimmune diseases in which they are found. Surprisingly, the present disclosure demonstrates that a 3E10 antibody or an antigen-binding fragment thereof, in complex with a therapeutic nucleic acid can target and deliver the therapeutic nucleic acid to cystic epithelial cells of the kidney.

A. Definitions

[0047] The terminology used in the present disclosure is for the purpose of describing particular aspects only and is not intended to be limiting.

[0048] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and/or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. Unless the context requires otherwise, it will be further understood that the terms “includes,” “comprising,” or any variation thereof, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” Additionally, where the terms “comprising,” “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, alternatives reciting “consisting of’ or “consisting essentially of’ are intended to be encompassed within such disclosures.

[0049] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0050] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/- 10%.

[0051] “Polynucleotide” or “nucleic acid” means a linear polymer of natural or modified nucleosidic monomers linked by phosphodiester bonds or analogs thereof. The term “polynucleotide” usually refers to polymers comprising from about 100 monomers to many thousands of monomers, e.g., 10,000 monomers, or more. Polynucleotides may be natural or synthetic. Polynucleotides can include deoxyribonucleosides, ribonucleosides, and nonnatural analogs thereof, provided that they are capable of specifically binding to a target genome by way of a regular pattern of monomer-to-monomer interactions, such as Watson- Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.

[0052] As used herein, the term “subject” means any individual who is the target of administration. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The term does not denote a particular age or sex.

[0053] The term “effective amount” refers to an amount that is non-toxic to a subject or a majority of normal cells, but is an amount of the active agent that is sufficient to provide a desired effect. This amount can vary from subject to subject, depending on the species, age, and physical condition of the subject, the severity of the disease that is being treated, the particular conjugate, or more specifically, the particular active agent used, its mode of administration, and the like.

[0054] The term “pharmaceutically acceptable” refers to the fact that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. For example, the carrier, diluent, or excipient or composition thereof can be administered to a subject along with the 3E10 antibody or antigen-binding fragment thereof and the therapeutic agent of the disclosure without causing any undesirable biological effects or interacting in an undesirable manner with any of the other components of the pharmaceutical composition in which it is contained. Pharmaceutical compositions including the 3E10 antibody or antigen-binding fragment thereof and the therapeutic agent can be administered by any suitable means, for example, parenterally, such as by subcutaneous, intravenous, intramuscular, intrathecal, or intraci sternal injection or infusion techniques (e.g., as sterile injectable aqueous or nonaqueous solutions or suspensions) in dosage formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents. In some aspects the conjugate is administered parenterally or intravenously.

[0055] As used herein, the term “treat,” and grammatical variants thereof, refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0056] The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses polyclonal antibodies, monoclonal antibodies, antibody fragments (such as Fab, Fab’, F(ab’)2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.

[0057] Traditional antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present disclosure is directed to antibodies that generally are based on the IgG class, which has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. In general, IgGl, IgG2 and IgG4 are used more frequently than IgG3. It should be noted that IgGl has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M).

[0058] The light chain generally comprises two domains, the variable light domain (containing the light chain CDRs and together with the variable heavy domains forming the Fv region), and a constant light chain region (often referred to as CL or CK). The heavy chain comprises a variable heavy domain and a constant domain, which includes a CH1- optional hinge-Fc domain comprising a CH2-CH3.

[0059] The terms “antigen binding domain” or “ABD” mean a set of six Complementary Determining Regions (CDRs) that, when present as part of a polypeptide sequence or sequences, specifically binds a target antigen. As is known in the art, these CDRs are generally present as a first set of variable heavy CDRs (vhCDRs or VHCDRs, which are used interchangeably throughout this disclosure) and a second set of variable light CDRs (vlCDRs or VLCDRs which are used interchangeably throughout this disclosure), each comprising three CDRs: vhCDRl, vhCDR2, vhCDR3 for the heavy chain and vlCDRl, vlCDR2 and vlCDR3 for the light chain. The CDRs are present in the variable heavy and variable light domains, respectively, and together form an Fv region. Thus, in some cases, the six CDRs of the antigen binding domain are contributed by a variable heavy and a variable light domain. In a “Fab” format, the set of 6 CDRs are contributed by two different polypeptide sequences, the variable heavy domain (vh or VH; containing the vhCDRl, vhCDR2 and vhCDR3) and the variable light domain (vl or VL; containing the vlCDRl, vlCDR2 and vlCDR3), with the C-terminus of the vh domain being attached to the N- terminus of the CHI domain of the heavy chain and the C-terminus of the vl domain being attached to the N-terminus of the constant light domain (and thus forming the light chain).

[0060] For all positions discussed in the present disclosure that relate to antibodies, unless otherwise noted, amino acid position numbering is according to the EU index. The EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody. Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof. See SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat et al.; Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, the contents of which are incorporated herein by reference.

[0061] “Target antigen,” as used herein, means the molecule that is bound specifically by the antigen binding domain comprising the variable regions of a given antibody.

[0062] “Fab” or “Fab region” as used herein, means a polypeptide that comprises the VH, CHI, VL, and CL immunoglobulin domains, generally on two different polypeptide chains (e.g. VH-CH1 on one chain and VL-CL on the other). Fab can refer to this region in isolation, or this region in the context of an antibody of the disclosure. In the context of a Fab, the Fab comprises an Fv region in addition to the CHI and CL domains.

[0063] “Fv” or “Fv fragment” or “Fv region” as used herein, means a polypeptide that comprises the VL and VH domains of an antibody binding domain. Fv regions can be formatted as both Fabs (as discussed above, generally two different polypeptides that also include the constant regions as outlined above) and scFvs, where the vl and vh domains are combined (generally with a linker as discussed herein) to form an scFv.

[0064] “Single chain Fv” or “scFv” as used herein, means a variable heavy domain covalently attached to a variable light domain, generally using a scFv linker as discussed herein, to form a scFv or scFv domain. A scFv domain can be in either orientation from N- to C-terminus (vh-linker-vl or vl-linker-vh). In the sequences depicted in the sequence listing and in the figures, the order of the vh and vl domain is indicated in the name, e.g. H.X L.Y means N- to C-terminal vh-linker-vl, and L.Y H.X is vl-linker-vh.

[0065] “Fc” or “Fc region” or “Fc domain” as used herein, means the polypeptide comprising the CH2-CH3 domains of an IgG molecule, and in some cases, inclusive of the hinge. In EU numbering for human IgGl, the CH2-CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230. Thus the definition of “Fc domain” includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. An “Fc fragment” in this context can contain fewer amino acids from either or both of the bland C-termini but still retains the ability to form a dimer with another Fc domain or Fc fragment as can be detected using standard methods, generally based on size (e.g. nondenaturing chromatography, size exclusion chromatography, etc.). Human IgGFc domains are of particular use in the present disclosure, and can be the Fc domain from human IgGl, IgG2 or IgG4.

