WO2025064905A1 - Therapeutic constructs and related methods to treat rbm20-related cardiomyopathy - Google Patents

Abstract

The present disclosure relates to compositions and methods for the treatment of cardiomyopathy. Several embodiments provided for herein relate to virally-mediated transfer of a gene to host cells to induce expression of an encoded polypeptide, protein or other product to host in order to ameliorate one or more symptoms of the cardiomyopathy. In several embodiments, the methods and compositions relate to recombinant adeno-associated virus particles encoding human RBM20 in order to treat cardiomyopathies, including dilated cardiomyopathy. Further embodiments related to incorporation and/or administration of a silencing element that downregulates expression of a protein product of a pathogenically mutated gene, such as RBM20. In some embodiments, the virally delivered RBM20 is engineered to be resistant to the silencing element.

Description

AAVAN.095WO PATENT THERAPEUTIC CONSTRUCTS AND RELATED METHODS TO TREAT RBM20-RELATED CARDIOMYOPATHY RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 63/539578, entitled “THERAPEUTIC CONSTRUCTS AND RELATED METHODS TO TREAT RBM20-RELATED CARDIOMYOPATHY” and filed on September 20, 2023, which is hereby incorporated by reference herein in its entirety. INCORPORATION BY REFERENCE OF MATERIAL IN SEQUENCE LISTING [0002] This application incorporates the material provided in the accompanying XML file entitled SequenceListing_AAVAN095WO.xml, created September 19, 2024, which is 1,998,951 bytes in size. FIELD [0003] Aspects of the present disclosure relate to compositions and methods of use of gene therapy vectors comprising RBM20 and variants thereof for use in therapeutic applications. Additionally, embodiments disclosed herein relate to silencing and co-expression of silencing elements designed to reduce transient expression of non-functional mutated RBM20 and variants thereof. BACKGROUND [0004] Cardiomyopathy refers to a variety of conditions that adversely affect cardiac muscle. Cardiomyopathies can reduce the efficiency of circulation of blood resulting from one or more of enlargement of the heart (e.g., enlarged chambers), changes in the thickness of the heart wall (e.g., thinning), and/or stiffening of the heart muscle. Dilated cardiomyopathy (DCM) is a leading cause of heart failure and death. The etiology of DCM is genetically heterogeneous, however, mutations in NA-binding protein 20 (RBM20), a regulator of splicing, are implicated. Gene therapy is contemplated as a method of treating DCM. SUMMARY [0005] Inheritance of mutations in RBM20, a regulator of splicing, is an important cause of dilated cardiomyopathy, a type of heart muscle disease that causes the ventricles to grow larger. RBM20 encodes a 1227 amino acid protein containing two zinc finger domains, a glutamate-rich region, a leucine-rich region, an RNA-Recognition Motif (RRM)-type RNA binding domain and an arginine-/serine-rich region (RS-domain). [0006] Aspects of the application provide compositions and methods for delivering gene expression constructs to a subject. Aspects of the application provide compositions and methods for delivering gene expression constructs to a host cell. Aspects of the application relate to methods and compositions for expressing nucleic acids from recombinant adeno- associated virus (rAAV) vectors. [0007] There is provided, in several embodiments, a recombinant adeno-associated virus (rAAV) comprising an expression cassette, the expression cassette comprising an RBM20 coding sequence and one or more silencing elements, wherein the one or more silencing elements reduces expression of endogenous RBM20 (both pathogenic and wild type alleles), wherein the RBM20 coding sequence comprises one or more modifications that render the RBM20 coding sequence resistant to silencing by the one or more silencing elements, thereby allowing for silencing of endogenous RBM20 (both pathogenic and wild type alleles) and expression of exogenous RBM20. In some embodiments, the construct has a promoter that is operably linked to the RBM20 coding sequence. [0008] There is also provided for herein a method for the treatment of cardiomyopathy, comprising administering to a subject having cardiomyopathy a recombinant adeno-associated virus (rAAV) comprising an RBM20 coding sequence one or more silencing elements. According to several embodiments, the one or more silencing elements to induce degradation of nucleic acids encoding endogenous RBM20 (both pathogenic and wild type nucleic acids in the subject’s cells). In several embodiments, the RBM20 coding sequence within the rAAV comprises one or more modifications that render the RBM20 coding sequence within the rAAV resistant to, insulated from, and/or unaffected by the function of the silencing elements. In some embodiments, the construct has a promoter that is operably linked to the RBM20 coding sequence. [0009] There is also provided, in several embodiments, a recombinant adeno- associated virus (rAAV) comprising an expression cassette, the expression cassette comprising n RBM20 coding sequence and one or more silencing elements, wherein the one or more silencing elements reduces expression of endogenous RBM20 (both pathogenic and wild type alleles), wherein the RBM20 coding sequence optionally comprises one or more modifications, wherein the one or more optional modifications confer resistance to silencing expression of non-endogenous RBM20 encoded by the RBM20 coding sequence. In some embodiments, the construct has a promoter that is operably linked to the RBM20 coding sequence. [0010] In several embodiments, the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to one or more of SEQ ID NOs: 4, 10, and 40. In several embodiments, the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 4, 10, and 40. [0011] In several embodiments, there is provided a method for the treatment of cardiomyopathy, comprising administering to a subject having cardiomyopathy (or a host cell with one or more physiological signs of cardiomyopathy) a recombinant adeno-associated virus (rAAV) comprising an RBM20 coding sequence and one or more silencing elements, wherein the one or more silencing elements induce degradation of nucleic acids encoding RBM20, and wherein the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, or 52; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, and 52. In some embodiments, the AAV- expressed RBM20 corrects splicing defects. In some embodiments, the construct has a promoter that is operably linked to the RBM20 coding sequence. [0012] In several embodiments, the one or more silencing elements encodes one or more siRNA sequences. In several embodiments, the one or more silencing elements encodes one or more shRNA sequences. In several embodiments, siRNA and shRNA are used in combination. In several embodiments, expression of the one or more siRNA and/or shRNA is driven by a first promoter sequence. Depending on the embodiment, RBM20 expression is driven by the first promotor sequence or by a second promoter sequence. In several embodiments, the orientation of transcription of the RBM20 coding sequence and the one or more silencing elements is reversed with respect to one another. In other embodiments, the RBM20 coding sequence and one or more silencing elements are in frame with one another but are separately expressed. In some embodiments, the construct has a promoter that is operably linked to the RBM20 coding sequence. [0013] In several embodiments, the RBM20 coding sequence is optionally CpG depleted, in whole or partially. [0014] In several embodiments, the RBM20 coding sequence comprises one or more modifications to the coding sequence, wherein the one or more modifications confer resistance to RNA-based degradation of the RBM20 coding sequence. In several embodiments, the one or more modifications to the RBM20 coding sequence do not alter the encoded RBM20 amino acid sequence. In several embodiments, the one or more modifications to the RBM20 coding sequence is selected from AATCAGGTGTTGAGTAAGGTA (SEQ ID NO: 58), AATTCAACTGCAGTATACAAT (SEQ ID NO: 59), AAAGTGACAAACTATATTTTA (SEQ ID NO: 60), and combinations thereof. In some embodiments, the AAV-expressed RBM20 corrects splicing defects. In some embodiments, the construct has a promoter that is operably linked to the RBM20 coding sequence. [0015] In several embodiments, the RBM20 coding sequence is selected from a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 16, 22 and 46. In several embodiments, the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 16, 22, and 46. [0016] In several embodiments, the RBM20 coding sequence further comprises a single amino acid modification. In several embodiments, the modification comprises a R634Q mutation in the encoded RBM20 amino acid sequence. In several embodiments, the modified RBM20 coding sequence comprises a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 28, 34, and 52. In several embodiments, the modified RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 28, 34, and 52. In some embodiments, the AAV-expressed RBM20 corrects splicing defects. [0017] In several embodiments, the one or more silencing elements are selected from one or more of SEQ ID NOs: 55 (CACTTTGGAGAGGACTTGGTT), 56 (GTTATAAACAGCTGTGCTGTT), or 57 (GAGGATGTAATTAGTGACCTT). [0018] In several embodiments, expression of the RBM20 is driven by a second promoter sequence, the second promoter sequence is selected from the group consisting of JeT, CK8, CMV, MHCK7, aMHC, mCMV, TNNT2, and EF1alpha. Combinations of promoters may also be used. [0019] In several embodiments, a first promoter sequence driving expression of the one or more silencing elements comprises a PolIII promoter. In several embodiments, a first promoter sequence driving expression of the one or more silencing elements comprises a PollII promoter. In several embodiments, the first promoter sequence is selected from the group consisting of H1, U6, and 7SK (or combinations thereof). [0020] In several embodiments, the expression cassette further comprises a Kozak sequence. In several embodiments, when present, the Kozak sequence is a consensus optimized Kozak sequence. [0021] In several embodiments, the expression cassette comprises a sequence having at least about 85% sequence identity to SEQ ID NOs: 1-6, 7-12, 13-18, 19-24, 25-30, 31-36, 37-42, 43-48, or 49-54, arranged in order. [0022] In several embodiments, the rAAV is serotype 9, or derived from serotype 9. [0023] In several embodiments, the rAAV is serotype rh74, or derived from serotype rh74. [0024] In several embodiments, the one or more silencing elements are within an expression cassette within the rAAV, wherein the expression cassette also includes the RBM20 coding sequence. In alternative embodiments, the one or more silencing elements are administered via a second rAAV. In several embodiments, the one or more silencing elements are administered by local or by systemic injection. [0025] In several embodiments, the methods and compositions provided for herein are for the treatment of dilated cardiomyopathy. [0026] Also provided for herein is a recombinant adeno-associated virus (rAAV) comprising an expression cassette, the expression cassette comprising an RBM20 coding sequence, an optional intron, and an optional Kozak sequence. [0027] In several embodiments, RBM20 expression is driven by a promoter sequence. In several embodiments, the promoter sequence is selected from the group consisting of JeT, CK8, CMV, MHCK7, aMHC, mCMV, TNNT2, and EF1alpha. [0028] In several embodiments, the RBM20 coding sequence is CpG depleted (either partially or fully). [0029] In several embodiments, when present, the Kozak sequence is a consensus optimized Kozak sequence. [0030] In several embodiments, the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to one or more of SEQ ID NOs: 4, 10, 40, and 67. In several embodiments, the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 4, 10, 40, 46, and 67. [0031] In several embodiments, the RBM20 coding sequence comprises one or more modifications to the RBM20 coding sequence do not alter the encoded RBM20 amino acid sequence. In several embodiments, the one or more modifications to the coding sequence is selected from AATCAGGTGTTGAGTAAGGTA (SEQ ID NO: 58), AATTCAACTGCAGTATACAAT (SEQ ID NO: 59), AAAGTGACAAACTATATTTTA (SEQ ID NO: 60), and combinations thereof. [0032] In several embodiments, the RBM20 coding sequence is selected from a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 16, 22 and 46. In several embodiments, the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 16, 22, and 46. [0033] In several embodiments, the RBM20 coding sequence further comprises a single amino acid modification, wherein the modification comprises a R634Q mutation in the encoded RBM20 amino acid sequence. In several such embodiments, the modified RBM20 coding sequence comprises a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 28, 34, and 52. In several such embodiments, the modified RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 28, 34, and 52. [0034] In several embodiments, the expression cassette includes an intron. In several embodiments, the expression cassette comprises a sequence having at least about 85% sequence identity to SEQ ID NOs: 1-6, 7-12, 13-18, 19-24, 25-30, 31-36, 37-42, 43-48, or 49- 54, arranged in order. In several embodiments, the expression cassette comprises a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 1-6, 7-12, 13-18, 19-24, 25-30, 31-36, 37-42, 43-48, or 49-54, arranged in order. [0035] In several embodiments, the rAAV is serotype 9, or derived from serotype 9. [0036] In several embodiments, the rAAV is serotype rh74, or derived from serotype rh74. [0037] In several embodiments, there is provided a method for the treatment of cardiomyopathy, comprising administering to a subject having cardiomyopathy or a host cell with one or more physiological signs of cardiomyopathy) a recombinant adeno-associated virus (rAAV) comprising an RBM20 coding sequence, wherein the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to one or more of SEQ ID NO: 4, 10 and 40, or, optionally, wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 4, 10, and 40. In some embodiments, the AAV-expressed RBM20 corrects splicing defects. [0038] In several embodiments, the RBM20 coding sequence comprises one or more modifications to the coding sequence do not alter the encoded RBM20 amino acid sequence. In several embodiments, the one or more modifications to the coding sequence is selected from aatcaggtgttgagtaaggta (SEQ ID NO: 58), aattcaactgcagtatacaat (SEQ ID NO: 59), aaagtgacaaactatatttta (SEQ ID NO: 60), and combinations thereof. [0039] In several embodiments, the RBM20 coding sequence is selected from a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 16, 22 and 46. In several embodiments, the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 16, 22, and 46. [0040] In several embodiments, the RBM20 coding sequence further comprises a single amino acid modification, wherein the modification comprises a R634Q mutation in the encoded RBM20 amino acid sequence. In several such embodiments, the modified RBM20 coding sequence comprises a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 28, 34, and 52. In several such embodiments, the modified RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 28, 34, and 52. [0041] In several embodiments, the cardiomyopathy is dilated cardiomyopathy. [0042] In several embodiments, there is provided the use of a rAAV according to the present disclosure for treatment of cardiomyopathy, which is optionally dilated cardiomyopathy. [0043] In several embodiments, there is provided the use of a rAAV according to the present disclosure in the preparation of a medicament for the treatment of cardiomyopathy, which is optionally dilated cardiomyopathy. [0044] Some aspects of the present invention contemplate a method of treating a disease or condition in a subject, comprising administering an rAAV according to any embodiment of the present disclosure to the subject. In some embodiments, the subject is a human subject. Some aspects of the present invention contemplate a method of treating a disease or condition in a host cell, comprising administering an rAAV according to any embodiment of the present disclosure to the host cell. In some embodiments, the host cell is a human host cell. In some embodiments, the AAV-expressed RBM20 corrects splicing defects. [0045] Accordingly, in some embodiments a recombinant nucleic acid described in this application (e.g., in an rAAV) is administered to a subject (e.g., a human patient) or a host cell (e.g., a human cell) having one or more signs or symptoms of a disease or disorder (e.g., described herein) to treat or assist in the treatment of the disease or disorder. [0046] In some embodiments of any of the RBM20 expression constructs described herein the siRNAs targeted to the RBM20 mRNA are targeted to regions where there are only a few CpGs and/or disease-causing mutations. In some embodiments of the constructs comprising the miRNA backbones, the miRNA backbones are selected based on percentage similarity to the siRNA. In some embodiments the components of the different constructs are interchangeable between constructs. In some embodiments, the constructs are resistant to cellular silencing mechanisms including epigenetic mechanisms. In some embodiments, the expression of RBM20 and/or the siRNAs are controlled through transcriptional mechanisms and/or are inducible. In some embodiments, the AAV- expressed RBM20 corrects splicing defects. In some embodiments, the AAV- expressed RBM20, alters and/or changes alternative splicing. In some embodiments, the altered alternative splicing is caused by a mutated RBM20. In some embodiments, the RBM20 gene is configured to regulate overexpression of RBM20. BRIEF DESCRIPTION OF THE DRAWINGS [0047] FIG. 1 illustrates a non-limiting embodiment of a vector designed to overexpress RBM20. [0048] FIG. 2 illustrates a partial vector illustration of JET-wt.huRBM20. [0049] FIG. 3 illustrates a partial vector illustration of CK8-INT-wt.huRBM20. [0050] FIG. 4 illustrates a partial vector illustration of JeT-Myc-wt.hurbm20. [0051] FIG.5 illustrates a non-limiting embodiment of a vector designed to express a silencing element and RBM20 sequence resistant to the silencing element. [0052] FIG. 6 illustrates an additional non-limiting embodiment of a vector designed to express a silencing element and RBM20 sequence resistant to the silencing element. [0053] FIG. 7 illustrates a partial vector illustration of JET - huRBM20_siRNA_CpG_Opt. [0054] FIG. 8 illustrates a partial vector illustration of CK8-Int- huRBM20_siRNA_CpG_Opt. [0055] FIG. 9 illustrates a partial vector illustration of JeT- huRBM20_siRNA_CpG_Opt_R634Q. [0056] FIG. 10 illustrates a partial vector illustration of CK8-Int-huRBM20- siRNA_CpG_Opt_R634Q. [0057] FIG. 11 illustrates a partial vector illustration of JeT-Myc- huRBM20_siRNA_CpG_Opt. [0058] FIG. 12 illustrates a partial vector illustration of JeT-Myc- huRBM20_siRNA_CpG_Opt_R634Q. [0059] FIG. 13 shows data related to expression of a non-limiting embodiment of a rAAV in a first non-limiting cell type. [0060] FIG. 14 shows data related to expression of a non-limiting embodiment of a rAAV in a second non-limiting cell type. [0061] FIG. 15 shows Western blot analysis related to RBM20 protein expression in some embodiments of rAAV expression vectors in iCell cardiomyocytes. [0062] FIG. 16 shows biodistribution analysis related to vector genomic DNA levels in mouse hearts. [0063] FIGs. 17A-17C shows immunofluorescence localization analysis of human RBM20 protein in in wild-type C57BI/6 mouse heart expressing TNNT2₄₀₀-huRBM20 vector (FIG.17A), in wild-type C57BI/6 mouse heart expressing TNNT2400-huRBM20-NMyc vector (FIG. 17B), and wild-type C57BI/6 mouse heart expressing TNNT2400-huRBM20-siRNA- NMyc vector (FIG. 17C). [0064] FIG. 18 show relative RBM20 mRNA levels in some embodiments of hiPS cardiomyocytes expressing siRNAs targeting RBM20 mRNA for knockdown. [0065] FIGs. 19A-19B show relative mRNA levels of genes targeted by RBM20 for splicing in some embodiments of hiPS cardiomyocytes expressing siRNAs targeting RBM20 mRNA for knockdown. [0066] FIGs. 20A-20C show relative mRNA levels of off-target genes in hiPS cardiomyocytes in some embodiments of cells expressing each of the siRNA antisense strands as identified through NCBI nucleotide BLAST. [0067] FIG. 21 shows luciferase expression in some embodiments HEK293T cells expressing dual luciferase reporter plasmids showing the expression of guide/antisense strands from different primary miRNA scaffolds. DETAILED DESCRIPTION [0068] The present disclosure relates to sequences, methods and compositions that are useful for delivering gene constructs to treat diseases or disorders that involve RBM20. Described herein are compositions and methods of using and making said compositions to generate viral vectors comprising RBM20. Also described herein are sequences and compositions and methods of using and making compositions to generate viral vectors comprising RBM20 and one or more silencing element(s) to reduce, or eliminate, pathogenic and wild-type RBM20 alleles in a subject or host cell prior to the RBM20 and the one or more silencing element(s) being introduced to the subject or host cell. In some embodiments, the silencing elements are introduced, either as a separate vector, or embedded within an existing vector encoding a gene therapy target, and may be advantageous for a number of reasons, including decreased dosing or reduced timing between doses, and enhanced physiological function when the wild type gene is expressed. TERMS [0069] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. [0070] The term “AAV” is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, unless otherwise indicated. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”), which refers to AAV comprising a polynucleotide sequence not of AAV origin (e.g., a transgene). The term “AAV” includes, but is not limited to AAV serotype 1 (AAV-1), AAV serotype 2 (AAV-2), AAV serotype 3 (AAV-3), AAV serotype 4 (AAV-4), AAV serotype 5 (AAV-5), AAV serotype 6 (AAV-6), AAV serotype 7 (AAV-7), AAV serotype 8 (AAV-8), AAV serotype 9 (AAV-9), serotype rh10 AAV, serotype rh74 AAV, or a pseudotyped rAAV (e.g., AAV2/9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9) or derivatives (e.g., engineered formats) of any of the forgoing, such as by way of peptide insertion(s) and/or point mutation(s), or combinations thereof. AAV further includes any modified AAV described or disclosed in WO2021/072197 and WO2019/207132, both of which are incorporated herein by reference, as well as other modified AAV known in the art, such as modified AAV9 P1 capsid protein with peptide RGDLGLS inserted between residues Q588 and A589 with a polynucleotide. [0071] The term “AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least AAV capsid protein and an encapsidated polynucleotide. [0072] The term “adeno-associated virus (AAV) capsid” refers to the three- dimensional proteinaceous shell encapsidating, e.g., enclosing, the viral genetic material. The AAV capsid is a non-enveloped, icosahedral 60-mer of three repeating monomers: VP1, VP2, and VP3. The AAV capsid determines the properties of viral particles, including tissue tropism and antigenic properties. [0073] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including therapeutic proteins and other peptides, e.g., linkers, tags, capsid proteins, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some respects, the polypeptide may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. [0074] Amino acid substitutions may also refer to one or more changes in a polypeptide sequence. The changes may include replacement of one amino acid in a polypeptide with another amino acid, insertion of one or amino acids, and/or deletion of one or more amino acids, or any combination thereof. Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class. [0075] A “nucleic acid” sequence refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence. The term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl- methyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1- methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy- aminomethyl- 2-thiouracil, beta-D-mannosylqueosine, 5’-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5-methyluracil, N- uracil-5-oxyacetic acid methylester, uracil-5- oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. [0076] The term “polynucleotide,” refers to a polymeric form of nucleotides of any length, including DNA, RNA, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. [0077] The term “isolated” when referring to a nucleotide sequence, means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. Thus, an “isolated nucleic acid molecule which encodes a particular polypeptide” refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not materially affect the basic characteristics of the composition. [0078] The terms “percent (%) amino acid sequence identity” and “percent identity” and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) refer to the percentage of amino acid residues in a candidate sequence (e.g. , the engineered AAV capsid) that are identical with the amino acid residues in the non- engineered capsid reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. [0079] For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated “upstream,” “downstream,” “3’,” or “5’” relative to another sequence, it is to be understood that it is the position of the sequences in the “sense” or “coding” strand of a DNA molecule that is being referred to as is conventional in the art. [0080] The term “recombinant,” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature and/or a combination of polynucleotides and viral proteins that is not found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct. [0081] The term “gene,” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the genes with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art. [0082] The term “transgene,” as used herein, refers to a nucleic acid sequence to be positioned within a viral vector and encoding a polypeptide, protein or other product of interest. In some embodiments, one rAAV vector, or engineered rAAV vector may comprise a sequence encoding one or more transgenes (which can optionally be the same gene, or different genes). For example, one rAAV vector may comprise the coding sequence for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 transgenes. [0083] The terms “gene transfer” or “gene delivery” refer to methods or systems for inserting DNA, such as a transgene, into host cells, such as those of a subject afflicted with a cardiomyopathy. In several embodiments, gene transfer yields transient expression of non- integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes). In additional embodiments, gene transfer results in integration of transferred genetic material into the genomic DNA of host cells. [0084] The terms “regulatory element” or “regulatory sequence”, or variations thereof, refer to a nucleotide sequence that participates in functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulatory elements can be enhancing or inhibitory in nature, depending on the embodiment. Non-limiting examples of regulatory elements include transcriptional regulatory sequences such as promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like. These elements collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell, though not all of these sequences need always be present. It shall be appreciated that the structural components of a rAAV vector as provided for herein may be listed in individual paragraphs solely for clarity and may be used together in combination. For example, any regulatory element or other component can be used in combination with any transgene (or transgenes) provided for herein. [0085] A “promoter” is a polynucleotide that interacts with an RNA polymerase and initiates transcription of a coding region (e.g., a transgene) usually located downstream (in the 3′ direction) from the promoter. [0086] A “tissue-specific promoter”, as used herein, refers to promoters that can only function in a specific type of tissue, e.g., the heart. Thus, a “tissue- specific promoter” is not able to drive the expression of the transgenes in other types of tissues. [0087] The term “operably linked” refers to an arrangement of elements wherein the components are configured to perform a function. For example, regulatory sequences operably linked to a coding sequence result in the expression of the coding sequence. Depending on the embodiment, a regulatory sequence need not be contiguous with the coding sequence. Thus, for example, one or more untranslated, yet transcribed, sequences can be present between a promoter sequence and a coding sequence, with those two sequences still being considered “operably linked”. [0088] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” [0089] An “expression vector” is a vector comprising a region of nucleic acid (e.g., a transgene) which encodes a gene product (e.g., a polypeptide or protein) of interest. As disclosed herein, vectors are used for achieving expression, e.g., stable expression, of a protein in an intended target cell. An expression vector may also comprise control elements operatively linked to the transgene to facilitate expression of the encoded protein in the target cell. A combination of one or more regulatory elements and a gene or genes to which they are operably linked for expression may be referred to herein as an “expression cassette.” [0090] A Kozak sequence is a functional sequence motif that is positioned near or at the translational initiation site of eukaryotic mRNAs. Kozak sequences mediate ribosome assembly and translation initiation and help regulate that a protein is correctly translated in the correct reading frame. [0091] A “subject” refers to mammal that is the object of treatment using a method or composition as provided for herein. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and humans. In some embodiments, the subject is human. [0092] The terms “treating,” “treatment,” “therapeutic,” or “therapy” do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and/or therapy. To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. [0093] The term “effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect in a host cell or in a subject, such as reducing the frequency or severity of at least one sign or symptom of a disease or disorder exhibited by the host cell and/or experienced by a subject. [0094] As used herein, a “composition” refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof. [0095] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products. [0096] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects, embodiments, and variations described herein include “comprising,” “consisting,” and/or “consisting essentially of” aspects, embodiments and variations. [0097] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range. [0098] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. [0099] “Gene silencing” refers to the suppression of gene expression, e.g., transgene, heterologous gene and/or endogenous gene expression. Gene silencing may be mediated through processes that affect transcription and/or through processes that affect post- transcriptional mechanisms. In some embodiments, gene silencing occurs when siRNA initiates the degradation of the mRNA of a gene of interest in a sequence-specific manner via RNA interference. In some embodiments, gene silencing may be allele-specific. “Allele- specific” gene silencing refers to the specific silencing of one allele of a gene. [0100] “Knock-down,” “knock-down technology” refers to a technique of gene silencing in which the expression of a target gene is reduced as compared to the gene expression prior to the introduction of the RNAi molecule, which can lead to the inhibition of production of the target gene product. The term “reduced” is used herein to indicate that the target gene expression is lowered by 1-100%. For example, the expression may be reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or even 99%. Knock-down of gene expression can be directed by the use of dsRNAs or siRNAs. For example, “RNA interference (RNAi),” which can involve the use of siRNA, has been successfully applied to knockdown the expression of specific genes in plants, D. melanogaster, C. elegans, trypanosomes, planaria, hydra, and several vertebrate species including the mouse. In several embodiments, RNAi is effective in human subjects. [0101] “RNA interference (RNAi)” is the process of sequence-specific, post- transcriptional gene silencing initiated by siRNA. RNAi is seen in a number of organisms such as Drosophila, nematodes, fungi and plants, and is believed to be involved in anti-viral defense, modulation of transposon activity, and regulation of gene expression. During RNAi, RNAi molecules induce degradation of target mRNA with consequent sequence-specific inhibition of gene expression. [0102] A “small interfering” or “short interfering RNA” or siRNA is a RNA duplex of nucleotides that is targeted to a gene interest. A “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule. siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, the length of the duplex is 19 — 25 nucleotides in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3′ or 5′ overhang portions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. The “sense” and “antisense” sequences can be used with or without a loop region to form siRNA molecules. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example, double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, post- transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetic silencing. For example, siRNA molecules of the invention can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic modulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression. In another non-limiting example, modulation of gene expression by siRNA molecules of the invention can result from siRNA mediated cleavage of RNA (either coding or non-coding RNA) via RISC, or alternately, translational inhibition as is known in the art. [0103] The siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal. [0104] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, e.g., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. [0105] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or’” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,”’ or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shal1 only be interpreted as indicating exclusive alternatives (for example, “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. [0106] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the Est of elements, but not necessarily including at least one of each and every element specifica11y listed within the list of e1ernents and not excluding any combinations of e1ernents in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. [0107] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, e.g., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi—closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentia1ly of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”. [0108] In some embodiments, “administering” or “administration” means providing a material to a subject or a host cell in a manner that is pharmacologically useful. [0109] To “’treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject or host cell. The compositions described above or elsewhere herein are typically administered to a subject or host cell in an effective amount, that is, an amount capable of producing a desirable result. The desirable result will depend upon the active agent being administered. [0110] In some embodiments, a single composition comprising rAAV particles as disclosed herein is administered only once. In some embodiments, a subject or host cell may need more than 1 administration of an rAAV composition (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times). For example, a subject or host cell may need to be provided a second administration of any one of the rAAV compositions as disclosed herein 1 day, 1 week, l month, 1 year, 2 years, 5 years, or 10 years after the subject or host cell was administered a first composition. In some embodiments, a first composition of rAAV particles is different from the second composition of rAAV particles. In some embodiments, the administration of the composition is repeated at least once (e.g., at least once, at least twice, at least thrice, at least four times, at least five times, at least six times, at least 10 times, at least 25 times, or at least 50 times), and wherein the time between a repeated administration and a previous administration is at least l month (e.g., at least l month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least l 2 months). In some embodiments, the administration of the composition is repeated at least once, and wherein the time between a repeated administration and a previous administration is at least 1 year (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 30 years). [0111] In some embodiments, the administration of the composition is facilitated by AAV capsids such as AAV1-9, and/or rh74 (or modified versions thereof) e.g., with AAV2 ITRs, or other capsids that sufficiently deliver to affected tissues. Nucleic acids [0112] In some embodiments, a nucleic acid (e.g., an expression cassette) is provided in a viral vector (e.g., an rAAV vector). In some embodiments, the nucleic acid comprises a promoter and sequence corresponding to a gene of interest (e.g., RBM20 according to several embodiments) that is capable of being expressed from the nucleic acid. In some embodiments, the nucleic acid is a rAAV genome comprising a DNA molecule, wherein the DNA molecule comprises sequences that encode an RNA molecule. [0113] In some embodiments, the nucleic acid is sufficiently small to be effectively packaged in an AAV viral particle (e.g., the gene construct may be around 0.5-5 kb long, for example around 4.9 kb, 4.8 kb, 4.7 kb, 4.6 kb, 4.5 kb, 4.4 kb, 4.3 kb, 4.2 kb, 4.1 kb, 4 kb, 3.5 kb, or 3 kb long). [0114] In some embodiments, the nucleic acid comprises a silencing element that corresponds to a siRNA that targets a chromosomal allele encoding RBM20. As used herein, “siRNA,” or small interfering RNA refers to RNAs that reduce the level of its binding target for example, via an Argonaute-mediated pathway. Examples of siRNAs are known in the art, and may include, for example, siRNAs that originate from short hairpin RNAs that are cleaved by Drosha and Dicer and recruit Argonaute to their targets for subsequent cleavage and/or regulation of translation. Examples of siRNAs are known in the art, and a review of RNAi can be found, for example, reviewed in Wilson, R.C. and Doudna, J. A., (2013), Annual Review of Biophysics: Molecular Mechanisms of RNA Interference, 42:1, 217-39. [0115] The RNA-binding motif protein 20 (RBM20) gene provides instructions for making a protein that regulates splicing; mutations in the RBM20 gene are known to cause dilated cardiomyopathy (DCM). Promoters and Other Elements [0116] In some embodiments, the rAAV vector comprises one or more regions comprising a sequence that facilitates expression of the heterologous nucleic acid, e.g., expression regulatory sequences operatively linked to the heterologous nucleic acid. A promoter drives transcription of the nucleic acid sequence that it regulates, thus, it is typically located at or near the transcriptional start site of a gene. A promoter may have, for example, a length of 10, 25, 50 or 100 to 1000 nucleotides. In some embodiments, a promoter is operably linked to a nucleic acid, or a sequence of a nucleic acid (nucleotide sequence). A promoter is considered to be “operably linked” to a sequence of nucleic acid that it regulates when the promoter is in a correct functional location and orientation relative to the sequence such that the promoter regulates (e.g., to control (“drive”) transcriptional initiation and/or expression of) that sequence. Numerous such sequences are known in the art. [0117] Promoters that may be used in accordance with the present disclosure may comprise any promoter that can drive the expression of the transgenes in the heart of the subject or a cardiac host cell. In some embodiments, the promoter may be a tissue-specific promoter. A “tissues-specific promoter”, as used herein, refers to promoters that can only function in a specific type of tissue, e.g., the heart. Thus, a “tissue- specific promoter” is not able to drive the expression of the transgenes in other types of tissues. In some embodiments, the promoter that may be used in accordance with the present disclosure is a cardiac -restricted promoter. Non-limiting examples of tissue-specific promoters and/or regulatory elements that may be used include (1) desmin, creatine kinase, myogenin, alpha myosin heavy chain, and natriuretic peptide, CK6, CK8, MHCK7, miniMCK, myoglobin, and SPc5-12 specific for muscle cells, and (2) albumin, alpha-1- antitrypsin, hepatitis B virus core protein promoters, specific for liver cells. Non-limiting examples of cardiac-restricted promoter selected from cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT). In treating cardiomyopathies as provided for herein, cardiac-restricted promoters are advantageous at least due to the reduced possibility of off-target expression of the transgene(s), thereby effectively increasing the delivered dose to the heart and enhancing therapy. Non-limiting examples of expression regulatory sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such regulatory sequences is contemplated herein (e.g., a promoter and an enhancer). [0118] Alternatively, the promoter may be, without limitation, a promoter from one of the following genes: a-myosin heavy chain gene, 6- myosin heavy chain gene, myosin light chain 2v (MLC-2v) gene, myosin light chain 2a gene, CARP gene, cardiac a-actin gene, cardiac m2 muscarinic acetylcholine gene, atrial natriuretic factor gene (24ND), cardiac sarcoplasmic reticulum Ca-ATPase gene, skeletal a-actin gene; or an artificial cardiac promoter derived from MLC-2v gene. In several embodiments, an engineered, synthetic promoter is used. In several embodiments, a JeT promoter is used. [0119] To achieve appropriate expression levels of the nucleic acid, protein, or polypeptide of interest, any of a number of promoters suitable for use in the selected host cell may be employed. The promoter may be, for example, a constitutive promoter, tissue-specific promoter, inducible promoter, or a synthetic promoter. For example, constitutive promoters of different strengths can be used. An rAAV vector described herein may include one or more constitutive promoters, such as viral promoters or promoters from mammalian genes that are generally active in promoting transcription. Non-limiting examples of constitutive viral promoters include the Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad El A and cytomegalovirus (CMV) promoters. Non-limiting examples of non-viral constitutive promoters include various housekeeping gene promoters, as exemplified by the β-actin promoter, including the chicken β-actin promoter (CβA). Inducible promoters and/or regulatory elements may also be contemplated for achieving appropriate expression levels of the protein or polypeptide of interest. Nonlimiting examples of suitable inducible promoters include those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone- inducible genes, such as the estrogen gene promoter. Another example of an inducible promoter is the tetVP16 promoter that is responsive to tetracycline. [0120] Synthetic promoters are also contemplated herein. A synthetic promoter may comprise, for example, regions of known promoters, regulatory elements, transcription factor binding sites, enhancer elements, repressor elements, and the like. As discussed above, in several embodiments, the JeT promoter is used. [0121] Enhancer elements can function in combination with other regulatory elements to increase the expression of a transgene. In several embodiments, the enhancer elements are upstream (positioned 5’) of the transgene. Non-limiting embodiments of enhancer elements include nucleotide sequences comprising, for example, a 100 base pair element from Simian virus 40 (SV40 late 2XUSE), a 35 base pair element from Human Immunodeficiency Virus 1(HIV-1 USE), a 39 base pair element from ground squirrel hepatitis virus (GHV USE), a 21 base pair element from adenovirus (Adenovirus L3 USE), a 21 base pair element from human prothrombin (hTHGB USE), a 53 base pair element from human C2 complement gene (hC2 USE), truncations of any of the foregoing, and combinations of the foregoing. In some embodiments the enhancer is derived from the a-myosin heavy chain (aMHC) gene. In some embodiments the aMHC enhancer comprises a nucleic acid sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to: CCTTCAGATTAAAAATAACTAAGGTAAGGGCCATGTGGGTAGGGGAGGTGG TGTGAGACGGTCCTGTCTCTCCTCTATCTGCCCATCGGCCCTTTGGGGAGGA GGAATGTGCCCAAGGACTAAAAAAAGGCCCTGGAGCCAGAGGGGCGAGGG CAGCAGACCTTTCATGGGCAAACCTCAGGGCTGCTGTC SEQ ID NO: 61. [0122] Non-limiting polyadenylation signals include nucleotide sequences comprising, for example, a 624 base pair polyadenylation signal from human growth hormone (hGH), a 135 base pair polyadenylation signal from simian virus 40 (sV40 late), a 49 base pair synthetic polyadenylation signal from rabbit beta-globin (SPA), a 250 base pair polyadenylation signal from bovine growth hormone (bGH), truncations of any of the foregoing, and combinations of the foregoing. [0123] In some embodiments of the disclosed rAAV vectors, the two or more transgenes are operably controlled by a single promoter. In some embodiments, each of the two or more transgenes are operably controlled by a distinct promoter. [0124] In several embodiments, a plurality of promoter elements may be used. For example, in one embodiment a promoter drives expression of the transgene and an additional promoter drives expression of another component. In several embodiments, an additional promoter is used to drive expression of a silencing element as provided for herein. In several embodiments, a PolIII promoter, or a PolII promoter is used to drive expression of a silencing element. In several embodiments, an additional promoter element is selected from the group consisting of H1, U6, and 7SK. In still additional embodiments, a single promotor may be used to drive expression of both the transgene and the one or more silencing elements. [0125] In some embodiments, the rAAV vectors of the present disclosure further comprise an Internal Ribosome Entry Site (IRES). An IRES is a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the greater process of protein synthesis. Usually, in eukaryotes, translation can be initiated only at the 5’ end of the mRNA molecule, since 5’ cap recognition is required for the assembly of the initiation complex. In some embodiments, the IRES is located between the transgenes. [0126] In such embodiments, the proteins encoded by different transgenes are translated individually (e.g., versus translated as a fusion protein). [0127] In some embodiments, the rAAV vectors of the present disclosure comprise at least, in order from 5’ to 3’, a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, a promoter operably linked to a first transgene, an IRES operably linked to a second transgene, a polyadenylation signal, and a second AAV inverted terminal repeat (ITR) sequence. [0128] In some embodiments, the rAAV vectors of the present disclosure further comprise a polyadenylation (pA) signal. Introns [0129] In some embodiments, an rAAV of the present invention comprises a nucleic acid encoding an RNA, wherein the RNA comprises at least one intron (e.g., 1-5, 5- 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, or more) that may be regulated by an intracellular factor. In some aspects, an intron for which splicing may be regulated is an intron for which splicing levels differ by at least 5%, for example at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99%, or 100% under two different conditions (e.g., in different tissues, in response to intracellular levels of one or more RNA binding proteins, in the context of an autoregulated gene, etc.). That is the splicing levels for an intron of interest are measured in two different conditions, and the splicing level is compared between the conditions and expressed as a percentage change. For example, if the splicing level in condition A is 80%, and the splicing level in condition B is 85%, the splicing levels between conditions A and B differ by 5%. Likewise, if the splicing level in condition A is 80%, and the splicing level in condition B is 75%, the splicing levels between conditions A and B also differ by 5%. [0130] In some embodiments, an intron contains functional splice donor and acceptor sites (e.g., naturally occurring or engineered splice donor and/or acceptor sites), and one or more regulatory regions that can control intron splicing (e.g., whether splicing occurs or whether which of one or more alternative splicing events occurs). In some embodiments, a regulatory region can bind to one or more intracellular factors (e.g., RNA binding proteins) that regulate intron splicing. In some embodiments, an intracellular factor comprises a protein, an RNA, or a protein-RNA complex. In some embodiments, the protein comprises an RNA- binding protein. In some embodiments, an intron for which splicing is regulated comprises one or more RNA binding protein sites (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10-20). As used herein, an “RNA binding protein site” refers to an RNA sequence or structure that interacts with an “RNA binding protein” as defined below. In some embodiments, the binding site(s) can be in an intron, an exon, or both. An “RNA binding protein” (RBP) refers to a protein that binds to double- or single-stranded RNA in cells and participates in forming ribonucleoprotein complexes. RBPs contain various structural motifs, such as RNA recognition motif (RRM), dsRNA binding domain, zinc finger and others. [0131] In some embodiments, an RNA binding protein is a sequence-specific RNA binding protein. In some embodiments, a useful sequence-specific RNA binding protein binds to a target sequence with a binding affinity (e.g., Kd) of 0.01-1000 nM or less (e.g., 0.01 to 1, 1-10, 10-50, 50-100, 100-500, 500-1,000 nM). In some embodiments, an RNA binding protein has serine/arginine domains that act as splicing enhancers, or glycine-rich domains that act as splicing repressors. In some embodiments, an RNA binding protein acts as an intronic splicing enhancer, intronic splicing silencer, exonic splicing enhancer, or exonic splicing silencer. Different types of sequence-specific RNA binding proteins can be used. In some embodiments, a sequence-specific RNA binding protein is one that contains zinc fingers, RNA recognition motifs, KH domains, deadbox domains, or dsRBDs. Non-limiting examples of RBPs that contain zinc fingers include: MBNL, TIS11, or TTP. Non-limiting examples of RBPs that contain RNA recognition motifs include hnRNPs and SR proteins, RbFox, PTB, Tra2beta. Non- limiting examples of RNA binding proteins that contain KH domains include Nova, SF1, and FBP. Non-limiting examples of RNA binding proteins that contain deadbox domains are DDX5, DDX6, and DDX17. Non-limiting exampes of RNA binding proteins that contain dsRBDs include ADAR, Staufen, and TRBP. Further examples of these types of RNA binding proteins and their respective sequence specific binding motifs are known in the art, and can be found, for example, in Perez-Perri, J. I., et al., (2018), Nat. Comm., 9:4408; Van