[0066] A “variant Fc domain” contains amino acid modifications as compared to a parental Fc domain. Thus, a “variant human IgGl Fc domain” is one that contains amino acid modifications (generally amino acid substitutions, although in the case of ablation variants, amino acid deletions are included) as compared to the human IgGl Fc domain. In general, variant Fc domains have at least about 80, about 85, about 90, about 95, about 97, about 98 or about 99 percent identity to the corresponding parental human IgG Fc domain (using the identity algorithms discussed below, with one aspect utilizing the BLAST algorithm as is known in the art, using default parameters). Alternatively, the variant Fc domains can have from 1 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acid modifications as compared to the parental Fc domain. Additionally, as discussed herein, the variant Fc domains herein still retain the ability to form a dimer with another Fc domain as measured using known techniques as described herein, such as nondenaturing gel electrophoresis.

[0067] “Heavy chain constant region” as used herein, means the CHl-hinge-CH2-CH3 portion of an antibody (or fragments thereof), excluding the variable heavy domain; in EU numbering of human IgGl this is amino acids 118-447. By “heavy chain constant region fragment” herein is meant a heavy chain constant region that contains fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another heavy chain constant region. [0068] “Variable region” or “variable domain” as used herein, means the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the VK, V , and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively, and contains the CDRs that confer antigen specificity. Thus, a “variable heavy domain” pairs with a “variable light domain” to form an antigen binding domain (“ABD”). In addition, each variable domain comprises three hypervariable regions (“complementary determining regions,” “CDRs”) (vhCDRl, vhCDR2 and vhCDR3 for the variable heavy domain and vlCDRl, vlCDR2 and vlCDR3 for the variable light domain) and four framework (FR) regions, arranged from aminoterminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4.

[0069] In some aspects, the antibody is a bispecific antibody. Bispecific antibodies and other binding proteins having a first heavy chain and a first light chain from 3E10 and a second heavy chain and a second light chain from a monoclonal antibody that specifically binds a second target are discussed in Weisbart, et al., Mol. Cancer Ther., 11 (10):2169-73 (2012), and Weisbart, et al., Int. J. Oncology, 25: 1113-8 (2004), and U.S. Patent Application No. 2013/0266570, which are specifically incorporated by reference in their entireties. In some aspects, the second target is specific for a target cell-type, tissue, organ etc. Thus the second heavy chain and second light chain can serve as a targeting moiety that targets the complex to the target cell-type, tissue, organ.

[0070] Bispecific antibodies can be used to direct therapeutic nucleic acid cargo to cells that express a particular antigen. These antibodies possess two binding sites directed at two different antigens or two different epitopes on the same antigen. For example, in some aspects the bispecific can comprise one arm for ENT2 engagement and another arm for a second target. Bispecific antibody design can include a variety of antibody designs with multiple binding arms. Techniques for making bispecific antibodies are common in the art (Millstein et al., 1983, Nature 305:537-539; Brennan et al., 1985, Science 229:81; Suresh et al, 1986, Methods in Enzymol. 121 : 120; Traunecker et al., 1991, EMBO J. 10:3655- 3659; Shalaby et al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J. Immunol. 148: 1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; and U.S. Patent 5,731,168). Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147:60 (1991)). [0071] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

B. Renal Diseases

[0072] The methods disclosed herein can be used to treat one or more renal diseases. In some aspects, the renal disease or disorder is caused by hyperproliferative cells in the kidney. Accordingly, in some aspects, the renal disease or disorder comprises a hyperproliferative disease or disorder of the kidney. Hyperproliferative diseases and disorders of the kidney are known and can be caused by a variety of factors. In some aspects the hyperproliferative disease or disorder of the kidney comprises an inherited genetic disease or disorder, wherein the progeny inherit one or more disease causing gene mutations from the parents. In some aspects the hyperproliferative disease or disorder is not a cancer. Nonlimiting examples of such diseases and disorders include polycystic kidney disease (PKD), von Hippel Lindau Syndrome, Tuberous Sclerosis, and recessive forms of cystic disease, such as Autosomal Recessive Polycystic Kidney Disease (ARPKD), Bardet Biedel Syndrome and Nephronophthisis.

[0073] Alternatively, in some aspects, the hyperproliferative disease or disorder of the kidney is not an inherited genetic disease or disorder. In some aspects the hyperproliferative disease or disorder of the kidney comprises a neoplasm and/or cancer of the kidney. Kidney cancer typically results from an acquired mutation in a gene that causes aberrant and uncontrolled cell proliferation, clonal expansion, and/or potential metastasis to other tissues. Nonlimiting examples of kidney cancers include renal cell carcinoma (RCC), clear cell renal cell carcinoma, non-clear cell renal cell carcinomas including papillary renal cell carcinoma, chromophobe renal cell carcinoma, collecting duct RCC, multilocular cystic RCC, medullary carcinoma, mucinous tubular and spindle cell carcinoma, neuroblastoma- associated RCC. Other types of kidney cancer include transitional cell carcinoma, urothelial carcinoma, Wilms tumor and renal sarcoma. In some aspects, the hyperproliferative disease or disorder of the kidney can be a benign (z.e., non-cancer) tumor, which does not metastasize. Nonlimiting examples of such benign tumors include angiomyolipomas and oncocytomas.

[0074] In some aspects, the renal disease is polycystic kidney disease (PKD). In some aspects, the renal disease is autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1, which encodes polycystin-1 (PCI), cause -78% of autosomal ADPKD. PCI is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. Mutations in PDK2, which encodes polycystin-2 (PC2) can also cause ADPKD, but are less common. ADPKD is a disease that causes fluid-filled cysts to develop in the kidney, eventually leading to kidney failure. In some aspects, atypical ADPKD can be caused by rare mutations in DnaJ heat shock protein family (HSP4) member Bl l (DNAJB11) or intraflagellar transport 140 (IFT140). In some aspects, the renal disease is autosomal recessive polycystic kidney disease (ARPKD). Mutations in ciliary IPT domain containing fibrocystin/polyductin (PHKD1) cause ARPKD. ARPKD is a recessive form of PKD that causes poor kidney function due to fluid filed kidney cysts. In some aspects, the 3E10 antibody or antigen binding fragment thereof can deliver the therapeutic nucleic acid cargo to epithelial cells of a kidney. In some aspects, the 3E10 antibody or antigen binding fragment thereof can deliver the therapeutic nucleic acid cargo to cystic epithelial cells of a kidney.

[0075] In some aspects, the present disclosure provides a complex comprising a 3E10 antibody and a therapeutic nucleic acid cargo for administration to a subject suffering from a renal disease, where the administration of the complex can treat the renal disease. In some aspects, the present disclosure provides a complex comprising a 3E10 antibody and a therapeutic nucleic acid cargo for administration to a subject suffering from a renal disease, where the administration of the complex can reduce one or more symptoms of the renal disease. In some aspects, the complex can be administered to a subject to suppress the cystic phenotype of PKD. In some aspects, the complex can be administered to a subject to suppress the cystic phenotype of ADPKD. In some aspects, the complex can be administered to a subject with PKD to preserve renal function. In some aspects, the complex can be administered to a subject with PKD to halt disease progression.

C. Methods of Treatment

[0076] In some aspects, the present disclosure provides methods for using 3E10 to enhance delivery of at least one therapeutic nucleic acid cargo. In some aspects, more than one therapeutic nucleic acid cargo can be delivered. In some aspects, an effective amount of a 3E10 antibody or antigen-binding fragment thereof and one or more therapeutic nucleic acid cargos are combined in a complex to facilitate delivery. The complexes can be systemically administered to deliver the therapeutic nucleic acid in vivo upon contact with cells. In some aspects, the cell contacted with the complex expresses ENT2.

[0077] 3E10 antibodies and antigen-binding fragments thereof can non-covalently bind to nucleic acids. In some aspects, a 3E10 antibody and a therapeutic nucleic acid cargo form a non-covalent complex. For example, in some aspects, the interaction between the 3E10 antibody and the therapeutic nucleic acid cargo is non-covalent. In some aspects, the 3E10 antibody or antigen-binding fragment thereof is contacted with a therapeutic nucleic acid cargo whose delivery into cells is desired prior to administration and/or contact with a cell. For example, a therapeutic nucleic acid cargo and a 3E10 antibody or antigen-binding fragment thereof can be mixed in solution for sufficient time for the nucleic acid cargo and antibody to form non-covalent complexes. In other aspects, a therapeutic nucleic acid cargo and a 3E10 antibody or antigen-binding fragment thereof can be administered to or added to cells separately, and the complexes are formed in the presence of the cells. Therefore, the non-covalent complexes can be formed in vitro or in vivo, before contacting a cell.

[0078] In some aspects, a 3E10 antibody and a therapeutic nucleic acid cargo form a covalent complex. In some aspects, a covalent complex can comprise a linker. A “linker” is any chemical moiety that is capable of linking a compound, for example, a therapeutic nucleic acid cargo, to a cell-binding agent such as a 3E10 antibody or a fragment thereof, in a stable, covalent manner. Linkers can be susceptible to acid-induced cleavage, light- induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers, and hydrophilic forms thereof as described herein and known in the art.

[0079] A therapeutic nucleic acid cargo encoding a polypeptide of interest or functional nucleic acid, can be delivered into cells using a 3E10 antibody for expression of a polypeptide in the cells. The compositions and methods can be used over a range of different applications.

[0080] In some aspects, one or more particular cell types or tissue is the target of the disclosed complexes. In some aspects, the target cell is a kidney cell. In some aspects, the 3E10 antibody or antigen binding fragment thereof can deliver a therapeutic nucleic acid cargo to epithelial cells of a kidney. In some aspects, the 3E10 antibody or antigen binding fragment thereof can deliver a therapeutic nucleic acid cargo to cystic epithelial cells of a kidney.

[0081] For example, an effective amount or therapeutically effective amount of the composition can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, or to otherwise provide a desired pharmacologic and/or physiologic effect, for example, reducing, inhibiting, or reversing one or more of the pathophysiological mechanisms underlying a disease or disorder. The formulation of the composition is made to suit the mode of administration. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions containing the complexes. The precise dosage will vary according to a variety of factors such as subject- dependent variables (e.g., age, immune system health, clinical symptoms etc.).

[0082] In some aspects, in vivo delivery of nucleic acid cargo to cells is used for the treatment of a disease or disorder in a subject. For example, the composition can be administered directly to a subject for in vivo therapy.

[0083] In general, methods of administering compounds, including antibodies, are well known in the art. In particular, the routes of administration already in use for nucleic acid therapeutics, along with formulations in current use, provide preferred routes of administration and formulation for the therapeutic nucleic acid cargo described above.

[0084] The compositions can be administered by a number of routes including, but not limited to, intravenous, intraperitoneal, intraamniotic, intramuscular, subcutaneous, or topical (sublingual, rectal, intranasal, pulmonary, rectal mucosa, and vaginal), and oral (sublingual, buccal). Administration of the formulations may be accomplished by any acceptable method that allows the complexes to reach their targets. The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being treated. Accordingly, in some aspects, the compositions can be administered systemically to target kidney cells. Compositions and methods for in vivo delivery are also discussed in WO 2017/143042.

[0085] The methods can also include administering an effective amount of the antibody- nucleic acid complex composition to an embryo or fetus, or the pregnant mother thereof, in vivo. In some methods, compositions are delivered in utero by injecting and/or infusing the compositions into a vein or artery, such as the vitelline vein or the umbilical vein, or into the amniotic sac of an embryo or fetus. See, e.g., Ricciardi, et al., Nat Commun. 2018 Jun 26;9(1):2481. doi: 10.1038/s41467-018-04894-2, and WO 2018/187493. In some aspects, the methods exclude administering an effective amount of the antibody-nucleic acid complex composition to an embryo or fetus, or the pregnant mother thereof.

D. 3E10 Antibodies

[0086] As used herein, the term “cell-penetrating antibody” refers to an immunoglobulin protein, fragment, variant thereof, or fusion protein based thereon that is transported into the cytoplasm and/or nucleus of living mammalian cells. Cell-penetrating antibodies for use in the compositions and methods described herein can be anti-DNA antibodies. The cell-penetrating antibody can bind single stranded DNA and/or double stranded DNA. The cell-penetrating antibody can be an anti-RNA antibody (e.g., the antibody specifically binds RNA). In some aspects, the antibody is transported into the cytoplasm of the cells without the aid of a carrier or conjugate. In some aspects, the cell-penetrating antibody is transported in the nucleus with or without a carrier or conjugate. Although the cellpenetrating molecules are generally referred to herein as “cell-penetrating antibodies,” it will be appreciated that fragments, including antigen-binding fragments, variants, binding proteins and fusion proteins such as scFv, di-scFv, tri-scFv, and other single chain variable fragments, and other cell-penetrating molecules disclosed herein are also expressly provided for use in compositions and methods disclosed herein. Autoantibodies to doublestranded deoxyribonucleic acid (dsDNA) are frequently identified in the serum of patients with systemic lupus erythematosus (SLE) and are often implicated in disease pathogenesis. Therefore, in some aspects, cell-penetrating antibodies (e.g., cell-penetrating anti-DNA antibodies) can be derived or isolated from patients with SLE or animal models of SLE.

[0087] In some aspects, the present disclosure relates to the use of 3E10 antibodies, and derivatives thereof, for delivering therapeutic agents to a subject. Although generally referred to herein as “3E10” or “3E10 antibodies,” it will be appreciated that fragments and binding proteins, including antigen-binding fragments, variants, and fusion proteins such as scFv, di-scFv, tr-scFv, and other single chain variable fragments, and other cellpenetrating, nucleic acid transporting molecules disclosed herein, are encompassed by the phrase and are also expressly provided for use in compositions and methods disclosed herein. Thus, the antibodies and other binding proteins are also referred to herein as cellpenetrating.

[0088] In some aspects, the 3E10 antibody is transported into the cytoplasm and/or nucleus of the cells without the aid of a carrier or conjugate. For example, the monoclonal antibody 3E10 and active fragments thereof that are transported in vivo to the nucleus of mammalian cells without cytotoxic effect are disclosed in U.S. Patent Nos. 4,812,397 and 7,189,396 to Richard Wei sb art.