Nostrand, E. L., et al., (2020), Nature, 583, 711–19; and Corley, M., et al., (2020), Cell, (20): 30159-3, the contents of which are hereby incorporated by reference with respect to RNA protein binding sites and RNA binding proteins. [0132] In some embodiments, splicing of a regulated intron (e.g., an auto-regulated intron) in a gene of a subject can be affected by the presence in the subject of one or more mutations (e.g., genomic mutations) that alter binding of splice regulatory proteins to a regulatory region of the regulated intron. In some embodiments, such mutations can include the presence of other sequences that can alter (e.g., increase or decrease) binding to splice regulatory proteins. Accordingly, in some embodiments a composition of the application can be delivered to a subject or host cell that has a condition associated with aberrant splice regulation of one or more genes in order to restore, at least partially, normal levels of splice regulation. In some embodiments, a gene comprising one or more introns for which splicing is appropriately regulated is provided in an rAAV. In some embodiments, an intron is an engineered intron. In some embodiments, the engineered intron comprises a donor and acceptor splice site, and a functional branch point to which the donor splice site can be joined in the first trans-esterification reaction of splicing. In some embodiments, an intron comprises a truncated version of a natural intron. By “truncated natural intron”, it is meant that the naturally occurring, full-length intron is shortened (e.g., truncated) via the removal of nucleotides from either the 5’ or 3’ (or both) end. In some embodiments, a recombinant intron (e.g., a synthetic intron) can be used. In some embodiments, a chimeric intron is used. In some embodiments, a recombinant intron is a truncated version of a natural intron. However, in some embodiments a recombinant intron can be designed to include functional splice donor and acceptor sites and a functional branch point in addition to one or more regulatory regions that are derived from different introns, or that are non-naturally occurring sequences (e.g., sequence variants of naturally occurring sequences, consensus sequences, or de-novo designed sequences). Accordingly, in some embodiments a recombinant intron is not a truncated version of a naturally occurring intron, but contains one or more sequences from a naturally occurring intron. [0133] In some embodiments, a truncated intron is truncated at its 5’ end. In some embodiments, 1-10,000 nucleotides are truncated from the 5’ end (e.g., 1-50, 50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000, 10,000-20,000, 20,000-50,000, 50,000-100,000, or a subset of any of the foregoing, nucleotides are truncated from the 5’ end). In some embodiments, the 5’ splice site is retained in the truncated intron. In some embodiments, a different 5’ splice site is included in the truncated intron. In some embodiments, a truncated intron is truncated at its 3’ end. In some embodiments, 1-10,000 nucleotides are truncated from the 3’ end (e.g., 1-50, 50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000, 10,000-20,000, 20,000-50,000, 50,000-100,000, or a subset of any of the foregoing, nucleotides are truncated from the 3’ end). In some embodiments, the 3’ splice site is retained in the truncated intron. In some embodiments, a different 3’ splice site is included in the truncated intron. In some embodiments, a truncated intron is truncated at one or more internal locations. In some embodiments, 1-10,000 internal nucleotides are removed (e.g., 1-50, 50-100, 100-500, 500- 1,000, 1,000-5,000, 5,000-10,000, 10,000-20,000, 20,000-50,000, or 50,000-100,000 internal nucleotides are removed). In some embodiments, the splice regulatory region is retained in the truncated intron. In some embodiments, a different splice regulatory region is included in the truncated intron. In some embodiments, a truncated intron comprises one or more 5’, 3’, and/or internal deletions. It should be understood that the extent of truncation may depend on the size of the intron and the size of the gene. A truncation may require removal of sufficient intron sequences to result in a recombinant gene construct that is small enough to be packaged in a recombinant virus of interest (e.g., in an rAAV virus). However, an engineered (e.g., truncated) intron typically includes one or more sequences required for efficient splicing and/or regulated (e.g., autoregulated) splicing. In some embodiments, a recombinant (e.g., truncated) intron retains a donor site (e.g., towards the 5’ end of the truncated intron), a branch site (e.g., towards the 3’ end of the truncated intron), an acceptor site (e.g., at the 3’ end of the truncated intron), and a splice regulatory sequence. In some embodiments, the intron comprises a 5’ splice donor site. In some embodiments, the 5’ splice donor site is a GU or an AU. In some embodiments, the intron comprises a 3’ splice acceptor site. In some embodiments, the 3’ splice acceptor site is an AG or an AC. In some embodiments, a regulatory sequence comprises a response element within an AG exclusion zone of the truncated intron. In some embodiments, the truncated intron retains sequence motifs bound by the encoded protein. In some embodiments, an engineered (e.g., truncated) intron may include one or more human, non-human primate, and/or other mammalian or non-mammalian intron splice-regulatory sequences. In some embodiments, the regulatory sequences may have 80%-100% (e.g., 80-85%, 85%-90%, greater than 90%, 90%-95%, 95%-100%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100%) sequence identify with a wild-type regulatory sequence. In some embodiments, an engineered intron is approximately 50 to 4000 nucleotides long. In some embodiments, an engineered intron is approximately 50 to 100, 75-125, 100- 150, 125-175, 200-250, 225-275, 300-350, 325-375, 400-450, 425-475, 500-550, 525-575, 600-650, 625-675, 700-750, 725-775, 800-850, 825-875, 900-950, 925-975, 950-1000, 1025- 1075, 1050 to 1100, 1075-1125, 1100-1150, 1125-1175, 1200-1250, 1225-1275, 1300-1350, 1325-1375, 1400-1450, 1425-1475, 1500-1550, 1525-1575, 1600-1650, 1625-1675, 1700- 1750, 1725-1775, 1800-1850, 1825-1875, 1900-1950, 1925-1975, 1950-2000, 2025-2075, 2050 to 2100, 2075- 2125, 2100-2150, 2125-2175, 2200-2250, 2225-2275, 2300-2350, 2325- 2375, 2400-2450, 2425- 2475, 2500-2550, 2525-2575, 2600-2650, 2625-2675, 2700-2750, 2725-2775, 2800-2850, 2825- 2875, 2900-2950, 2925-2975, 2950-3000, 3025-3075, 3050 to 3100, 3075-3125, 3100-3150, 3125-3175, 3200-3250, 3225-3275, 3300-3350, 3325-3375, 3400-3450, 3425-3475, 3500-3550, 3525-3575, 3600-3650, 3625-3675, 3700-3750, 3725- 3775, 3800-3850, 3825-3875, 3900-3950, 3925-3975, or 3950-4000 nucleotides long, or any integer contained therein (e.g., 51, 52, 53, 54, 55, etc.). Accordingly, in some embodiments a recombinant nucleic acid for which splicing is regulated is a construct that can produce either i) a protein coding transcript of interest (e.g., a functional protein coding sequence) or ii) a transcript that that does not encode a full length protein coding sequence depending on whether an intron is removed or not (e.g., if the splicing event results in inclusion of a start codon, or if the splicing event results in inclusion of a stop codon). [0134] In some embodiments, a recombinant nucleic acid for which splicing is regulated can produce alternative splice products each of which can encode a protein, but the proteins have different functions (e.g., the alternatively spliced RNA products encode protein isomers). For example, in some embodiments the splicing events could generate cardiac- versus skeletal muscle-specific isoforms, or isoforms with differing functions or properties. In some embodiments one or more tissue specific RNA protein binding motifs can be included in a recombinant nucleic acid (e.g., within an intron or an exon) in order to regulate splicing (e.g., to promote splicing in a first tissue or cell type relative to a second tissue or cell type). In some embodiments, a recombinant nucleic acid for which splicing is regulated is a synthetic construct configured to regulate expression of an RNA by including a nonsense mediated decay (NMD) exon within the RNA, wherein the NMD exon is flanked by introns for which alternative splicing is regulated. In some embodiments, an NMD exon is an exon that encodes at least one stop codon that is in frame with a previous exon, wherein the stop codon is at least 50 nucleotides upstream (5’) from the 5’ splice site of the exon. In some embodiments, if the NMD exon is included in the spliced RNA, it causes degradation of the RNA via nonsense- mediated decay. In some embodiments, if the NMD exon is spliced out, the resulting transcript is stable, and in some embodiments encodes a functional (e.g., full-length) protein of interest. In some embodiments, a recombinant nucleic acid for which splicing is regulated is a synthetic construct configured to regulate expression of a protein by including a 5’ exon comprising an amino terminal amino acid encoding sequence (e.g., an ATG or part of the ATG) and/or translation control sequences, wherein the 5’ exon is separated from subsequent exon(s) by an intron for which splicing is regulated. In some embodiment, if the intron is spliced out of the RNA transcript, the recombinant 5’ exon is spliced in frame to the subsequent exon(s) and the resulting spliced transcript encodes a protein that is expressed. In some embodiments, if the intron is not spliced out of the RNA transcript, the recombinant 5’ exon is not spliced to the subsequent exon(s) and as a result a protein is not expressed from the transcript. In some embodiments, an intron for which splicing is regulated can be included within a gene that encodes a regulatory RNA (e.g., an siRNA). In some embodiments, intron(s) for which splicing is regulated and that encode regulatory RNA(s) can be included in a recombinant nucleic acid encoding an RNA transcript. It will be understood by those of skill in the art that a nucleic acid may comprise a single intron or multiple introns. In some embodiments, the nucleic acid comprises one intron. In some embodiments, the nucleic acid comprises two introns. In some embodiments, the nucleic acid comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 introns. Exons [0135] In some embodiments, a nucleic acid comprises an exon. In some embodiments, the exon is flanked by one or more introns (e.g., one or more auto-regulated introns), that may in some embodiments be truncated as described herein. In some embodiments, the exon is naturally occurring. In some embodiments, the exon is a recombinant exon. In some embodiments, the exon is an alternatively regulated exon. In some embodiments, the alternatively regulated exon is flanked by one or more introns. In some embodiments, the retention of one or more introns causes the transcript to remain in the nucleus and unable to be exported into the cytoplasm, preventing translation of any protein product. In some embodiments, exons that are naturally flanked by non-regulated introns are fused into continuous coding regions (e.g., a recombinant exon) without any intervening introns. In some embodiments, each exon that is auto-regulated has two or more flanking introns, which may or may not be truncated as described herein (e.g., “flanking” referring to an intron located immediately upstream (5’) or immediately downstream (3’) of an associated exon). In some embodiments, the exon is a regulatory exon. It will be understood by those of skill in the art that a nucleic acid may comprise a single exon or multiple exons. In some embodiments, the nucleic acid comprises one exon. In some embodiments, the nucleic acid comprises two exons. In some embodiments, the nucleic acid comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 exons. In several embodiments, the transgene (as described elsewhere) comprises one or more exons joined together to provide a coding sequence. The Transgene [0136] A transgene may be employed to correct, reduce, eliminate, or otherwise ameliorate gene deficiencies, which may include deficiencies in which normal genes are expressed at less than normal levels, are expressed at normal or near-normal levels but having a gene product with abnormal activity, or deficiencies in which the functional gene product is not expressed. In several embodiments, the transgene sequence encodes a therapeutic protein or polypeptide which is to be expressed in a host cell. In several embodiments, the transgene is modified (e.g., at the nucleic acid level) to endow the transgene to have reduced susceptibility to one or more silencing elements (described elsewhere herein). In several embodiments, the modifications do not result in changes to the amino acid sequence of the transgene. Embodiments of the present disclosure also include using multiple transgenes. [0137] RNA binding motif protein 20 is encoded by the RBM20 gene. Mutations in or perturbations in the function of RBM20 are known to be causative of DCM (Dilated Cardiomyopathy). RBM20 is a major regulator of heart-specific alternative splicing of the TTN gene, which is found to be most frequently mutated in patients with idiopathic DCM (approximately 20–25%). The TTN gene has the largest number of exons (364 in humans) and titin, a sarcomeric protein encoded by the TTN gene, is the largest known protein in mammals. In an RBM20 mutant rat strain lacking nearly all the RBM20 exons, the shortest cardiac titin isoform N2B is not expressed. Therefore, RBM20 is a key regulator of TTN pre-mRNA processing in the heart and may cause DCM phenotypes through altered splicing of the RBM20-regulated genes. Missense mutations in a highly conserved RSRSP stretch, within an arginine/serine (RS)-rich region and not in the RNA binding domains are the most frequent disease alleles. In some embodiments of the disclosed rAAV vectors, the transgene is RBM20 cDNA, such as human RBM20 cDNA. In some embodiments, the transgene is an RBM20 coding sequence that has been codon-optimized for expression in a mammalian cell. In some embodiments, the transgene is an RBM20 coding sequence that has been codon optimized for expression in human cells. In several embodiments, the RBM20 coding sequence is modified (e.g., at the nucleic acid level) to endow the RBM20 coding sequence with reduced susceptibility to one or more silencing elements (described elsewhere herein). In several embodiments, the modifications do not result in changes to the amino acid sequence encoded by the RBM20 coding sequence. In several embodiments, the RBM20 coding sequence is CpG depleted, partially or fully. [0138] In some embodiments, any of the disclosed rAAV vectors contain multiple transgenes. In some embodiments, the rAAV vector discloses two or more transgenes. Silencing Elements [0139] In some embodiments, the rAAV also encodes an inhibitory molecule (e.g., an inhibitory RNA, for example an siRNA or shRNA) that reduces, inhibits, or otherwise eliminates expression (e.g., transcription and/or translation) of endogenous RBM20 (e.g., both pathogenic and wild type alleles) expressed from the genome of the subject. In some embodiments, the subject or host cell having received the silencing element(s) would then express the RBM20 delivered to the subject or host cell over the endogenous RBM20, including by way of a rAAV as provided for herein. Expression Cassette [0140] The expression cassette is composed of, at a minimum, a transgene and its regulatory sequences. Where the cassette is designed to be expressed from a rAAV, the expression cassette further contains 5’ and 3’ AAV ITRs. These ITR's may be full-length, or one or both ITRs may be truncated. In one embodiment, the rAAV is pseudotyped, e.g., the AAV capsid is from a different source AAV than the AAV which provides the ITRs. In one embodiment, the ITRs of AAV serotype 2 are used. In additional embodiments, the ITRs of AAV serotype 1 are used. However, ITRs from other suitable sources may be selected. [0141] Figure 1 depicts a non-limiting example of a rAAV expression cassette that may be used to overexpress wild-type RBM20, for example in a therapeutic approach that relies on overexpression of the transgene (“overexpression only”). The expression cassette includes a promoter and a polyadenylation site. In one embodiment, the promoter is a constitutive cardiac specific promoter. In other embodiments, the promoter is a constitutive muscle-specific promoter. In another embodiment, the promoter is a constitutive ubiquitous promoter. The expression cassette may also include other regulatory sequences. In one embodiment, the expression cassette includes an intron. In one embodiment, the RBM20 coding sequence in the expression cassette is codon optimized. In additional embodiments the RBM20 sequence is CpG depleted or both codon-optimized and CpG depleted. [0142] Figure 2 depicts a non-limiting embodiment of a construct described herein. In several embodiments, such a construct is employed in the “overexpression only” therapeutic approach. At the 5’ end is an inverted terminal repeat (ITR) sequence, ITR-L-Flip, followed by a JeT promoter and a Kozak sequence. Following the Kozak sequence is the human wild type RBM20 (wt.huRbm20) coding sequence, a polyadenylation signal, and ITR- R-flop sequence. The locations in the RBM20-coding sequence that the small interfering RNAs (siRNA) listed in Table 10 target are shown. Additionally, a multiplicity of further promoter or regulatory sequences may comprise a construct to alter or change the expression of RBM20. [0143] Figure 3 depicts a non-limiting embodiment of a construct described herein. In several embodiments, such a construct is employed in the “overexpression only” therapeutic approach. At the 5’ end is an ITR sequence, ITR-L-Flip, followed by a CK8 promoter, a chimeric intron, and a Kozak sequence. Following the Kozak sequence is the human wild type RBM20 (wt.huRbm20) coding sequence, a polyadenylation signal, and ITR- R-flop sequence. The locations in the RBM20-coding sequence that the small interfering RNAs (siRNA) listed in Table 10 target are shown. Additionally, a multiplicity of further promoter or regulatory sequences may comprise a construct to alter or change the expression of RBM20. [0144] Figure 4 depicts an embodiment of a non-limiting example of a construct with a Myc-tag coding sequence described herein. In several embodiments, such a construct is employed in the “overexpression only” approach. At the 5’ end is an ITR sequence, ITR-L- Flip, followed by a JeT promoter and a Kozak sequence. Following the Kozak sequence is Myc-tag coding sequence, human wild type RBM20 (wt.huRbm20) coding sequence, a polyadenylation signal, and ITR-R-flop sequence. The locations in the RBM20-coding sequence that the small interfering RNAs (siRNA) listed in Table 10 target are shown. Additionally, a multiplicity of further promoter or regulatory sequences may comprise a construct to alter or change the expression of RBM20. [0145] Figure 5 depicts a non-limiting embodiment of a construct described herein. In several embodiments, such a construct is employed in a therapeutic approach to express both silencing elements and wild type RBM20 that is resistant to siRNA degradation (“silencing and overexpression”). At the 5’ end is an ITR sequence, followed by a short hairpin RNA, a Pol II promoter, an intron, followed by a siRNA-resistant RBM20 coding sequence and a 3’ ITR sequence. The expression cassette may include Pol III promoters for expressing shRNAs. Non-limiting examples of Pol III promoters include H1 and U6. The expression cassette may include Pol II promoters for expressing the siRNA-resistant wild type RBM20 coding sequence and introns. The expression cassette may also include other regulatory sequences. In some embodiments, the expression cassette includes an intron. In some embodiments, the shRNA encoding the siRNA is in an intron. In some embodiments, the RBM20 coding sequence in the expression cassette is codon optimized. In additional embodiments the RBM20 coding sequence is CpG depleted or both codon-optimized and CpG depleted. [0146] Figure 6 depicts a non-limiting embodiment of a construct described herein. In several embodiments, such a construct is employed in the “silencing and overexpression” therapeutic approach. At the 5’ end is an ITR sequence, followed by a promoter (such as a PolIII or Pol II promoter), an intron that includes a pri-miRNA scaffold that an contains an shRNA, followed by siRNA-resistant RBM20 coding sequence and a 3’ ITR sequence. The expression cassette may also include Pol II promoters for expressing a siRNA-resistant wild type RBM20 coding sequence and introns. The expression cassette may also include other regulatory sequences. In some embodiments, the expression cassette includes an intron. In some embodiments, the shRNA encoding the siRNA is in an intron. In some embodiments, the RBM20 coding sequence in the expression cassette is codon optimized. In additional embodiments the RBM20 coding sequence is CpG depleted or both codon-optimized and CpG depleted. [0147] Figure 7 depicts a non-limiting embodiment of a construct described herein. At the 5’ end is an ITR sequence, ITR-L-Flip, followed by a JeT promoter and a Kozak sequence. Following the Kozak sequence is a human RBM20 (huRbm20) coding sequence, modified to deplete CpGs and confer resistance to the siRNAs in Table 10, a polyadenylation signal, and ITR-R-flop sequence. The locations of RBM20-coding sequences modified to confer resistance to degradation by the small interfering RNAs (siRNA) are indicated in the diagram and the sequences are listed in Table 11. Additionally, a multiplicity of further promoter or regulatory sequences may comprise a construct to alter or change the expression of RBM20. [0148] Figure 8 depicts an embodiment of a construct described herein. At the 5’ end is an ITR sequence, ITR-L-Flip, followed by a CK8 promoter, a chimeric intron, and a Kozak sequence. Following the Kozak sequence is a human RBM20 (huRbm20) coding sequence, modified to deplete CpGs and confer resistance to one or more siRNAs in Table 10, a polyadenylation signal, and ITR-R-flop sequence. The locations of RBM20-coding sequences modified to confer resistance to degradation by small interfering RNAs (siRNA) are indicated in the diagram and exemplary sequences are listed in Table 11. Additionally, a multiplicity of promoter or regulatory sequences may comprise a construct to alter or change the expression of RBM20. [0149] Figure 9 depicts an embodiment of a construct described herein. At the 5’ end is an ITR sequence, ITR-L-Flip, followed by a JeT promoter and a Kozak sequence. Following the Kozak sequence is a human RBM20 (huRbm20) coding sequence, which comprises the R634Q mutation and modifications to deplete CpGs and confer resistance to the siRNAs in Table 10, a polyadenylation signal, and ITR-R-flop sequence. The locations of RBM20-coding sequences modified to confer resistance to degradation by the small interfering RNAs (siRNA) are indicated in the diagram and the sequences are listed in Table 11. Additionally, a multiplicity of promoter or regulatory sequences may comprise a construct to alter or change the expression of RBM20. [0150] Figure 10 depicts an embodiment of a construct described herein. At the 5’ end is an ITR sequence, ITR-L-Flip, followed by a CK8 promoter, a chimeric intron, and a Kozak sequence. Following the Kozak sequence is the human RBM20 (huRbm20) coding sequence, which contains the R634Q mutation and is modified to deplete CpGs and confer resistance to the siRNAs in Table 10, a polyadenylation signal, and ITR-R-flop sequence. The locations of the RBM20-coding sequences modified to confer resistance to degradation by the small interfering RNAs (siRNA) are indicated in the diagram and the sequences are listed in Table 11. Additionally, a multiplicity of promoter or regulatory sequences may comprise a construct to alter or change the expression of RBM20. [0151] Figure 11 depicts an embodiment of a construct with a Myc-tag encoding sequence as described herein. At the 5’ end is an ITR sequence, ITR-L-Flip, followed by a JeT promoter and a Kozak sequence. Following the Kozak sequence is Myc-tag coding sequence, human RBM20 (huRbm20) coding sequence, modified to deplete CpGs and confer resistance to the siRNAs in Table 10, a polyadenylation signal, and ITR-R-flop sequence. The locations of the RBM20-coding sequences modified to confer resistance to degradation by the small interfering RNAs (siRNA) are indicated in the diagram and the sequences are listed in Table 11. Additionally, a multiplicity of promoter or regulatory sequences may comprise a construct to alter or change the expression of RBM20. [0152] Figure 12 depicts an embodiment of a construct described herein. At the 5’ end is an ITR sequence, ITR-L-Flip, followed by a JeT promoter and a Kozak sequence. Following the Kozak sequence is Myc-tag coding sequence, human RBM20 (huRbm20) coding sequence, which contains the R634Q mutation and is modified to deplete CpGs and confer resistance to the siRNAs in Table 10, a polyadenylation signal, and ITR-R-flop sequence. The locations of the RBM20-coding sequences modified to confer resistance to degradation by the small interfering RNAs (siRNA) are indicated in the diagram and the sequences are listed in Table 11. Locations of restriction enzyme sites in the construct are also shown. Additionally, a multiplicity of promoter or regulatory sequences may comprise a construct to alter or change the expression of RBM20. [0153] In some embodiments of any of the RBM20 expression constructs described herein the siRNAs targeted to the RBM20 mRNA are targeted to regions where there are only a few CpGs and/or disease-causing mutations. In some embodiments of the constructs comprising the miRNA backbones, the miRNA backbones are selected based on