[0089] In some aspects, the antibody may bind and/or inhibit Rad51. See for example, the antibody described in Turchick, et al., Nucleic Acids Res., 45(20): 11782-11799 (2017), WO 2020/047344, and WO 2020/047353, each of which is specifically incorporated by reference herein, in its entirety.

[0090] In some aspects, the 3E10 antibody is a monoclonal 3E10, or a variant, derivative, fragment, fusion, or humanized form thereof that binds the same or different epitope(s) as 3E10.

[0091] In some aspects, the 3E10 antibody is a deposited variant. A deposit according to the terms of the Budapest Treaty of a hybridoma cell line producing monoclonal antibody 3E10 was received on September 6, 2000, and accepted by, American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209, USA, and given Patent Deposit Number PTA-2439. In some aspects, the antibody may have the same or different epitope specificity as monoclonal antibody 3E10 produced by ATCC No. PTA 2439 hybridoma. The antibody can have the paratope of monoclonal antibody 3E10. The antibody can be a single chain variable fragment of 3E10, or a variant, e.g., a conservative variant thereof. For example, the antibody can be a single chain variable fragment of 3E10 (3E10 Fv), or a variant thereof.

[0092] In some aspects of the present disclosure, the amino acid residue corresponding with D31 of the heavy chain CDR1 is substituted with N. It is known in the art that mutation of aspartic acid at residue 31 of CDR1 to asparagine increases the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N). Additional exemplary variants include mutation of aspartic acid at residue 31 of CDR1 to arginine (3E10-D31R), which modeling indicates expands cationic charge, or lysine (3E10-D31K) which modeling indicates changes charge orientation. Thus, in some aspects, the 3E10 binding protein includes a D31R or D31K substitution. Additional exemplary variants include mutation of arginine (R) 96 to asparagine (N), and/or serine (S) 30 to aspartic acid (D) alone or in combination with D31N, D31R, or D31K. All of the sequences disclosed herein having the residue corresponding to 3E10 D31 or N31, are expressly disclosed with a D31R or D3 IK or N31R or N3 IK substitution.

[0093] In some aspects, the antibody or antigen binding fragment thereof comprises (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO: 12), (b) a VL CDR2 comprising the amino acid sequence of YASYLES (SEQ ID NO: 13), and (c) a VL CDR3 comprising the amino acid sequence of QHSREFPWT (SEQ ID NO: 14), and (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 9), (e) a VH CDR2 comprising the amino acid sequence of YISSGSSTIYYADTVKG (SEQ ID NO: 10), and (f) a VH CDR3 comprising the amino acid sequence of RGLLLDY (SEQ ID NO: 11). In some aspects, the antibody or antigen binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence that is identical to SEQ ID NO: 19. In some aspects, the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is identical to SEQ ID NO: 18. In some aspects, the antibody or antigen binding fragment thereof comprises a full length light chain (LC) comprising an amino acid sequence that is identical to SEQ ID NO:2. In some aspects, the antibody or antigen binding fragment thereof comprises a full length heavy chain (HC) comprising an amino acid sequence that is identical to SEQ ID NO:1.

[0094] The murine version of the 3E10 antibody is described in Zack, et al., Immunology and Cell Biology, 72:513-520 (1994) (which is incorporated by reference herein, in its entirety).

[0095] Amino acid variants of the 3E10 antibody are also known in the art, for example, as described in Zack, et al., J. Immunol., 157(5):2082-8 (1996). For example, amino acid position 31, in CDR1 of the heavy chain variable region of 3E10, influences nucleic acid binding and the antibody’s ability to penetrate nuclei. Substitution of of the ‘wild-type’ (e.g., relative to the original murine antibody) aspartic acid by asparagine (the ‘D31N’ mutation) improves nucleic acid binding and nuclei penetration of the antibody, relative to the ‘wild type’ murine antibody. See, for example, Zack, et al., Immunology and Cell Biology, 72:513-520 (1994); Weisbart, et al., J. Autoimmun., 11, 539-546 (1998); and Weisbart, Int. J. Oncol., 25, 1867-1873 (2004) (which are incorporated by reference herein, in their entireties).

[0096] Sequences for the 3E10 antibody and antigen binding fragments thereof, with the D31N substitution, are disclosed herein. In some aspects, the 3E10 antibodies and binding fragments thereof disclosed herein include the D31N substitution. In some aspects, other amino acids are substituted at position 31 in the 3E10 antibodies and binding fragments thereof disclosed herein. For example, D31R, D31K, or D31L substitutions are incorporated in some aspects of the present disclosure.

[0097] Other 3E10 light chain sequences are known in the art. See, for example, Zack, et al., J. Immunol., 15;154(4):1987-94 (1995); GenBank: L16981.1 - Mouse Ig rearranged L- chain gene, partial cds; GenBank: AAA65681.1 - immunoglobulin light chain, partial [Mus musculus]).

Humanized 3E10 Antibodies

[0098] The term “humanized antibody” refers to forms of non-human (e.g. murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non- human species (e.g. mouse, rat, rabbit, and hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321 :522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239: 1534-1536). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539 or 5,639,641.

[0099] Generally, a humanized antibody is the result of a process in which the sequence of a parental antibody from a non-human species is modified to increase the overall similarity of the parental antibody to human antibodies, while retaining antigen binding activity of the parental antibody. Generally, the process involves identifying a human antibody, sometimes referred to as a scaffold antibody, and then either (i) replacing amino acids in the parent (non-human) antibody with equivalent amino acids from the scaffold (human) antibody, e.g., framework amino acids having little to no effect on antigen binding or (ii) replacing amino acids in the scaffold (human) antibody with equivalent amino acids from the parent (non-human) antibody, e.g., CDRs and other amino acids with significant effects on antigen binding. Various methods for humanization are known in the art, including framework-homology-based humanization, germline humanization, complementary determining regions (CDR)-homology-based humanization, and specificity determining residues (SDR) grafting. For a review of these methods see, for example, Safdari Y. et al., Biotechnology and Genetic Engineering Reviews, 29:2, 175-86 (2013).

[0100] Humanized 3E10 antibody or cell-penetrating fragment thereof sequences are known in the art. For example, useful humanized 3E10 antibody, antigen binding fragments, and variants thereof are disclosed in US 2023/0303719, WO 2015/106290, WO 2016/033324, WO 2019/018426, and WO 2019/018428, each of which is incorporated by reference herein.

Sequences

[0101] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of XYGMH (SEQ ID NO: 20), wherein X = D, N, R, L, or K and corresponds to an amino acid residue at position 31 of the 3E10 heavy chain.

[0102] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) CDR1 comprising SEQ ID NO: 15 or SEQ ID NO: 9, a VH CDR2 comprising SEQ ID NO: 10, a VH CDR3 comprising SEQ ID NO: 11; a light chain variable region (VL) CDR1 comprising SEQ ID NO: 12, a VL CDR2 comprising SEQ ID NO: 13, and a VL CDR3 comprising SEQ ID NO: 14. [0103] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain variable domain (VH) comprising SEQ ID NO: 16; or SEQ ID NO: 18, and a light chain variable domain (VL) comprising SEQ ID NO: 17 or SEQ ID NO: 19.

[0104] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 3 or SEQ ID NO: 1, and a light chain comprising SEQ ID NO: 4 or SEQ ID NO: 2.

[0105] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 5, and a light chain comprising SEQ ID NO: 6.

[0106] In some aspects, a therapeutic nucleic acid cargo comprises SEQ ID NO: 8.

[0107] In some aspects, the therapeutic nucleic acid encodes a protein comprising SEQ ID NO: 7.

[0108] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 1, and a light chain comprising SEQ ID NO: 2.

[0109] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 3, and a light chain comprising SEQ ID NO: 4.

[0110] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises heavy chain comprising SEQ ID NO: 5, and a light chain comprising SEQ ID NO: 6.

[OHl] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 21, and a light chain comprising SEQ ID NO: 27.

[0112] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 22, and a light chain comprising SEQ ID NO: 28.

[0113] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 23, and a light chain comprising SEQ ID NO: 29.

[0114] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 24, and a light chain comprising SEQ ID NO: 30.

[0115] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 25, and a light chain comprising SEQ ID NO: 31.

[0116] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 26, and a light chain comprising SEQ ID NO: 32.

[0117] In some aspects, a 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising any one of the humanized heavy chain sequences disclosed herein, in combination with a light chain comprising any one of the humanized light chain sequences disclosed herein.

E. Therapeutic Nucleic Acid Cargo

[0118] As used herein, a “therapeutic nucleic acid cargo” refers to any nucleic acid that has a biological, or a therapeutic effect. In some aspects, a therapeutic nucleic acid cargo has a therapeutic effect in a cell. In some aspects, a therapeutic nucleic acid cargo can be DNA or RNA. In some aspects, a therapeutic nucleic acid cargo can be single stranded or double stranded. In some aspects, a therapeutic nucleic acid cargo can be cDNA. In some aspects, a therapeutic nucleic acid cargo is mRNA, tRNA, or rRNA. In some aspects, the therapeutic nucleic acid cargo can be an mRNA.

[0119] In some aspects, a therapeutic nucleic acid cargo can be an isolated nucleic acid. An “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome. The term “isolated” as used herein with respect to nucleic acids also includes the combination with any non-naturally- occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.

[0120] The nucleic acid sequences encoding polypeptides include genomic sequences. Also disclosed are mRNA/cDNA sequence wherein the exons have been deleted. Other nucleic acid sequences encoding polypeptides, such polypeptides that include the aboveidentified amino acid sequences and fragments and variants thereof, are also disclosed. Nucleic acids encoding polypeptides may be optimized for expression in the expression host of choice. Codons may be substituted with alternative codons encoding the same amino acid to account for differences in codon usage between the organism from which the nucleic acid sequence is derived and the expression host. In this manner, the nucleic acids may be synthesized using expression host-preferred codons.

[0121] Therapeutic nucleic acid cargo can be in sense or antisense orientation, or can be, for example, complementary to a reference sequence encoding a polypeptide.

[0122] In some aspects, a therapeutic nucleic acid cargo includes or is composed of nucleotide analogs that have been chemically modified to improve stability, half-life, or specificity or affinity for a target receptor, relative to a DNA or RNA counterpart. The chemical modifications include chemical modification of nucleobases, sugar moieties, nucleotide linkages, or combinations thereof. As used herein ‘modified nucleotide” or “chemically modified nucleotide” defines a nucleotide that has a chemical modification of one or more of the heterocyclic base, sugar moiety or phosphate moiety constituents. In some aspects, the charge of the modified nucleotide is reduced compared to DNA or RNA of the same nucleobase sequence.

[0123] Typically, nucleoside analogs support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence-specific fashion between the oligonucleotide analog molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single- stranded DNA). In some aspects, the analogs have a substantially uncharged, phosphorus containing backbone.

[0124] In some aspects, for example where the therapeutic nucleic acid cargo is a RNA, the RNA can be modified to increase RNA stability. In some aspects, for example where the therapeutic nucleic acid cargo is a mRNA, a mRNA can be modified to increase RNA stability. In some aspects, a mRNA can be modified to increase the stability of a mRNA in the cytosol of a cell. In some aspects, the bases in a mRNA are modified. In some aspects, a mRNA can be modified to comprise modified bases. In some aspects, a mRNA can be methylated. In some aspects, a mRNA comprises a n6-methyladenosine (m6a) modification. In some aspects, a mRNA comprises a pseudouridine ( ) modification. In some aspects, a mRNA comprises a 5-methylcytidine (m5C) modification. In some aspects, a mRNA comprises a 2’-0 methylation modification.

[0125] In some aspects, a therapeutic nucleic acid cargo can improve or ameliorate a disease or disorder by modulating gene and/or protein expression. In some aspects, a therapeutic nucleic acid cargo can be a cDNA. In some aspects, a therapeutic nucleic acid cargo can be an mRNA. In some aspects, a therapeutic nucleic acid cargo can encode an amino acid peptide.

[0126] One non-limiting example of a therapeutic nucleic acid cargo is a nucleic acid that encodes the C-terminal tail (CTT) of polycystin-1 (PCI). Therefore, in some aspects, a therapeutic nucleic acid cargo can be a cDNA or mRNA that encodes the PCI CTT. Transgenic expression of the 200-aa PCI C-terminal tail (CTT) in the Pkdl;Pax8;TetO- Crel (Pkdl-KO) mouse model of ADPKD suppresses cystic phenotype and preserves renal function (Ma, M. et al. Nat Genet 2013 45, 1004-12). This suppression depends upon an interaction between the C-terminal tail of PCI and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (NNT) (Onuchic, L. et al. Nat Commun 2023 30; 14(1): 1790). This interaction modulates tubular/cyst cell proliferation, metabolism, mitochondrial function, and the redox state. Accordingly, in some aspects, a therapeutic nucleic acid cargo comprises the sequence set forth in SEQ ID NO: 8, which encodes the amino acid sequence set forth in SEQ ID NO: 7. In some aspects, the nucleic acid cargo can be modified, for example, to increase stability, and therefore, life span, in the cytosol. Without wishing to be bound by any one theory, it is contemplated that a nucleic acid cargo could be modified by, for example, modifying or removing the PEST sequence to increase stability. In some aspects, a therapeutic nucleic acid cargo can encode PCI, or a different fragment thereof than the sequence disclosed in SEQ ID NO: 7.

[0127] In some aspects, a therapeutic nucleic acid cargo can encode a peptide. For example, the therapeutic nucleic acid cargo can encode a peptide toxin. Peptide toxins can be useful for the treatment of neoplastic diseases and disorders. In some aspects, a cargo can be a chemical cargo. For example, the chemical cargo can deliver mRNAs encoding missing proteins in genetic disease. For example, the chemical cargo can deliver mRNAs encoding peptide toxins in neoplastic diseases.