percentage similarity to the siRNA. In some embodiments the components of the different constructs are interchangeable between constructs. In some embodiments, the constructs are resistant to cellular silencing including via epigenetic mechanisms. In some embodiments, the expression of RBM20 and/or the siRNAs are controlled through transcriptional mechanisms and/or are inducible. In some embodiments, the AAV- expressed RBM20 corrects splicing defects. In some embodiments, the AAV- expressed RBM20, alters and/or changes alternative splicing. In some embodiments, the altered alternative splicing is caused by a mutated RBM20. In some embodiments, the RBM20 gene is configured to regulate overexpression of RBM20. Subjects [0154] Aspects of the disclosure relate to methods for use with a subject (e.g., a mammal). In some embodiments, a mammalian subject is a human, a non-human primate, or other mammalian subject. In some embodiments, the subject has one or more mutations associated with cardiomyopathy. Aspects of the disclosure relate to methods for use with a host cell or host cells. In some embodiments the host cells are mammalian cells. Unless otherwise indicated to the contrary, reference to a subject includes reference to a host cell from a subject (including both subjects with and without cardiomyopathy). [0155] In some embodiments, a subject suffers from or is at risk of developing a disease or condition associated with cardiomyopathy resulting in one or more symptoms of disease or disorder. A non-limiting example of this disease includes dilated cardiomyopathy. [0156] In a non-limiting example, compositions or methods of this application are administered to a subject resulting in regulated overexpression of RBM20. [0157] In another non-limiting example, compositions or methods of this application are administered to a subject resulting in the regulated degradation of endogenous nucleic acids encoding RBM20 (e.g., both pathogenic and wild type alleles). [0158] In another non-limiting example, compositions or methods of this application are administered to a subject resulting in the regulated degradation of nucleic acids encoding RBM20. [0159] In another non-limiting example, compositions or methods of this application are administered to a subject resulting in both the regulated overexpression of RBM20 and the regulated degradation of endogenous nucleic acids encoding RBM20 (e.g., both pathogenic and wild type alleles). [0160] A non-limiting example of symptoms of cardiomyopathy include heart failure, sudden cardiac death, arrhythmias, myocardial insufficiency, left-ventricular non- compaction, left-ventricular dilation, ventricular tachycardia, and cardiac electrical abnormalities. [0161] Accordingly, in some embodiments one or more nucleic acid constructs can be delivered to a subject having one or more symptoms of cardiomyopathy. In some embodiments, an rAAV composition provided herein is administered to a subject having cardiomyopathy. [0162] In some embodiments, wherein in the nucleic acid construct is an RBM20 construct, the RBM20 constructs ameliorates one or more symptoms associated with cardiomyopathy. In some embodiments, wherein in the nucleic acid construct is a RBM20 construct, treatment with a disclosed RBM20 construct reduces disease symptoms in a subject. [0163] In some embodiments, wherein in the nucleic acid construct is a RBM20 construct, treatment with the disclosed RBM20 constructs reduces heart failure in a subject. [0164] In some embodiments, wherein in the nucleic acid construct is a RBM20 construct, treatment with the disclosed RBM20 construct prevents sudden cardiac death in a subject. [0165] In some embodiments, wherein in the nucleic acid construct is a RBM20 construct, treatment with the disclosed RBM20 constructs reduces arrhythmias in a subject. [0166] In some embodiments, wherein in the nucleic acid construct is a RBM20 construct, treatment with the disclosed RBM20 constructs reduces myocardial insufficiency in a subject. [0167] In some embodiments, wherein in the nucleic acid construct is a RBM20 construct, treatment with the disclosed RBM20 constructs reduces left-ventricular non- compaction in a subject. [0168] In some embodiments, wherein in the nucleic acid construct is a RBM20 construct, treatment with the disclosed RBM20 constructs reduces left-ventricular dilation in a subject. [0169] In some embodiments, wherein in the nucleic acid construct is a RBM20 construct, treatment with the disclosed RBM20 constructs reduces ventricular tachycardia in a subject. [0170] In some embodiments, wherein in the nucleic acid construct is a RBM20 construct, treatment with the disclosed RBM20 constructs reduces cardiac electrical abnormalities in a subject. Methods of producing rAAV particles [0171] Methods of producing rAAV particles and nucleic acid vectors are known in the art and commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158- 167; and U.S. Patent Publication Nos. US 2007/0015238 and US 2012/0322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid containing the nucleic acid vector sequence may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VPl, VP2, and VP3, including a modified VP3 region as described herein), and transfected into a producer cell line such that the rAAV particle can be packaged and subsequently purified. [0172] In some embodiments, the one or more helper plasmids includes a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising a Ela gene, a Elb gene, a E4 gene, a E2a gene, and a VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2 and the cap gene is a cap gene derived from AAV2 and includes modifications to the gene in order to produce a modified capsid protein described herein. Helper plasmids, and methods of making such plasmids, are known in the art and commercially available (see, e.g., pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E/R585E), and pDP8.ape plasmids from Plasmidfactory, Bielefeld, Germany; other products and services available from Vector Bio labs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adeno associated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, l l072-l l081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VPl N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, Vol. 16, 301185-1188). [0173] In some embodiments, treatment reduces or corrects one or more other symptoms of cardiomyopathy. [0174] A non-limiting, rAAV particle production method comprises one or more helper plasmids are produced or obtained, which comprise rep and cap ORFs for the desired AA V serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The cap ORF may also comprise one or more modifications to produce a modified capsid protein as described herein. HEK293 cells (available from ATCC®) are transfected via CaP04-mediated transfection, lipids or polymeric molecules such as Polyethylenimine (PEI) with the helper plasmid(s) and a plasmid containing a nucleic acid vector described herein. The HEK293 cells are then incubated for at least 60 hours to allow for rAAV particle production. Alternatively, in another example Sf9-based producer stable cell lines are infected with a single recombinant baculovirns containing the nucleic acid vector. As a further alternative, in another example HEK293 or BHK cell lines are infected with a HSY containing the nucleic acid vector and optionally one or more helper HSY s containing rep and cap ORFs as described herein and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to allow for rAAV particle production. T11e rAAV particles can then be purified using any method known the art or described herein, e.g., by iodixano1 step gradient, CsC1 gradient, chromatography, or polyethylene glycol (PEG) precipitation. [0175] As used herein, an “RNA binding protein site” refers to an RNA sequence or structure that interacts with an “RNA binding protein” as defined below. In some embodiments, the binding site(s) can be in an intron, an exon, or both. An "RNA binding protein" (RBP) refers to a protein that binds to double- or single-stranded RNA in cells and participates in forming ribormc1eoprotein complexes. RBPs contain various structural motifs, such as RNA recognition motif (RRM), dsRNA binding domain, zinc finger and others. Methods of Delivering AAV [0176] In several embodiments, there is provided a method of treating a disease or condition in a subject comprising administering an rAAV of the present disclosure to a subject. Accordingly, provided herein is a method of delivering the disclosed rAAV particles. In some embodiments, rAAV particles are delivered by administering any one of the compositions disclosed herein to a subject. In some embodiments, “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful and alters the disease state or condition. In some embodiments, rAAV particles are delivered to one or more tissues and cell types in a subject. In some embodiments, rAAV particles are delivered to one or more of cardiac muscle, skeletal muscle, CNS, and immune cells. In some embodiments, an rAAV particle is administered to the subject parenterally. In some embodiments, an rAAV particle is administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, enterally, via inhalation, topically, or by direct injection to one or more cells, tissues, or organs. In some embodiments, an rAAV particle is administered to the subject by injection into the hepatic artery or portal vein. [0177] In several embodiments, the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject is reduced and/or eliminated. The compositions described above or elsewhere herein are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result. The desirable result will depend upon the active agent being administered. For example, an effective amount of rAAV particles may be an amount of the particles that are capable of transferring an expression construct to a host organ, tissue, or cell. A therapeutically acceptable amount may be an amount that is capable of treating a disease, e.g., RMB20-related cardiomyopathy. As is well known in the medical and veterinary arts, dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently. In some embodiments, a single composition comprising rAAV particles as disclosed herein is administered only once. In some embodiments, a subject may need more than 1 administration of an rAAV composition (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times). For example, a subject may need to be provided a second administration of any one of the rAAV compositions as disclosed herein 1 day, 1 week, 1 month, 1 year, 2 years, 5 years, or 10 years after the subject was administered a first composition. In some embodiments, a first composition of rAAV particles is different from the second composition of rAAV particles. In some embodiments, the administration of the composition is repeated at least once (e.g., at least once, at least twice, at least thrice, at least four times, at least five times, at least six times, at least 10 times, at least 25 times, or at least 50 times), and wherein the time between a repeated administration and a previous administration is at least 1 month (e.g., at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 12 months). In some embodiments, the administration of the composition is repeated at least once, and wherein the time between a repeated administration and a previous administration is at least 1 year (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 years). In some embodiments, the administration of the composition is facilitated by AAV capsids such as AAV1-9, e.g., with AAV2 ITRs, or other capsids that sufficiently deliver to affected tissues. Compositions described herein may further comprise a pharmaceutical excipient, buffer, or diluent, and may be formulated for administration to host cell ex vivo or in situ in an animal, and particularly a human being. Such compositions may further optionally comprise a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof. Such compositions may be formulated for use in a variety of therapies, such as for example, in the amelioration, prevention, and/or treatment of conditions such as peptide deficiency, polypeptide deficiency, peptide overexpression, polypeptide overexpression, including for example, conditions which result in diseases or disorders as described herein. [0178] Formulations comprising pharmaceutically acceptable excipients and/or carrier solutions are well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intra- articular, and intramuscular administration and formulation. [0179] Typically, these formulations may contain at least about 0.1% of the therapeutic agent (e.g., therapeutic rAAV particle or preparation) or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 90% or more of the weight or volume of the total formulation. Naturally, the amount of therapeutic agent(s) in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art when preparing such pharmaceutical formulations. Additionally, a variety of dosages and treatment regimens may be desirable. [0180] In certain circumstances, it will be desirable to deliver the therapeutic rAAV particles or preparations in suitably formulated pharmaceutical compositions disclosed herein; either subcutaneously, intracardially, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells (e.g., cardiomyocytes and/or other heart cells), tissues, or organs. In some embodiments, the therapeutic rAAV particles or the composition comprising the therapeutic rAAV particles of the present invention are delivered systemically via intravenous injection, particularly in those for treating a human. In some embodiments, the therapeutic rAAV particles or the composition comprising the therapeutic rAAV particles of the present invention are injected directly into the heart of the subject. Direct injection to the heart may comprise injection into one or more of the myocardial tissues, the cardiac lining, or the skeletal muscle surrounding the heart, e.g., using a needle catheter. In several embodiments, direct injection to human heart is preferred, for example, if delivery is performed concurrently with a surgical procedure or interventional procedure whereby access to the heart is improved. In some embodiments, the interventional procedure includes any procedure wherein coronary or pulmonary perfusion is altered. In some embodiments, the interventional procedure includes one or more of percutaneous administration, catheterization, or coronary retroperfusion. [0181] The pharmaceutical formulations of the compositions suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the formulation is sterile and fluid to the extent that easy syringability exists. In some embodiments, the form is stable under the conditions of manufacture and storage, and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, vegetable oils or other pharmaceutically acceptable carriers such as those that are Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In fact, there is virtually no limit to other components that may also be included, as long as the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The therapeutic rAAV particles or preparations may thus be delivered along with various other pharmaceutically acceptable agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. [0182] The amount of therapeutic rAAV particle or preparation, and/or therapeutic rAAV vector compositions and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically- effective amounts of the compositions of the present disclosure may be achieved by a single administration, such as for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. In some circumstances, it may be desirable to provide multiple or successive administrations of the rAAV particle or preparation, and/or rAAV vector compositions, either over a relatively short, or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions. [0183] Toxicity and efficacy of the compositions utilized in methods of the present invention may be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD₅₀ (the dose lethal to 50% of the population). The dose ratio between toxicity and efficacy (the therapeutic index) may be expressed as the ratio LD50/ED50. Those compositions that exhibit large therapeutic indices are preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of compositions as described herein lies generally within a range that includes an ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. [0184] Other aspects of the present disclosure relate to methods and preparations for use with a subject, such as human or non-human subjects, a host cell in situ in a subject, or a host cell derived from a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a companion animal. “A companion animal”, as used herein, refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a human subject. [0185] In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers) are added to the pharmaceutical compositions including a therapeutic, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a subject, such as a human. [0186] A pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the therapeutic and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingredient (API, therapeutic product) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance. [0187] Excipients include, but are not limited to: absorption enhancers, anti- adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents. [0188] Pharmaceutical compositions can contain other additional components commonly found in pharmaceutical compositions. Such additional components can include, but are not limited to, anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.). [0189] The carrier can be, but is not limited to, a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. A carrier may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. A carrier may also contain isotonic agents, such as sugars, polyalcohols, sodium chloride, and the like into the compositions. [0190] Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the subject from a pharmacological/toxicological point of view. The phrase pharmaceutically acceptable refers to molecular entities, compositions, and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a subject. In some embodiments, a pharmaceutically acceptable compound is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans. [0191] The rAAVs or pharmaceutical compositions as described herein, may be formulated for administration to host cell ex vivo or in situ in an animal, and particularly a human being. The rAAVs or pharmaceutical compositions can be administered by a variety of routes. Administration routes included, but are not limited to, intravenous, intra-arterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and/or local delivery to a target tissue. In some embodiments, a plurality of injections, or other administration types, are provided, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more injections. Routes of administration may be combined, if desired. Depending on the embodiment, the first and second rAAV need not be administered the same number of times (e.g., the first rAAV may be administered 1 time, and the second vector may be administered three times). In some embodiments, the dosing is intramuscular administration. [0192] In some embodiments, the number of rAAV particles administered to a subject may be on the order ranging from about 10⁶ to about 10¹⁴ particles/mL or about 10³ to about 10¹³ particles/mL, or any values in between for either range, such as for example, about 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ particles/mL. In some embodiments, the number of rAAV particles administered to a subject may be on the order ranging from about 10⁶ to about 10¹⁴ vector genomes (vgs)/mL or 10³ to 10¹⁵ vgs/mL, or any values in between for either range, such as for example, about 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ vgs/mL. In some embodiments, between about 0.5 and about 5 rAAV vector genomes per cell are administered. In some embodiments, between about 0.5 and about 2 rAAV vector genomes per cell are administered. In some embodiments, between about 1 x 10¹³ and about 3 x 10¹⁵ vector genomes per kilo (vgs/kg) are administered, such as about 2 x 10¹³, about 4 x 10¹³, about 6 x 10¹³, about 8 x 10¹³, about 2 x 10¹⁴, about 4 x 10¹⁴, about 6 x 10¹⁴, about 8 x 10¹⁴, about 2 x 10¹⁵, or any amount between the forgoing. In some embodiments, dosing is based on the mass of the subject’ s cardiac muscle. In some embodiments, dosing is based on body weight. In some embodiments, dosing is based on body surface area. The rAAV particles can be administered as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In some embodiments, doses ranging from about 0.0001 mL to about 10 mL are delivered to a subject. [0193] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, intravitreal, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). In several embodiments, the rAAV formulation will comprise, consist of, or consist essentially of active rAAV ingredient, a mono-basic buffer (e.g., sodium phosphate mono-basic buffer, a di-basic salt (e.g., sodium phosphate di-basic), a sodium-based tonicifier (e.g., sodium chloride tonicifier), a non-sodium tonicifier (e.g., magnesium chloride hexahydrate tonicifier), a surfactant (e.g., poloxamer 188 surfactant), and water. In several embodiments, the rAAV formulation will comprise, consist of, or consist essentially of active rAAV ingredient, sodium phosphate mono-basic buffer, sodium phosphate di-based, sodium chloride tonicifier, magnesium chloride hexahydrate tonicifier, poloxamer 188 surfactant, and water. In several embodiments, the active rAAV ingredient is present in the formulation according to the vector genome amounts provided for herein. In several embodiments, the mono-basic buffer (e.g., sodium phosphate mono-basic buffer) is present in the formulation at a concentration between about 0.2 mg/mL and about 0.5 mg/mL. In several embodiments, the di-basic salt (e.g., sodium phosphate di-basic) is present in the formulation at a concentration between about 1.5 mg/mL and about 4 mg/mL. In several embodiments, the sodium-based tonicifier (e.g., sodium chloride tonicifier) is present in the formulation at a concentration between about 8 mg/mL and about 12 mg/mL. In several embodiments, the non- sodium tonicifier (e.g., magnesium chloride hexahydrate tonicifier) is present in the formulation at a concentration between about 0.1 mg/mL and about 0.35 mg/mL. In several embodiments, the surfactant (e.g., poloxamer 188 surfactant) is present in the formulation at a concentration between about 0.05 mg/mL and about 0.8 mg/mL. In several embodiments, water is present to bring the volume of the formulation (e.g., a dosage unit) to 1 mL. [0194] Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by, e.g., FDA Office of Biologics standards. [0195] Sterile injectable solutions are prepared by incorporating the rAAV particles or preparations in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum- drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [0196] The amount of rAAV particle or preparation and time of administration of such particle or preparation will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically effective amounts of the rAAV particles or preparations of the present disclosure may be achieved by a single administration, such as for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. Alternatively, in some circumstances, it may be desirable to provide multiple or successive administrations of the rAAV particle or preparation, either over a relatively short, or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions. [0197] If desired, rAAV particles may be administered in combination with other agents as well, such as, e.g., proteins or polypeptides or various pharmaceutically active agents, including one or more administrations of therapeutic polypeptides, biologically active fragments, or variants thereof. In fact, there is virtually no limit to other components that may also be included, as long as the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The rAAV particles or preparations may thus be delivered along with various other pharmaceutically acceptable agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. [0198] In some embodiments, treatment of a subject with a rAAV particles as described herein achieves one, two, three, four, or more of the following effects, including, for example: (i) reduction or amelioration the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against the progression of a disease or symptom associated therewith; (iv) regression of a disease or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy. In some embodiments, the disease or symptom is caused by hypertrophic cardiomyopathy or dilated cardiomyopathy. In some embodiments, the disease or symptom is dilated cardiomyopathy. In some embodiments, the disease or symptom is idiopathic dilated cardiomyopathy. [0199] As is apparent to those skilled in the art in view of the teachings of this specification, an effective amount of viral vector to be added can be empirically determined. Administration can be administered in a single dose, a plurality of doses, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosages of administration are well known to those of skill in the art and will vary with the viral vector, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. [0200] The following tables provide the sequence of various non-limiting examples constructs within the scope of the present disclosure. It shall be appreciated that any single element of one construct can be used in place, or in addition to, a corresponding element of another construct (e.g., exchange of promoter A for promoter B). Likewise, any of the elements in the tables can be used with any of the elements (or combinations of elements) disclosed elsewhere herein. Table 1: JET cg gt ta tc c gt t g g cc ca c gc gc aa ac gt tg tc ct ct ta c