F. Pharmaceutical Compositions

[0128] The compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0129] The compositions including a therapeutic nucleic acid cargo complexed with 3E10 antibody are preferably employed for therapeutic uses in combination with a suitable pharmaceutical carrier. Such compositions include an effective amount of the composition, and a pharmaceutically acceptable carrier or excipient.

[0130] The compositions may be in a formulation for administration topically, locally or systemically in a suitable pharmaceutical carrier. Remington’s Pharmaceutical Sciences, 15th Edition by E. W. Martin (Mark Publishing Company, 1975), discloses typical carriers and methods of preparation. The complexes may also be encapsulated in suitable biocompatible particles formed of biodegradable or non-biodegradable polymers or proteins or liposomes for targeting to cells. Such systems are well known to those skilled in the art. In some aspects, the complexes are encapsulated in nanoparticles. [0131] Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, optionally with an added preservative. The compositions may take such forms as sterile aqueous or nonaqueous solutions, suspensions and emulsions, which can be isotonic with the blood of the subject in certain aspects. Examples of nonaqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oil such as olive oil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or fixed oils including synthetic mono or di-glycerides. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3- butandiol, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, and electrolyte replenishers (such as those based on Ringer’s dextrose). The materials may be in solution, emulsions, or suspension (for example, incorporated into particles, liposomes, or cells). Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Trehalose, typically in the amount of 1-5%, may be added to the pharmaceutical compositions. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.

[0132] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, and surface-active agents. Carrier formulation can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. Those of skill in the art can readily determine the various parameters for preparing and formulating the compositions without resort to undue experimentation.

[0133] The compositions may be delivered in a manner which enables tissue-specific uptake of the agent and/or nucleotide delivery system, using invasive devices such as vascular or urinary catheters, and using interventional devices such as stents having drug delivery capability and configured as expansive devices or stent grafts.

[0134] Other delivery systems suitable include time-release, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations in many cases, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems such as polylactic and/or polyglycolic acids, poly anhydrides, polycaprolactones, copolyoxalates, polyesteramides, poly orthoesters, polyhydroxybutyric acid, and/or combinations of these. Microcapsules of the foregoing polymers containing nucleic acids are described in, for example, U.S. Patent No. 5,075,109. Other examples include non-polymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid based-systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. The formulation may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. In some aspects, the system may allow sustained or controlled release of the composition to occur, for example, through control of the diffusion or erosion/degradation rate of the formulations containing the complexes.

[0135] The complexes can be delivered to the target cells using a particle delivery vehicle. Nanoparticles generally refers to particles in the range of between 500 nm to less than 0.5 nm, preferably having a diameter that is between 50 and 500 nm, more preferably having a diameter that is between 50 and 300 nm. Cellular internalization of polymeric particles is highly dependent upon their size, with nanoparticulate polymeric particles being internalized by cells with much higher efficiency than micoparticulate polymeric particles. For example, Desai, et al. have demonstrated that about 2.5 times more nanoparticles that are 100 nm in diameter are taken up by cultured Caco-2 cells as compared to microparticles having a diameter on 1 mM (Desai, et al., Pharm. Res., 14: 1568-73 (1997)). Nanoparticles also have a greater ability to diffuse deeper into tissues in vivo.

Examples

[0136] It is understood that the examples and aspects described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

Example 1: 3E10 Antibody Delivers mRNA to Kidney and Tumor Tissue.

[0137] C57B1/6 mice bearing flank pancreatic ductal adenocarcinoma (KPC) tumors were injected with three doses of GFP-encoding mRNA alone or GFP-encoding mRNA in complex with a humanized 3E10 antibody comprising a HC set forth in SEQ ID NO: 55 and a LC set forth in SEQ ID NO: 68 (V66/mRNA). Each dose was administered 24 hours apart. The GFP-encoding mRNA was dosed at 20 pg/dose and the V66 was dosed at 100 pg/dose. Tumor and healthy tissues were harvested 24 hours after the last dose and imaged by In Vivo Imaging System (IVIS). As shown in FIG. 1, a GFP signal was detected in the kidneys and tumors of V66/mRNA injected mice, but not in mice injected with the mRNA alone. This Example demonstrates that 3E10 delivers mRNA to tumor and healthy kidney tissue in vivo.

Example 2: ENT2 Colocalizes with the Plasma Membrane Marker Na, K+-ATPase in the mouse kidney tubules.

[0138] The observed V66-mediated mRNA delivery to kidney tissue suggests high expression of ENT2 in kidney cells. To confirm this, ENT2 mRNA expression was tested in renal tubules from WT mice and Pkdl^fl/fl;Pax8^rtTA;TetO-Cre (Pkdl-KO) mice, a conditional mouse model of autosomal dominant polycystic kidney disease, which is induced by treating the mice with doxycycline. Pkdl encodes polycystin-1 (PCI) and mutations in Pkdl are the cause of approximately 78% of autosomal dominant polycystic kidney disease (ADPKD). Typically, for example, 10-week old mice are used for the experiment with doxycycline treatment starting at 6 weeks of age to induce Cre recombinase expression and consequent inactivation of the Pkdl gene, so the mice manifest early stage renal cyst development.

[0139] WT and Pkdl- Q mice were injected with the V66/GFP mRNA complex as described in Example 1, and 24 hours after the last V66/GFP mRNA dose their renal tubules were harvested and prepared for immunofluorescence (IF) analysis with antibodies to ENT2 and Na, K⁺-ATPase, a basolateral plasma membrane marker. As shown in FIG. 2, ENT2 was detected in all renal tubular epithelial cells, with the highest protein levels detected in distal tubular epithelial cells as compared to proximal tubules (PTs); which were identified using Lotus tetragonolobus lectin coupled to FITC. Moreover, IF imaging revealed that Na, K⁺-ATPase colocalizes with ENT2, particularly in the tubular epithelial cells with the highest ENT2 expression. Arrows indicate points of co-localization between ENT2 and Na, K⁺-ATPase. This Example confirms that tubular epithelial cells express high levels of ENT2 at their basolateral plasma membrane, facilitating the delivery of the V66/GFP mRNA complex to kidney cells. Example 3: 3E10 Mediated Delivery of IR700 to Mouse Kidneys

[0140] 3E10 delivery to kidney cells was quantified by IVIS imaging. One 200pg dose of

3E10-D3 IN labeled with fluorochrome IR700 was administered to 11-week-old Pkdl-\kQ) and age-matched WT mice. Age-matched WT mice that were not injected were used as a negative control. Kidneys were harvested 24 hours after dosing and imaged by IVIS. As shown in FIG. 3, the signal intensity of IR700 was measured by the total radiant efficiency (TRE). As expected, the negative control showed no IR700 signal, but IR700 was detected in the kidneys of all the 3E10-D31N injected mice, demonstrating 3E10 penetration into both healthy and cystic kidney cells. Interestingly, the kidneys of the early cystic (Pkdl- KO) mice had an approximately 50% higher IR700 signal intensity than the kidneys of the WT mice. This Example further demonstrates that 3E10 efficiently penetrates kidney cells in vivo and thus, could be used to target kidney tissue, particularly highly proliferative ADPKD tissue.