agatggtcagcctggcttcctgccatcctcggcctcaacctcgggc agtgtgacctatgaagggcactacagccacacagggcaggatggt tc a g g ta g g ca gc a ct g ct gc tg t tg t a t ca c aa tg tg gc g a ac gg a t g g c gg ct a a g g ta aa gc gt a

aggagtcaaggccgtagggaatggggctgcagaaatcagcctca agtcacccagagaactgccctctgcttccacaagctgtcccagtga aa g c g ca g c at ct ca ct a ct ga tg t g gg g

Table 2: CK8-INT- wt huRBM20 a a

t t a c tc ac g

a c a gc tc tt gc c c c a c a cc g g a ca

ggtcccatgtggcctccaccccacaaccagccctatgagctgtacgaccccgaggaaccaac ctcagacaggacacctccttccttcgggggtcggcttaacaacagcaaacagggttttatcggt a g g tg g tt c ta ca gg at a gc cc gg c cc cc ca ca tg a a tt c g a ct c a tg g gc c a a tc ct c ag g

11 Poly(A) aataaaagatccttattttcattggatctgtgtgttggttttttgtgt gg gc

huRBM20 siRNA g g gg cc g gc c c c c c a tc ac tg c a c c g tg g ag

cctggcttcctgccatcctctgcctcaacctctggcagtgtgacctat gaagggcactacagccacacagggcaggatggtcaagctgcctttt gt g a cc a ag a ct g g a a c c c ga tg tt tt gg g gg G c tc cc ac at g c gg gt a ac t a c at g t ta ag cc a g c

acccagagaactgccctctgcttccacaagctgtcccagtgacatgg atgtggaaatgcctggcctaaatctggatgctgagaggaagccagc g t c cc a gt g tt tc c ca c c a c ct g

Table 4: CK8- Int- gc c c ac ct ca tg t c gt ac a

ggtagccatgtcccagcctctcttcaatcaactgaggcatccatctgtgatcactggcccccatggccatgctgggg ttccccaacatgctgcagccatacccagtaccaggtttccctctaatgcaattgccttttcaccccccagccagacaa tg ct ct ca a ca cc g gg g ga tg aa g a ga a A ca g ca cc g cc gc a g a c ga ta tc tct g g a tt ct tt cc a gt

Table 5: JeT- huRBM20_si gc ac c ac ct ca t c gt ac a gg aa tg ct ct ca a ca cc g gg g ga tg aa g a ga a C ca g ca cc g cc gc

tggggccaaaggtcactagggcccctgagggagccaaggccaagcagaatgagaaaaataaaaccaagagaa ctgatagagaccaagaaggagctgatgatagaaaagaaaacacaatggcagagaatgaggctggaaaagagg a c ga ta tc tct g g a tt ct tt cc a gt

Int- gc ac c ac ct ca tg t c gt ac a gg aa tg

cctcagacccctggccagccagcagtcatcttgggcattggcaagactgggcctgctccagctacagcaggattct atgagtatggcaaagccagctctggccagacatatggccctgaaacagatggtcagcctggcttcctgccatcctct ca a ca cc g gg g ga tg aa g a ga a C ca g ca cc g cc gc a g a c ga ta tc tct g g a tt ct tt cc a gt

Table 7: JeT- Myc- gc ac c ac ct ca t cc tc g gt cc g ct tg gc aa a g aa g cc a gg a ct c ag gt c ct ct aa tc ga ac ga cc ga

gaccaagaaggagctgatgatagaaaagaaaacacaatggcagagaatgaggctggaaaagaggaacaggag ggcatggaagaaagccctcaatcagtgggcagacaggagaaagaagcagagttctctgatccggaaaacacaa ga g a cc t gg c cc g g tc cg gg gt

Table 8: JeT- Myc- gc ac c ac ct ca g ct ca g t cc ag ct tg

agtatggcaaagccagctctggccagacatatggccctgaaacagatggtcagcctggcttcctgccatcctctgc ctcaacctctggcagtgtgacctatgaagggcactacagccacacagggcaggatggtcaagctgccttttccaaa a ag a g c a gg ga ct at ga ag cc G a g ca ac ct g tg t aa a a gc ga aa gg a g g a ga tg a c gt

Table 9: JeT- Myc- gc ac c ac ct ca g ct ca g t cc ag ct tg c aa a ag a g c a gg ga ct at ga ag cc AT g a at ca ta g

aggaaagatgaggccaggctgagggaaagcagacacccccatccagatgactcaggcaaggaagatgggctgg ggccaaaggtcactagggcccctgagggagccaaggccaagcagaatgagaaaaataaaaccaagagaactg ac a ac tg ct g ga a a c c aa t c ct gt

Table 10: siRNA Sequences

Table 11: siRNA-Resistance Sequences

Table 12: Additional c c c a a c c c g gt g t a a tg g c at a c t ac a g c a ct t g c a gg at c c at g at c cg

aggtcccacactcccagcttcacctcctgcagctcttcccacagccctcc gggcccctcccgggctgactggggcaatggccgggactcctgggagca a a g c a t g a a a t at ac ag g c cc ag a gt g at c g a c tg g gc g c c gg a

[0201] It shall be appreciated that SEQ ID NO: 67 (or any other sequence disclosed or embodied herein) can be used in connection with any of the constructs laid out in the tables above or otherwise described herein. In other words, SEQ ID NO: 67, which is CpG deplete, but not engineered to be resistant to the siRNAs disclosed herein, may be used in a rAAV driven by a JeT promoter, a CK8 promotor, or any other promoter provided for herein. Table 13: siRNAs sequences targeting RBM20 SEQ ID NO: siRNA Identifier Sequence (5’>3’) 68 siControl UCUCCACGCAGUACAUUUCUUUU

q Target gene Primers Sequence (5’>3’) SEQ ID NO: RBM20 Forward GCATGTGAAAGGGAAGCTGC 72

Table 15: Primary-microRNA-scaffold sequences post incorporation of siRBM20_V4 into the primary-miRNA124 scaffold (pri-miR124). SEQ Primary- Sequence (5’>3’)

6 Pri- CTTTCTTTCACCTTTCCTTCCTTCCTTCCTCCTTTCCTTCCTCAGGAGAAAGGCCTCTCT miR124- CTCAAGCACAGCCGTTATTAACCATTTAAATGTCCATACAATGGTTATAAACAGCTG A T G A T G A

a e : _ - -u _s v _ eaut_ re . na ector SEQ ID 4 2 0 0 8 5 4 7 2 5 0 8 1 9

124 BsaI-Kan-R1 125 BsaI-KanP-F1

Table 17: 2__BB1_TNNT2-400-huRBM20_siRNA v4_Cardiac_CPG red SEQ ID

161 Col E1 ori 4886..5474 162 Single nt. change compared to pBR322 5327..5327

Table 18: 3_SGT-003_TNNT2-400-huRBM20_siRNA v4_Default SEQ 9 9 4

198 TNNT2-400 1855..2254 199 TSS 2203..2210 3 5 9 9 8 6 0 0 0 5 9 7 3 9 6

Table 19: 4_SGT-003_TNNT2-400-huRBM20_siRNA v4_Cardiac SEQ ID 9 9 9 4 0 3 5 9 8 6 0 0

235 albumin stuffer sequence #2 6301..7800 236 M13 rev 7819..7835 9 7 3 9 6

abe 0: 5_SG -003_ NN -00- u 0_s N v _ eaut_C G red SEQ ID 9 9 9 4 0 3 5 9 8 6 0 0 0 5 9 7 3 9 6

272 NheI-CI-F1 273 TNNT2-F

_ _ _ _ _ SEQ ID 9 9 9 4 0 3 5 9 8 6 0 0 0 5 9 7 3 9 6

Table 22: 7_SGT-003_TNNT2-400-huRBM20_siRNA v4 SEQ

304 ITR 1689..1829 305 not really ITR 1689..1699 9 4 0 3 5 9 9 6 8 5 5 6 0 0 0 5 9 7 3 9 6

Table 23: 8_SGT-003_TNNT2-400_NO INT_huRBM20_siRNA v4 SEQ 9 9 6 4 0 2

341 huRbm20 2273..5956 342 ORF 1 (frame 2) 2273..5956 3 5 2 2 3 7 7 7 2 6 4 0 6 3

Table 24: 9_SGT-003_TNNT2-400-huRBM20_R634Q SEQ ID 9 9 9 4 0 3 5 9 9 6 8 5 7 6 0 0 0

378 M13 rev 7819..7835 379 lac operator 7843..7859 7 3 9 6

a e : - _ - - - _ u _s v _ ar ac_ p _re -7e86cb3f SEQ ID NO: Sequence Name Location 9 9 9 8 0 4 3 1 5 5 5 0 4 2 8 4 1

Table 26: SGT-003_TNNT2-455-huRBM20_siRNA v4_Cardiac SEQ ID

411 ITR 1689..1829 412 not really ITR 1689..1699 9 8 0 4 3 1 5 5 5 0 4 2 8 4 1

Table 27: SGT-003_TNNT2-455-huRBM20_siRNA v4_Default SEQ ID NO: Sequence Location 9 9 9 8 0 4 3 1 5 5 0 4 2 8 4 1

Table 28: SGT-003_TNNT2-455-huRBM20_siRNA v4_Default_CPG red SEQ ID NO: Sequence Name Location 9 9 9 8 0 4 3 1 5 5 5 0 4 2 8 4 1

Table 29: SGT-003_TNNT2-455-huRBM20_siRNA v4_Default_stop_control SEQ ID NO: Sequence Name Location 9 9 9 8 0 4 3 7 1 0 0 0 0 0

481 stop 3358..3360 482 stop 3508..3510 0 3 0 0 0 0 0 0 0 1 5 5 0 4 2 8 4 1

_ SEQ ID NO: Sequence Name Location 9 9 9 8 0 0 7 1 1 1 6 0 8 4 0 7

Table 31: SGT-003_TNNT2-455-Nmyc-mouseRBM20 SEQ ID NO: Sequence Name Location 9 9 9 8 0 0 3 7 1 1 1 6 0 8 4 0 7

Table 32: shRBm20 SEQ ID Se uence

Table 33: BB1_Jet-huRBM20_Cardiac CodOpt_NMyc SEQ ID

548 Sp1 230..235 549 TATA box 250..256 0 8 8 6 3 2 5 0 3 8 6 9 7

Table 34: BB1_Jet-huRBM20_Default CodOpt_NMyc SEQ ID

BB1_Jet-huRBM20_Default CodOpt_NMyc 1..6877 ITR-L Flip 1..145 0 8 8 6 3 2 5 0 3 8 6 9 7

629 pcDNA31F Primer 630 PstIITR-F Primer

_ _ _ SEQ ID NO: Sequence Name Location 2 9 1 1 0 8 8 6 3 2 5 0 3 8 6 9

667 Random Stuffer 6858..6877 668 3F1oriF Primer

Table 36: BB1_Jet-huRBM20_siRNA v4_NMyc SEQ ID NO: Sequence Name Location 2

705 CAGCACAGCTGTTTATAAC 1784..1802 706 siRbm20-06 2019..2039 0 8 8 6 3 2 5 0 3 8 6 9 7

Table 37: BB1_Jet-huRBM20_siRNA_v4 SEQ ID

Sp1 170..175 CAT 178..182 2 2 2 9 0 8 8 6 3 2 5 0 3 8 6 9 7

786 PstIITR-F Primer 787 PstIITR-R Primer

_ _ SEQ ID NO: Sequence Name Location 6 4 4 2 9 8 1 6 9 4 2 5 3

824 colE1 ori-R Primer 825 F1 ori Primer

abe 39: _ NN -00- u 0_Cardac_N yc SEQ ID NO: Sequence Name Location 2 0 0 8 5 4 7 2 5 0 8 1 9

861 BsaI-Kan-R1 Primer 862 BsaI-KanP-F1 Primer

Table 40: BB1_TNNT2-400-huRBM20_Default_NMyc SEQ ID NO: Se uence Name Location 2 0 0 8 5 4

899 Single nt. change compared to pBR322 5357..5357 Arthrobacter sp. LS16 (noncoding 2 5 0 8 1 9

Table 41: BB1_TNNT2-400-huRBM20_NMyc SEQ ID

Kozak 716..721 huRbm20 722..4435 2 4 1 2 0 0 8 5 4 7 2 5 0 8 1 9

978 pTRbackbone2 Primer 979 TNNT2-F Primer

_ _ _ SEQ ID NO: Sequence Name Location 2 4 1 3 2 0 0 8 5 4 7 2 5 0 8 1 9

1016 Clone9-BsaI-F1 Primer 1017 colE1 ori-R Primer

Table 43: BB1_TNNT2-400-huRBM20_siRNA v4 SEQ ID NO: Sequence Name Location 2 4 1 1 2 0 0 8 5 4 7

Arthrobacter sp. LS16 (noncoding 1053 sequence) 5475..5622 5 0 8 1 9

Table 44: BB1_TNNT2-400-huRBM20_siRNA v4_NMyc SEQ ID

1089 Kozak 716..721 1090 huRbm20 722..4435 2 2 4 1 1 2 0 0 8 5 4 7 2 5 0 8 1 9

1131 PstIITR-R Primer 1132 pTRbackbone2 Primer

_ _ SEQ ID NO: Sequence Name Location 2 4 1 2 0 0 8 5 4 7 2 5 0 8 1 9

1168 colE1 ori-R Primer 1169 F1 ori Primer

Table 46: BB1_TNNT2-400-mouseRBM20_NMyc SEQ ID NO: Sequence Name Location 8 6 6 4 1 0 3 8 1 6 4

1205 E. coli LacZ gene fragment 7055..7127 1206 Random Stuffer 7136..7155

Table 47: BB1_TNNT2-400-huRBM20_siRNA v4 + miR33v4_passenger modified SEQ ID NO: Sequence Name Location

1243 Kozak 827..832 1244 huRbm20 833..4516 3 5 2 3 1 6 5 8 6 1 9 2

abe 8: _ NN -00- u 0_s N v + m v - 6 SEQ ID NO: Sequence Name Location 1257 BB1TNNT2-400-huRBM20siRNA v4 + miR124v4-R6 17394 7 9 6 7 5 0 9 2 0 5 3

1282 E. coli LacZ gene fragment 7294..7366

_ _ SEQ ID NO: Sequence Name Location 1283 BB1_TNNT2-400-huRBM20_siRNA v4 + miR208v4 1..7278 1 3 0 1 9 4 3 6 4 9 7 0

Table 50: SGT-003_TNNT2-455-huRBM20_wt SEQ ID NO: Sequence Name Location 8 29 99 09 48 60 44

1317 huRbm20 2461..6144 1318 TAGCCATGAGCCAGGACGCGGATCCCAGCGGTCCGGAGCAACCCGAC 2474..6141 41 73 10 01 55 55 55 90 14 52 88 64 51

EXAMPLES [0202] The present non-limiting embodiments are described by means of the following examples. The use of these and other examples anywhere in the specification is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified form. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the claims, along with the full scope of equivalents to which the claims are entitled. Example 1 – RNA Expression Analysis of RBM20 Encoding AAV Vectors [0203] In this example, RBM20 encoding rAAV vectors were evaluated with respect to their relative expression levels achieved in various cell types, based on their specific constituent elements. [0204] As discussed herein, RBM20 is a molecule that functions in the downstream splicing of numerous genes such as titin therefore nuclear localization of RBM20 is required for it to function in the splicing complex. Pathogenic mutations in RBM20 preclude localization to the nucleus and an inability to facilitate splicing of target molecules. Overexpressing a wild-type form of RBM20 may confer functional reconstitution. Constitutive expression of full-length human RBM20 was evaluated using a series of nine constructs (e.g., for expression by an AAV vector) (See Figures 2-4 and 9-14). The RBM20 gene was expressed as three different molecular species: (i) wild-type human RBM20 (no genetic modifications); (ii) human RBM20 with CpG depletions and specific siRNA sites modified to prevent down- regulation of ectopically expressed RBM20; and (iii) human RBM20 with all features described in (ii) and inclusion of the R634Q pathogenic allele. By way of non-limiting embodiments, each of these is expressed from the JeT promoter to provide constitutive and ubiquitous protein expression, or from the CK8 promoter to provide constitutive expression in muscle tissue. Additionally, a myc protein tag was added to the N-terminus to distinguish detection of the ectopically expressed RBM20 from endogenously expressed RBM20 protein. Expression of RBM20 from the AAV vector constructs was evaluated in two cell systems, HEK293T (human embryonic kidney cell line) and C2C12 (mouse myoblast cell line) cells, by transient transfection. [0205] In the HEK293T cell system the cells were cultured in 48-well plates to a confluency of 80%. Cells were co-transfected with 300 ng/well of each AAV vector DNA and 30 ng/well of a GFP expression plasmid to control transfection efficiency. Cells were transfected at a 1:3 ratio of plasmid DNA:Viafect reagent. Expression was evaluated from all constructs by RT-qPCR. Total RNA was prepared from transfected cells using the Zymo Direct-zol RNA microprep kit according to manufacturer’s instructions. Total RNA was reverse transcribed to cDNA using Applied Biosystems High-Capacity RNA-cDNA Kit. RBM20 cDNA was quantified by real time PCR with RBM20 specific primers. RBM20 RNA expression was confirmed for all expression cassettes evaluated (see Figures 13-14). [0206] In the C2C12 cell system the cells were cultured in 48-well plates to a confluency of 80% and remained undifferentiated for transfection and analysis. Following differentiation C2C12 cells were co-transfected and processed as described for HEK293T cells. RNA analysis was executed according to procedures outlined for HEK293T cells. RBM20 protein and RNA expression were confirmed for all expression cassettes evaluated (see, FIG. 14). [0207] Figure 13 shows expression of RBM20 mRNA from the rAAV expression cassettes in HEK293T cells: 1, JeT-wtRBM20; 2, CK8-wtRBM20; 3, JeT-RBM20 siRNAopt- CpGopt; 4, CK8-RBM20-siRNAopt-CpGopt; 5, JeT-RBM20-siRNAopt-CpG opt-R634Q; 6, CK8-RBM20-siRNAopt-CpGopt-R634Q; 7, JeT-Myc-wtRBM20; 8, JeT-Myc-RBM20- siRNAopt-CpGopt; 9, JeT-Myc-RBM20-siRNAopt-CpGopt-R634Q; 10-13, Different negative controls. [0208] Figure 14 shows expression of RBM20 mRNA from the rAAV expression cassettes in C2C12 cells: 1, JeT-wtRBM20; 2, CK8-wtRBM20; 3, JeT-RBM20siRNAopt- CpGopt; 4, CK8-RBM20siRNAopt-CpGopt; 5, JeT-RBM20siRNAopt-CpGopt-R634Q; 6, CK8-RBM20siRNAopt-CpGopt-R634Q; 7, JeT-Myc-wtRBM20; 8, JeT-Myc- RBM20siRNAopt-CpGopt; 9, JeT-Myc-RBM20siRNAopt-CpGopt-R634Q; 10-13, Different negative controls. [0209] Protein expression from these constructs was also detected by western blotting lysates of HEK293 cells, iCell cardiomyocytes, C2C12 myoblasts and C2C12 myotubes (data not shown). [0210] Taken together, these results show that all expression cassettes result in expression of RBM20 at levels higher than endogenous RBM20 expression. Example 2 – TNNT2 Constructs Tested for Protein Expression Following Plasmid Transfection and Transduction [0211] In this non-limiting example, RBM20 expression was evaluated in vitro and in vivo following transfections and transductions with TNNT2 vectors. Methods: [0212] Transfection: iCell Cardiomyocytes were plated in a 48 well plate (1.72x10^5 cells/well), after 4-24 hrs change the media to maintenance media. On day of transfection media was removed and 200uL of fresh maintenance media was added to all wells. Samples were normalized based on mass and copy number and a 3:1 ratio of Viafect:DNA was used. Table 51: Example Transfection Protocol plasm RBM20 Factor to make Stock RBM20 Total DNA “ uL)