Example 4: 3E10 Mediated Delivery of mRNA to Mouse Kidneys

[0141] To test whether 3E10 can deliver a nucleic acid payload to cystic mouse kidneys, 13 -week Pkdl- O mice were injected with three doses of GFP-encoding mRNA alone or GFP-encoding mRNA in complex with a humanized 3E10 antibody (V66/mRNA), as described in Example 1. Tissue was harvested 24 hours after the third dose and prepared for IVIS and IF analysis. As shown in FIG. 4, IVIS revealed GFP fluorescence only in cystic kidneys from mice that were treated with V66/GFP mRNA, not in cystic tissue from mice that received GFP mRNA alone, or untreated mice. As shown in FIG. 5, IF studies revealed GFP, detected with an anti-GFP antibody, present in a punctate cytoplasmic pattern in cystic epithelial cells of animals that received V66/GFP mRNA and not in controls. This Example demonstrates that V66-mediated delivery of GFP mRNA leads to the detection of GFP in cystic kidneys. Moreover, 3E10-D31N/V66 targets cystic epithelia and delivers GFP mRNA that drives protein expression in vivo. This strategy supports the delivery of therapeutic mRNAs or cDNAs in the context of renal diseases.

Example 5: 3E10 Mediated Delivery of CTT mRNA to Mouse Kidneys

[0142] To test the potential of using 3E10 to deliver a therapeutic nucleic acid to treat ADPKD, 10-week old WT and Pkdl-KO mice were injected with mRNA encoding the 200-aa PCI C-terminal tail (CTT) (SEQ ID NO: 8) with two HA tags added to the N- terminus (z.e., SEQ ID NO: 92) for detection, either in a complex with a 3E10 antibody (e.g., V66), or alone without the antibody, once per day for three days. The kidneys of the mice were harvested 24 hours after the final inj ection and prepared for immunofluorescence and Western Blot analysis to determine protein expression and potential impacts of this intervention on the development of the cystic phenotype. Antibodies directed against the HA epitope tag were used to localize PC1-CTT expression and an antibody against NNT was used to localize mitochondria. DAPI was also applied to reveal the localization of nuclei.

[0143] As shown in FIG. 6, IF revealed that mice that received HA-PC1-CTT mRNA and V66 in complex showed bright signal that co-localizes with mitochondria in the epithelial cells that line the dilated cystic renal tubules. No such signal is detected in the kidneys of mice that received HA-PC1-CTT mRNA without V66 or that were not treated. FIG. 7 shows similar results in additional exemplary mice.

[0144] As shown in FIG. 8, total cell lysates were prepared from the kidneys and analysed by Western Blotting using an antibody directed against the HA epitope tag. Bands corresponding to the polycystin-1 C-terminal tail and fragments derived from it (bracket) are detected in lysates from 3 of the 5 mice that were injected with mRNA encoding 2HA- PC1-CTT pre-complexed with V66, but not in lysates from any untreated mice or mice that received mRNA encoding 2HA-PC1-CTT without V66.

[0145] As shown in FIG. 9, the total cell lysates were immunoprecipitated using anti-HA antibody magnetic beads. The isolated immunoprecipitates were then analysed by Western Blot analysis for PC1-CTT as described above. Bands corresponding to the poly cystin- 1 C-terminal tail and fragments derived from it (bracket) are detected in immunoprecipitates from lysates from 3 of the 5 mice that were injected with mRNA encoding 2HA-PC1-CTT pre-complexed with V66, but not from lysates from untreated mice or from mice that received mRNA encoding 2HA-PC1-CTT without V66. Total lysates from HEK293 cells and Ml 13 cells transfected to express the polycystin-1 C-terminal tail were also run on this gel to provide a size comparison for the protein immunoprecipitated from the kidneys of treated mice.

[0146] Collectively, these data demonstrate that V66 can effectively deliver and produce expression of mRNA encoding the C-terminal tail of polycystin-1 in an orthologous mouse model of autosomal dominant polycystic kidney disease. [0147] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0148] The breadth and scope of the present invention should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a renal disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a complex comprising:

(a) a 3E10 antibody or antigen binding fragment thereof, wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of XYGMH (SEQ ID NO: 20), wherein X = D, N, R, L, or K and corresponds to an amino acid residue at position 31 of the 3E10 heavy chain; and

(b) a therapeutic nucleic acid cargo.

2. The method of claim 1, wherein the 3E10 antibody or antigen binding fragment thereof comprises:

(a) a heavy chain variable region (VH) CDR1 comprising SEQ ID NO: 15 or SEQ ID NO: 9, a VH CDR2 comprising SEQ ID NO: 10, and a VH CDR3 comprising SEQ ID NO: 11; and

(b) a light chain variable region (VL) CDR1 comprising SEQ ID NO: 12, a VL CDR2 comprising SEQ ID NO: 13, and a VL CDR3 comprising SEQ ID NO: 14.

3. The method of claim 1 or 2, wherein the 3E10 antibody or antigen binding fragment thereof comprises:

(a) a heavy chain variable domain (VH) comprising SEQ ID NO: 16; or SEQ ID NO: 18, and

(b) a light chain variable domain (VL) comprising SEQ ID NO: 17 or SEQ ID NO: 19; or

(c) a heavy chain variable domain (VH) comprising any one of the sequences set forth in SEQ ID NOs: 21-26, and

(d) a light chain variable domain (VL) comprising any one of the sequences set forth in SEQ ID NOs: 27-32.

4. The method of claim 1 or 2, wherein the 3E10 antibody or antigen binding fragment thereof comprises: (a) a heavy chain comprising SEQ ID NO: 3 or SEQ ID NO: 1, and

(b) a light chain comprising SEQ ID NO: 4 or SEQ ID NO: 2.

5. The method of claim 1 or 2, wherein the 3E10 antibody or antigen binding fragment thereof is humanized.

6. The method of claim 5, wherein the humanized 3E10 antibody or antigen binding fragment thereof comprises:

(a) a heavy chain comprising SEQ ID NO: 5 or SEQ ID NO: 55, and

(b) a light chain comprising SEQ ID NO: 6 or SEQ ID NO: 68.

7. The method of any one of claims 1-6, wherein the complex is a non-covalent complex.

8. The method of any one of claims 1-6, wherein the complex is a covalent complex.

9. The method of any one of claims 1-8, wherein the renal disease comprises a hyperproliferative disease or disorder of the kidney.

10. The method of any one of claims 1-9, wherein the renal disease comprises polycystic kidney disease, von Hippel Lindau disease, Tuberous Sclerosis, Bardet-Biedel Syndrome, nephronophthisis, or renal cell carcinoma.

11. The method of any one of claims 1-10, wherein the renal disease is polycystic kidney disease.

12. The method of any one claim 11, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

13. The method of claim 11, wherein the polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD).

14. The method of any one of claims 1-13, wherein the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to epithelial cells of a kidney.