TNNT2- 400_GFP-cl 6827 1.99 300.00 10 6.00 18.00 3.02 379.0

(volume was removed and replaced. On harvest day, cells are collected 6 days post transfection in 50uL of RIPA + PI, replicates are pooled and placed at -80C. Samples are thawed on ice and spun down at 4C and t supernatants collected and run on a Western blot. [0214] Transduction: iCell Cardiomyocytes were plated in a 48 well plate (1.72x10^5 cells/well), after 4-24 hrs change the media to maintenance media. Transduce the cells after 24 hrs in maintenance media. Transduction day - MOI = 2.00E+06 vg/cell. Remove media and add 200uL of fresh maintenance media to all wells. Normalize the samples to the lowest titer – so the sample with the lowest titer has no additional formulation buffer added. Ex. See table below Prep VC-24-0804 has no additional PBS added, but the other samples will have virus + PBS to hit the same total volume of 40.1uL Table 52: Example Transduction Protocol Desired MOI vg/cell Prep ID Titer (vg/mL) ul AAV ul PBS

[0215] Every other day 200uL of maintenance media was refreshed to the wells (volume was removed and replace. On harvest day, cells are collected 6 days post transfection in 50uL of RIPA + PI, replicates are pooled and placed at -80C.Thaw samples on ice and spin down at 4C and collect supernatant. Return to -80 or run western blot. Every other day 200uL of maintenance media was added to the wells. Harvest day, cells are collected 6 days post transfection in 50uL of RIPA + PI, replicates are pooled and placed at -80C. Thaw samples on ice and spin down at 4C and collect supernatant. Return to -80 or run western blot. [0216] qPCR: Use GenoGrinder to homogenize tissue and extract genomic DNA using the Qiagen DNeasy Blood and Tissue Kit. Scoop tissue into the appropriate lysis buffer according to the kit. Use Nano drop and normalize to 50ng/uL if possible. The aim is to load 100ng per well, however you can get that to happen. Table 53: Antibodies used for Western Blotting Target Manufacturer Concentration Species Dilution MW Secondary RBM20 Abcam (ab233147) 1 mg/mL rabbit 1:500 ~150 kDa α-Rb 800CW D

[0217] Cardiac specific promoters, including TNNT2-400 were used. These constructs were used to separate the different modifications such as RBM20 codon optimizations, CpG reductions, and the siRNAv4 modification, which is a genetic modification in the gene sequence, but not the amino acid sequence, of the ectopically expressed RBM20 which prevents knockdown of the ectopically expressed RBM20 by the siRNAv4 and the R634Q RBM20 mutation from each other across different constructs and to assess their effects on RBM20 protein expression. TNNT2-400 promoters were evaluated for driving RBM20 protein expression.

Table 54: Design for in vivo wild-type mouse protein expression and biodistribution study using TNNT2-400 constructs. Group Animal Dose In-Life AAV Treatment Dose Level (vg/kg) No. No. Regimen Time

[0218] [0229] Figure 15 shows RBM20 expression driven from the JeT and TNNT2- promoters following transfection of human iCell cardiomyocytes as detected by Western blot analysis. Human RBM20 protein expression was detected from all the new constructs (FIG. 15, top panel, lanes 3-7 and 9-15) using anti-RBM20 Antibody (Abcam). Myc-tagged RBM20 was detected from all Myc-tagged constructs using anti-myc antibody (Abcam) (FIG.15, lower panel, lanes 3, 5-8, 10, 12-16). These data confirm that the TNNT2- 400 promoter drives expression of human RBM20 protein in primary cardiomyocytes. Sequences used in this example and as analyzed in Figure 15 SEQ ID NOs in FIG. 15. [0219] Table 54 is the experimental outline for the in vivo wild-type mouse study using a set of TNNT2-400 constructs. Each of the indicated vectors was packaged into a SLB101 capsid. Data readout was for protein expression in the heart via histology using whole heart fixed in formalin for two animals out of each group using RBM20 antibody (#PA5- 58068, Thermo Fisher Scientific) and Myc antibody (#2276T, Cell Signaling Technology) (FIG. 17-17C) and biodistribution (FIG. 16). FIG. 16 shows that each of the vectors effectively transduced mouse hearts with biodistribution evaluated by quantifying vector DNA in mouse hearts. [0220] FIGS. 17A-17E show human RBM20 expression in mouse heart tissue. FIG.17Ashows human RBM20 expression from the TNNT2400-huRBM20 vector in wild-type C57Bl/6 mouse heart detected using the RBM20 antibody. RBM20 staining overlaps with DAPI staining of DNA in the nucleus in the merged image (see, arrows). Staining with the Myc antibody shows background Myc staining. [0221] FIG. 17B shows human RBM20 expression from the TNNT2400- huRBM20-Nmyc vector in wild-type C57Bl/6 mouse heart detected using the RBM20 and Myc antibodies. RBM20 and Myc staining overlap with DAPI staining of DNA in the nucleus in the merged image (see, arrows). [0222] FIG. 17C shows human RBM20 expression from the TNNT2₄₀₀- huRBM20_siRNAv4-NMyc vector in wild-type C57Bi/6 mouse heart detected using the RBM20 and Myc antibodies. RBM20 and Myc staining overlap with DAPI staining of DNA in the nucleus in the merged image (see, arrows). [0223] Taken together these data show that human RBM20 is expressed from the constructs described in this example. Example 3 –TNNT2 Constructs Tested for Protein Expression Following Plasmid Transfection [0224] The constructs described in this example include the TNNT-455 promoter, the SGT-003 backbone, and additional modifications. (Table 55). Table 55 indicates that the vectors can be packaged into viruses. Table 55: Constructs Packaged into SLB101 Capsid Description vg/mL

AAV-SLB101-TNNT2-455-huRBM20_siRNA_{v4_Default_stop_control}^5.36E+13

drive human RBM20 expression in heart tissue in vivo. Example 4 [0226] In this non-limiting example, knockdown/silencing of RBM20 in cardiomyocytes differentiated from human induced pluripotent stem cells using siRNA was tested. Methods [0227] Human induced pluripotent stem cells maintenance: Human induced pluripotent stem cells (hiPSCs) were grown in 6-well plates, coated with a basement membrane matrix extracted from murine Engelbreth-Holm-Swarm (EHS) tumors and free of lactose dehydrogenase elevating virus (LDEV) (Geltrex LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix, #A1413302, Gibco Life Tech), and with daily change of Essential8 cell culture medium (Essential8 complete, #A15169-01, Life Tech; supplemented with Essential8 supplement, #A1517-01-10ml, Life Tech) and incubated at 37ºC, 5% CO2. Cells were passaged at 80-100% confluency with TrypLE Express Enzyme (#12605010, Gibco Life Tech). Thereafter cells were re-plated in Essential8 complete medium with 2 μM thiazovivin (#420220, Sigma-Aldrich). After 24 hours, Essential8 complete medium with 2 μM thiazovivin medium was replaced with Essential8 complete medium. [0228] Cardiomyocyte differentiation from human induced pluripotent stem cells hiPSCs: Three separate differentiations were performed where hiPSCs were plated both at a confluence of 75,000 and 100,000 cells/well in a 6-well plate coated with a basement membrane matrix Geltrex 4 days prior to starting with the differentiation protocol. Daily replacement of Essential8 medium was performed. Throughout differentiation, cells were incubated at 37ºC, 5% CO2. [0229] After day 4 (d4), when the cells reached 80-90% confluency, Essential8 medium was changed for culture medium RPMI-1640-Medium-GlutaMAX Supplement- HEPES (#72400-021, Gibco Life Tech), supplemented with 0.5 mg/mL human recombinant albumin (#A9731, Sigma-Aldrich), 0.2 mg/mL l-ascorbic acid 2-phosphate (#A8960, Sigma- Aldrich) and 4 μM of CHIR99021, a potent inhibitor of glycogen synthase kinase 3 (#361559, Sigma-Aldrich). At day 6 (d6), medium was replaced for RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with 0.5 mg/mL human recombinant albumin, 0.2 mg/mL l-ascorbic acid 2-phosphate and 5 μM of IWP2, an inactivator of Porcn and/or inhibitor of Wnt production (#681671, Sigma-Aldrich). At day 8 (d8) and d12, medium was refreshed for RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with 0.5 mg/mL human recombinant albumin and 0.2 mg/mL l-ascorbic acid 2-phosphate. At day 14 (d14), medium was replaced for RPMI-1640-Medium-GlutaMAX Supplement-HEPES, supplemented with B-27 Supplement-serum free (#17504001, Gibco). At day 17 (d17), cardiomyocytes were selected by growing in RPMI-1640-Medium-HEPES, supplemented with 4 μl/mL 1M lactate and B-27 Supplement-serum free. At day 21 (d21), medium was replaced for RPMI-1640- Medium-GlutaMAX Supplement-HEPES, supplemented with B-27 Supplement-serum free and 2.5 mL of Penicillin-Streptomycin 5000 U/mL (#15070-063-100ml, Gibco Life Tech). [0230] siRNA Transfection in cardiomyocytes differentiated from human induced pluripotent stem cells. Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) as described above were plated at 80% confluency in a 12-well plate 96 hours before siRNA treatment. Cells were either transfected with 10 nM of siRNAs separately, a combination thereof or a control sequence. For control, a random RNA sequence which is non- complementary to RBM20 and thus does not target RBM20 transcripts was used (Table 56, Experimental layout; Table 13 of siRNAs sequences targeting RBM20, SEQ ID NO: 69-71). Each treatment group was subjected to a total of 10 nM siRNA per well. Each well was transfected using lipofectamine 3000 Transfection kit (#L3000001, Thermo Fisher) as per manufacturers’ instructions. hiPS-CMs were incubated at 37ºC, 5% CO2 for 96 hours prior RNA extraction. The different treatment groups are indicated in the table below. Table 56: Experimental layout of different treatment groups for RBM20 knockdown Experimental Treatment Groups Aim

y action (qPCR) After siRNA transfection and incubation, hiPS-CMs were detached from the plate with the use of TrypLE™ Select Enzyme (#A1217701, Thermo Fisher) and centrifuged at 400 x g for 5 minutes. RNA was extracted from the cells using TRIzol^TM Reagent (#15596026, Thermo Fisher Scientific) according to manufacturers’ instructions. Conversion to cDNA was achieved using the iScript™ cDNA Synthesis Kit (#1708890, Bio-Rad). QPCR was accomplished using the CFX96 Realtime PCR system (Bio-Rad) with iQ SYBR Green (Bio- Rad) according to manufacturers’ instructions, and using primers as provided in Table 14 (SEQ ID NO: 78-95). Housekeeping gene ribosomal protein L32 (RPL32) was used as internal control for normalizing and calculating the relative expression of RBM20 gene. [0232] Analysis of the off-target activity: The three tested siRNAs (Table 13; SEQ ID NO: 69-71) were assessed for their ability to silence transcripts of non-target (off- target) genes (FIG. 20A-20C). Prediction of the potential off-target genes was based on the antisense sequence of the siRNAs. The top three most potential off-target genes predicted by the NCBI nucleotide BLAST webtool was assessed for each siRNA using qPCR (Table 14, qPCR primers, SEQ ID NO: 78-95, FIG. 20A-20C). Thereafter, expression of the top three most potentially affected off-target genes was assessed using qPCR. [0233] Analysis of RBM20 knockdown. As shown in FIG. 18, different siRNAs against RBM20 were tested and their efficiency was assessed. It was observed that expression of RBM20 transcripts from an endogenous RBM20 gene of hiPS-CMs was successfully reduced when using siRNAs targeted against RBM20 (FIG. 18). Target sequences of each respective siRNA are provided in Table 13 (Table of siRNAs sequences targeting RBM20, SEQ ID NO: 69-71). All three tested siRNAs targeted against RBM20 transcripts successfully reduced RBM20 mRNA levels when compared to samples treated with siControl (SEQ ID NO: 68) a control siRNA that does not target RBM20. [0234] Figure 19 also shows that this knockdown of RBM20 impacts cellular function because it shows the effects on the downstream splice target genes in the absence of RBM20 (FIG. 19A-19B). RBM20 is a regulator of TTN isoform switching. The effect of RBM20 reduction on alternative splicing caused by the knockdown of RBM20 is demonstrated by the reduction in the TTN N2B isoform (FIG. 19A) and an increase in the TTN N2BA isoform (FIG. 19B), thus decreasing the ratio of TTN N2B:N2BA. [0235] Off-target activity of each siRNA was also assessed, as demonstrated in FIG. 20A-20C. siRBM20_V1 and siRBM20_V6 exhibited some off-target effects, whilst this is not evident for siRBM20_V4. Based on the efficient knockdown and absence of off-target activity, siRBM20_V4 was used as a candidate in the follow-up experiments for the production of guide/antisense strand incorporation into a primary-microRNA scaffold. [0236] Figure 18: Knockdown of RBM20 in iPS-CMs using siRNA. RBM20 specific siRNA knockdown in hiPS-derived cardiomyocytes using three different siRNAs targeted against RBM20 mRNA, or a combination of all three siRNAs. Depicted are mRNA expression levels of RBM20 (3 differentiations, n = 2-3 replicates for each differentiation) post siRNA transfection. siCombined refers to combination of all three siRNAs. Values were normalized to the housekeeping gene, RPL32. siControl was used as a control. A one-way ANOVA followed by Kruskal-Wallis nonparametric test was conducted (* P-value < 0.05, ** P-value < 0.01, **** P-value < 0.0001). [0237] Figure 19: Downstream splice target genes of RBM20 knockdown in iPS-CMs. Effects of downstream splice targets genes of RBM20 post RBM20 specific siRNA knockdown in hiPS-derived cardiomyocytes. (A) shows the mRNA expression levels of TTN N2B isoform post siRBM20 transfection. (B) shows the mRNA expression levels of TTN N2BA isoform post siRBM20 transfection. Values were normalized to the housekeeping gene, RPL32. siControl was used as a control Each shape in the graph (circles and triangle) is a technical repeat of the same differentiation with differentiations being biological repeats of each experiment. (3 differentiations, n = 2-3 replicates for each differentiation). (** P-value < 0.01, *** P-value < 0.001, **** P-value < 0.0001, ns: not significant). [0238] Figure 20: Off targets of the respective siRNAs in iPS-CMs. Top three potential off-target genes in hiPS-derived cardiomyocytes for each of the respective siRNAs antisense strand as identified through NCBI nucleotide BLAST. (A) shows mRNA expression levels of NOVA1, RNASE6 and UTP14A post siRBM20-V1 transfection. (B) shows mRNA expression levels of GAPVD1, ZNF827 and IYD post siRBM20_V4 transfection. (C) shows mRNA expression levels of MAST4, PEAK1 and NUDCD2 post siRBM20_V6 transfection. Values were normalized to the housekeeping gene, RPL32. Each shape in the graph (circles and triangle) is a technical repeat of the same differentiation with differentiations being biological repeats of each experiment (3 differentiations, n = 2-3 replicates for each differentiation). Significance has been assessed by a two-tailed Mann-Whitney test nonparametric test (* P-value < 0.05, ** P-value < 0.01, *** P-value < 0.001, ns: not significant). Example 5 [0239] In this non-limiting example, generation of a functional guide/antisense strand against RBM20 from the primary micro-RNA124 scaffold was demonstrated. [0240] Generation of primary-miRNA124 scaffold: The primary-miRNA124 scaffold was taken from the miRbase website (available at mirbase.org; accession number: MI0000443). The siRBM20_V4 siRNA sequence was added to the primary micro-RNA124 sequence. To mimic the structure of primary-miRNA124, non-complementary base pairs were introduced into the sense and antisense strands (wobbles). The primary-miR124-siRBM20_V4 constructs containing different combinations of wobbles were determined using the UNAFold website to ensure similar deltaG values to the original primary-miRNA124 (available at unafold.org/RNA_form.php). Three constructs that gave deltaG values most similar to the original primary-miRNA124 were chosen, namely primary-miR124-siRBM20_V4-R5, primary-miR124-siRBM20_V4-R6 and primary-miR124-siRBM20_V4-R7. Sequences for these constructs are provided in Table 15 (Table of primary-microRNA-scaffold sequences post incorporation of siRBM20_V4 into the primary-miRNA124 scaffold (pri-miR124) SEQ ID NO: 96-98). [0241] Dual luciferase reporter plasmid generation. For detection of the antisense and sense strand formation from the different primary-miRNA124 constructs, a dual reporter luciferase plasmid was used. The targeting sequence of the antisense strand was cloned in-frame with Renilla luciferase in the dual reporter luciferase plasmid, and firefly luciferase expression was used for transfection efficiency. For detection of the different primary- miRNA124 constructs containing different wobbles (non-complementary base pair matches) on the sense strand, the targeting sequence of the sense strand of the respective constructs Table 15 (pri-miR124-siRBM20_V4-R5 (SEQ ID NO: 96), pri-miR124-siRBM20_V4-R6 (SEQ ID NO: 97) and pri-miR124-siRBM20_V4-R7 (SEQ ID NO: 98)) were cloned in-frame with Renilla luciferase in a dual reporter luciferase plasmid. Renilla luciferase expression was normalized to firefly luciferase expression. [0242] Transfection of HEK293T cells. Cells were plated into 24-well plates and were at a confluency of 70-80% prior to transfection using lipofectamine 3000 Transfection kit (#L3000001, Thermo Fisher) as per manufacturers’ instructions. Cells were incubated at 37 ºC, 5% CO2 for 48 hours prior to collection for luciferase assays. The primary-miR124 scaffold control plasmid, containing an antisense strand that does not target RBM20, was used a control. Furthermore, as a positive control, a plasmid containing an shRNA containing an antisense strand that targets RBM20 driven by a U6 promoter was used (shRBM20 SEQ ID NO: 538). The different treatment groups are indicated in Table 57 below: Table 57: Experimental layout of different treatment groups for luciferase assay Experimental Aim Treatment Groups T h k f i 1 L if r id r rt r l mid-100n (N ff ld)