15. The method of any one of claims 1-14, wherein the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal cells expressing ENT2.

16. The method of any one of claims 1-15, wherein the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal cells expressing Na, K+-ATPase.

17. The method of any one of claims 1-16, wherein the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal cells expressing ENT2 and Na, K+-ATPase.

18. The method of any one of claims 1-17, wherein the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal cells expressing ENT2 and Na, K+-ATPase, wherein the ENT2 and NA, K+-ATPase colocalize on the plasma membrane.

19. The method of any one of claims 1-18, wherein the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to renal tubular epithelial cells.

20. The method of claim 19, wherein the renal tubular epithelial cells comprise proximal tubule, Loop of Henle, distal tubule, and/or collecting tubule cells.

21. The method of any one of claims 1-20, wherein the 3E10 antibody or antigen binding fragment thereof delivers the therapeutic nucleic acid cargo to cystic epithelial cells of a kidney.

22. The method of any one of claims 1-21, wherein the therapeutic nucleic acid cargo comprises a nucleotide sequence encoding polycystin-1 (PCI) protein, or a portion thereof.

23. The method of any one of claims 1-22, wherein the therapeutic nucleic acid cargo comprises polycystin-1 (PCI) C-terminal tail (CTT)-encoding nucleotide sequence.

24. The method of any one of claims 1-23, wherein the therapeutic nucleic acid cargo comprises the sequence set forth in SEQ ID NO: 8.

25. The method of any one of claims 1-24, wherein the sequence of the therapeutic nucleic acid cargo is

ATGGTGATCTTGCGCTGGCGGTATCACGCCCTTCGCGGAGAACTGTACAGACC GGCTTGGGAGCCTCAGGACTACGAGATGGTGGAACTGTTTCTGCGGCGGCTC AGACTTTGGATGGGGCTCTCCAAGGTCAAGGAGTTCAGGCACAAGGTCCGCT TCGAGGGGATGGAACCGTTGCCATCCCGGTCGAGCCGGGGATCTAAAGTGTC GCCCGATGTGCCACCCCCTTCCGCGGGCTCCGACGCCTCCCACCCGAGCACCA GCAGCTCCCAGCTCGACGGCCTGTCGGTGTCCCTGGGCCGGCTGGGTACCCGC TGCGAACCTGAACCCTCGAGACTGCAAGCCGTGTTCGAGGCCCTGCTGACTC AGTTCGACCGCCTGAACCAAGCAACCGAGGACGTGTACCAGCTGGAACAGCA GCTGCATTCGCTGCAAGGAAGGAGATCCTCCCGGGCGCCGGCTGGCTCATCA AGAGGTCCGAGCCCCGGACTGCGCCCCGCCCTCCCTTCCCGCCTCGCCCGGGC CTCCCGGGGAGTGGATCTCGCAACCGGACCGAGCCGAACCCCTCTGAGGGCC AAGAACAAGGTCCACCCCTCCTCAACT (SEQ ID NO: 8)

26. The method of claim 24 or 25, wherein the therapeutic nucleic acid encodes an amino acid polypeptide comprising the sequence set forth in SEQ ID NO: 7.

27. The method of claim 22, wherein the therapeutic nucleotide encodes an amino acid polypeptide comprising a PDK1 protein sequence as set forth in any one of SEQ ID NOs: 34-36, or a portion thereof.

28. The method of any one of claims 1-27, wherein administration of the complex to the subject suppresses cystic phenotype, thereby treating the subject.

29. The method of any one of claims 1-28, wherein administration of the non-covalent complex to the subject preserves and/or improves renal function, thereby treating the subject.

30. A method for treating a renal disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a non-covalent complex comprising a 3E10 antibody or antigen binding fragment thereof comprising SEQ ID NO: 1, and a light chain comprising SEQ ID NO: 2, and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8.

31. A method for treating a renal disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a non-covalent complex comprising a 3E10 antibody or antigen binding fragment thereof comprising SEQ ID NO: 3, and a light chain comprising SEQ ID NO: 4, and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8.

32. A method for treating a renal disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a non-covalent complex comprising a 3E10 antibody or antigen binding fragment thereof comprising SEQ ID NO: 5, and a light chain comprising SEQ ID NO: 6, and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8.

33. The method of any one of claims 30-32, wherein the therapeutic nucleic acid encodes a protein comprising SEQ ID NO: 7.

34. A complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo for use in treating a renal disease in a subject in need thereof, wherein the 3E10 antibody or antigen binding fragment thereof comprises: a heavy chain comprising SEQ ID NO: 1, and a light chain comprising SEQ ID NO: 2; a heavy chain comprising SEQ ID NO: 3, and a light chain comprising SEQ ID NO: 4; or a heavy chain comprising SEQ ID NO: 5, and a light chain comprising SEQ ID NO: 6, and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8.

35. The use of claim 34, wherein the complex is a non-covalent complex.

36. The use of claim 35, wherein the complex is a covalent complex.

37. A composition comprising a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of XYGMH (SEQ ID NO: 20), wherein X = D, N, R, L, or K and corresponds to an amino acid residue at position 31 of the 3E10 heavy chain.

38. A composition comprising a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable region (VH) CDR1 comprising SEQ ID NO: 15 or SEQ ID NO: 9, a VH CDR2 comprising SEQ ID NO: 10, and a VH CDR3 comprising SEQ ID NO: 11; and

(b) a light chain variable region (VL) CDR1 comprising SEQ ID NO: 12, a VL CDR2 comprising SEQ ID NO: 13, and a VL CDR3 comprising SEQ ID NO: 14.

39. A composition comprising a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable domain (VH) comprising SEQ ID NO: 16; or SEQ ID NO: 18, and

(b) a light chain variable domain (VL) comprising SEQ ID NO: 17 or SEQ ID NO: 19; or (c) a heavy chain variable domain (VH) comprising any one of the sequences set forth in SEQ ID NOs: 21-26, and

(d) a light chain variable domain (VL) comprising any one of the sequences set forth in SEQ ID NOs: 27-32.

40. A composition comprising a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 1, and a light chain comprising SEQ ID NO: 2.

41. A composition comprising a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 3, and a light chain comprising SEQ ID NO: 4.

42. A composition comprising a complex comprising a 3E10 antibody or antigen binding fragment thereof and a therapeutic nucleic acid cargo comprising SEQ ID NO: 8, wherein the 3E10 antibody or antigen binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 5, and a light chain comprising SEQ ID NO: 6.

43. The composition of any one of claims 37-42, wherein the complex is a non-covalent complex.

44. The composition of any one of claims 37-42, wherein the complex is a covalent complex.

45. The composition of any one of claims 37-44, wherein the therapeutic nucleic acid encodes an amino acid polypeptide comprising the sequence set forth in SEQ ID NO: 7.

46. A pharmaceutical composition comprising the composition of any one of claims 37-45 and a pharmaceutically acceptable excipient.

47. A pharmaceutical kit comprising the pharmaceutical composition of claim 46, and instructions for use thereof.