Pri-miR124- 2. Luciferase passenger R5 reporter plasmid-100ng

[0243] Results: As shown in FIG.21, the expression of the guide/antisense strand from the different primary-miR124-siRBM20_V4 constructs was detected for all tested constructs through the reduction of Renilla luciferase expression. [0244] Figure 21: Expression of guide/antisense strand post incorporation of siRBM20_V4 targeting sequence into the primary-miRNA124 scaffold (pri-miR124). The expression of guide/antisense strand from the different primary-miRNA124-siRBM20_V4 constructs is demonstrated through the reduction of Renilla luciferase expression. Renilla luciferase expression is normalized to firefly luciferase expression. No scaffold, only the luciferase reporter plasmid; Pri-miR124-scaffold control, contains a guide/antisense that does not target RBM20; positive control, contains a plasmid expressing the siRNA targeting RBM20 in a shRNA-scaffold driven by a U6 promoter. Example 6: Introduce into cells a vector containing the siRBM20_V4 (embedded in a miR124 scaffold) and the RBM20 expression construct, which is resistant to the siRBM20_V4. [0245] In this non-limiting example, cells will be transduced with a viral vector containing the siRBM20_V4 (embedded in a miR124 scaffold) and the RBM20 expression construct (which is resistant to the siRBM20_V4). A control plasmid will be used that is contains an siRNA non-complementary to RBM20 transcripts (either endogenous or engineered). hiPS-CMs will be incubated for at least 96hrs prior to mRNA or protein extraction. [0246] Human induced pluripotent stem cells (hiPSCs) were grown and maintained as described above. Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs were obtained as described above. [0247] Transduction of cardiomyocytes differentiated from human induced pluripotent stem cells: Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) as described above were plated at 80% confluency in a 12-well plate 96 hours before transduction. [0248] Expected results: We expect knockdown of endogenous RBM20 by the primary-miR124-siRBM20_V4 (primary-microRNA containing the siRBM20_V4) whilst the expression of RBM20 resistant construct to be unaffected by primary-miR124-siRBM20_V4 (SEQ ID NOs: 1231, 1257 and 1238). Results will be evaluated by measuring RBM20 protein and mRNA levels as described above. Function of the expressed RBM20 will be tested in the assays described above.

Claims

WHAT IS CLAIMED IS: 1. A recombinant adeno-associated virus (rAAV) comprising an expression cassette, the expression cassette comprising: an RBM20 coding sequence; optionally, a Kozak sequence; and one or more silencing elements, wherein the one or more silencing elements reduces expression of both pathogenic and wild type endogenous RBM20 expression, wherein the one or more silencing elements encodes one or more siRNA and/or shRNA sequences, wherein expression of the one or more siRNA and/or shRNA is driven by a first promoter sequence wherein the RBM20 expression is driven by the first promotor sequence or by a second promoter sequence, wherein the RBM20 coding sequence is optionally CpG depleted, wherein the RBM20 coding sequence optionally comprises one or more modifications, wherein the one or more optional modifications confer resistance to silencing expression of non-endogenous RBM20 encoded by the RBM20 coding sequence, and optionally wherein the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to one or more of SEQ ID NOs: 4, 10, and 40; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 4, 10, and 40.

2. The rAAV of Claim 1, wherein the RBM20 coding sequence is CpG depleted.

3. The rAAV of Claim 1 or 2, wherein the RBM20 coding sequence comprises one or more modifications to the coding sequence, wherein the one or more modifications confer resistance to RNA-based degradation of the RBM20 coding sequence.

4. The rAAV of Claim 1, 2, or 3, wherein the one or more modifications to the RBM20 coding sequence do not alter the encoded RBM20 amino acid sequence.

5. The rAAV of any one of Claims 1 to 4, wherein the one or more modifications to the RBM20 coding sequence is selected from AATCAGGTGTTGAGTAAGGTA (SEQ ID NO: 58), AATTCAACTGCAGTATACAAT (SEQ ID NO: 59), AAAGTGACAAACTATATTTTA (SEQ ID NO: 60), and combinations thereof.

6. The rAAV of any one of Claims 2 to 5, wherein the RBM20 coding sequence is selected from a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 16, 22 and 46; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 16, 22, and 46.

7. The rAAV of Claim 6, wherein the RBM20 coding sequence further comprises a single amino acid modification, wherein the modification comprises a R634Q mutation in the encoded RBM20 amino acid sequence, and wherein the RBM20 coding sequence comprises a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 28, 34, and 52; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 28, 34, and 52.

8. The rAAV of any one of Claims 1 to 5, wherein the one or more silencing elements are selected from one or more of SEQ ID NOs: 55 (CACTTTGGAGAGGACTTGGTT), 56 (GTTATAAACAGCTGTGCTGTT), or 57 (GAGGATGTAATTAGTGACCTT).

9. The rAAV of any one of Claims 1 to 8, wherein the second promoter sequence is selected from the group consisting of JeT, CK8, CMV, MHCK7, aMHC, mCMV, TNNT2, and EF1alpha.

10. The rAAV of any one of Claims 1 to 9, wherein the first promoter sequence is a PolIII promoter or a PolII promoter.

11. The rAAV Claim 10, wherein the first promoter sequence is selected from the group consisting of H1, U6, and 7SK.

12. The rAAV of any one of Claims 1 to 11, wherein the Kozak sequence is a consensus optimized Kozak sequence.

13. The rAAV of claim 1, wherein the expression cassette comprises a sequence having at least about 85% sequence identity to SEQ ID NOs: 1-6, 7-12, 13-18, 19-24, 25-30, 31-36, 37-42, 43-48, or 49-54, arranged in order.

14. The rAAV of any one of Claims 1 to 13, wherein the rAAV is serotype 9, or derived from serotype 9.

15. The rAAV of any one of Claims 1 to 13, wherein the rAAV is serotype rh74, or derived from serotype rh74. 16. A method for the treatment of cardiomyopathy, comprising: administering to a subject having cardiomyopathy: a recombinant adeno-associated virus (rAAV) comprising an RBM20 coding sequence; and one or more silencing elements, wherein the one or more silencing elements induce degradation of nucleic acids encoding both pathogenic and wild type endogenous RBM20 expression, and optionally wherein the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, or 52; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 4, 10,

16, 22, 28, 34, 40, 46, and 52.

17. The method of Claim 16, wherein the one or more silencing elements are within an expression cassette within the rAAV, wherein the expression cassette also includes the RBM20 coding sequence.

18. The method of Claim 16, wherein the one or more silencing elements are administered via a second rAAV.

19. The method of Claim 16, wherein the one or more silencing elements are administered by local or by systemic injection.

20. The method of any one of Claims 16 to 19, wherein the one or more silencing elements are selected from one or more of SEQ ID NOs: 55 (CACTTTGGAGAGGACTTGGTT), 56 (GTTATAAACAGCTGTGCTGTT), or 57 (GAGGATGTAATTAGTGACCTT).

21. The method of any one of Claims 16 to 20, wherein the RBM20 coding sequence comprises one or more modifications to the coding sequence, wherein the one or more modifications confer resistance to RNA-based degradation of the RBM20 coding sequence by the one or more silencing elements.

22. The method of Claim 21, wherein the one or more modifications to the RBM20 coding sequence do not alter the encoded RBM20 amino acid sequence.

23. The method of Claim 21 or 22, wherein the one or more modifications to the RBM20 coding sequence is selected from AATCAGGTGTTGAGTAAGGTA (SEQ ID NO: 58), AATTCAACTGCAGTATACAAT (SEQ ID NO: 59), AAAGTGACAAACTATATTTTA (SEQ ID NO: 60), and combinations thereof.

24. The method of any one of Claims 16 to 23, wherein the cardiomyopathy is dilated cardiomyopathy.

25. A recombinant adeno-associated virus (rAAV) comprising an expression cassette, the expression cassette comprising: an RBM20 coding sequence; a promoter sequence; and an optional intron a Kozak sequence; wherein the RBM20 expression is driven by the promoter sequence, wherein the AAV-expressed RBM20 corrects splicing defects, and wherein the RBM20 coding sequence is optionally CpG depleted, and wherein the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to one or more of SEQ ID NOs: 4, 10, 40, and 67; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 4, 10, 40, 46, and 67.

26. The rAAV of Claim 25, wherein the RBM20 coding sequence is CpG depleted.

27. The rAAV of Claim 25 or 26, wherein the RBM20 coding sequence comprises one or more modifications to the RBM20 coding sequence do not alter the encoded RBM20 amino acid sequence.

28. The rAAV of Claim 27, wherein the one or more modifications to the coding sequence is selected from AATCAGGTGTTGAGTAAGGTA (SEQ ID NO: 58), AATTCAACTGCAGTATACAAT (SEQ ID NO: 59), AAAGTGACAAACTATATTTTA (SEQ ID NO: 60), and combinations thereof.

29. The rAAV of any one of Claims 25 to 28, wherein the RBM20 coding sequence is selected from a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 16, 22 and 46; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 16, 22, and 46.

30. The rAAV of Claim 29, wherein the RBM20 coding sequence further comprises a single amino acid modification, wherein the modification comprises a R634Q mutation in the encoded RBM20 amino acid sequence, and wherein the RBM20 coding sequence comprises a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 28, 34, and 52; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 28, 34, and 52.

31. The rAAV of any one of Claims 25 to 30, wherein the promoter sequence is selected from the group consisting of JeT, CK8, CMV, MHCK7, aMHC, mCMV, TNNT2, and EF1alpha.

32. The rAAV of any one of Claims 25 to 31, wherein the Kozak sequence is a consensus optimized Kozak sequence.

33. The rAAV of Claims 25 to 32, wherein the expression cassette includes an intron.

34. The rAAV of claim 25, wherein the expression cassette comprises a sequence having at least about 85% sequence identity to SEQ ID NOs: 1-6, 7-12, 13-18, 19-24, 25-30, 31-36, 37-42, 43-48, or 49-54, arranged in order; optionally wherein the expression cassette comprises a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 1-6, 7-12, 13-18, 19- 24, 25-30, 31-36, 37-42, 43-48, or 49-54, arranged in order.

35. The rAAV of any one of Claims 25 to 34, wherein the rAAV is serotype 9, or derived from serotype 9.

36. The rAAV of any one of Claims 25 to 34, wherein the rAAV is serotype rh74, or derived from serotype rh74.

37. A method for the treatment of cardiomyopathy, comprising: administering to a subject having cardiomyopathy, wherein cardiac cells comprise splicing defects a recombinant adeno-associated virus (rAAV) comprising an RBM20 coding sequence; and wherein the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to one or more of SEQ ID NO: 4, 10 and 40; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 4, 10, and 40, and wherein the rAAV RBM20 coding sequence is expressed in cardiac cells and optionally wherein in rAAV-expressed RBM20 coding sequence reduces frequency of splicing defects of the cardiac cells.

38. The method of Claim 37, wherein the RBM20 coding sequence comprises one or more modifications to the coding sequence do not alter the encoded RBM20 amino acid sequence.

39. The method of Claim 38, wherein the one or more modifications to the coding sequence is selected from aatcaggtgttgagtaaggta (SEQ ID NO: 58), aattcaactgcagtatacaat (SEQ ID NO: 59), aaagtgacaaactatatttta (SEQ ID NO: 60), and combinations thereof.

40. The method of Claim 37, 38, or 39, wherein the RBM20 coding sequence is selected from a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 16, 22 and 46; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 16, 22, and 46.

41. The rAAV of Claim 40, wherein the RBM20 coding sequence further comprises a single amino acid modification, wherein the modification comprises a R634Q mutation in the encoded RBM20 amino acid sequence, and wherein the RBM20 coding sequence comprises a sequence having at least about 90% sequence identity to one or more of SEQ ID NOs: 28, 34, and 52; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 28, 34, and 52.

42. The method of any one of Claims 37 to 41, wherein the cardiomyopathy is dilated cardiomyopathy.

43. Use of a rAAV of any of one of the preceding claims for treatment of cardiomyopathy, which is optionally dilated cardiomyopathy.

44. Use of a rAAV of any of one of the preceding claims in the preparation of a medicament for the treatment of cardiomyopathy, which is optionally dilated cardiomyopathy.

45. A recombinant adeno-associated virus (rAAV) comprising an expression cassette, the expression cassette comprising: an RBM20 coding sequence; optionally, a Kozak sequence; and one or more silencing elements, wherein the one or more silencing elements reduces expression of both pathogenic and wild type endogenous RBM20 expression, wherein the one or more silencing elements encodes one or more siRNA and/or shRNA sequences, the one or more siRNA and/or shRNA linked by a first promoter sequence, the RBM20 coding sequence linked to the first promotor sequence or a second promoter sequence, wherein the RBM20 coding sequence is optionally CpG depleted, wherein the RBM20 coding sequence optionally comprises one or more modifications, wherein the one or more optional modifications confer resistance to silencing expression of non-endogenous RBM20 encoded by the RBM20 coding sequence, and optionally, wherein the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to one or more of SEQ ID NOs: 4, 10, and 40; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 4, 10, and 40. 46. A recombinant adeno-associated virus (rAAV) comprising an expression cassette, the expression cassette comprising: an RBM20 coding sequence; a promoter sequence; an optional intron a Kozak sequence; and the RBM20 coding sequence operably linked to the promoter sequence, wherein the RBM20 coding sequence is optionally CpG depleted, and optionally wherein the RBM20 coding sequence is selected from a sequence having at least about 85% sequence identity to one or more of SEQ ID NOs: 4, 10, 40, and 67; optionally wherein the RBM20 coding sequence is selected from a sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 4, 10, 40,

46, and 67, and wherein the AAV-expressed RBM20 corrects splicing defects.

47. A method for administering a recombinant adeno-associated virus (rAAV) to a host cell or subject, wherein said method comprises administering an rAAV of any one of claims 1-15 and 26-36.

48. A recombinant adeno-associated virus (rAAV) comprising an expression cassette, the expression cassette comprising an exogenous RBM20 coding sequence and one or more silencing elements, wherein the one or more silencing elements reduces or eliminates expression of endogenous RBM20, said RBM20 coding sequence resistant to silencing by the one or more silencing elements, thereby allowing for complete or partial silencing of endogenous RBM20 and expression of exogenous RBM20.

49. A method for the treatment of cardiomyopathy, comprising administering to a subject having cardiomyopathy a recombinant adeno-associated virus (rAAV) comprising an exogenous RBM20 coding sequence and one or more silencing elements, said one or more silencing elements capable of inducing degradation of nucleic acids encoding endogenous RBM20, said exogenous RBM20 coding sequence modified for resistant to, insulated from, and/or unaffected by silencing of said one or more silencing elements.

50. A recombinant adeno-associated virus (rAAV) comprising an expression cassette, the expression cassette comprising an RBM20 coding sequence and one or more silencing elements, wherein the one or more silencing elements reduces expression of endogenous RBM20, wherein the RBM20 coding sequence optionally comprises one or more modifications, wherein the one or more optional modifications confer resistance to silencing expression of non-endogenous RBM20 encoded by the RBM20 coding sequence.

51. A method for administering a recombinant adeno-associated virus (rAAV) to a host cell or subject, wherein said method comprises administering an rAAV of any one of Claims 47-50.