WO2025129079A1

WO2025129079A1 - Cardiac-specific promoters for gene therapy

Info

Publication number: WO2025129079A1
Application number: PCT/US2024/060141
Authority: WO
Inventors: Prasad R. KONKALMATT; Mohammad Ali MANDEGAR; Christopher A. Reid
Original assignee: Tenaya Therapeutics Inc
Current assignee: Tenaya Therapeutics Inc
Priority date: 2023-12-13
Filing date: 2024-12-13
Publication date: 2025-06-19
Anticipated expiration: 2026-06-13

Abstract

The present technology provides compositions and methods for gene therapy and/or cell therapy for treatment of heart diseases. In particular, the present technology provides cardiac-specific promoters and vectors for expressing transgenes (e.g., large transgenes) in cardiac cells, for example, with the AAV system. The present technology also provides expression cassettes, recombinant AAV (rAAV) viral genomes, rAAV viruses or virions, pharmaceutical compositions, and methods of use in treatment or prevention of diseases (e.g., heart disease, such as cardiomyopathy).

Description

CARDIAC-SPECIFIC PROMOTERS FOR GENE THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is an international PCT application claiming priority to US Provisional Application No. 63/609,724 filed December 13, 2023, the contents of which are incorporated herein by reference in their entirety.

ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (TENA_050_01 WO_SeqList_ST26.xml; Size: 383,848 bytes; and Date of Creation: December 13, 2024) are herein incorporated by reference in its entirety.

TECHNICAL FIELD

[0003] The present technology relates generally to the fields of compositions (e.g., promoters, vectors) and methods for gene therapy and cell therapy for diseases or disorders of the heart.

BACKGROUND

[0004] Heart diseases are a major cause of death worldwide. Various treatments for heart diseases have been proposed, including gene therapy that corrects abnormal or mutant genes causing or associated with disease or expresses genes that have a cardioprotective effect. Another approach is cell-based therapy using, for example, induced cardiomyocytes to replenish diseased cell population in the heart. Both therapies require manipulation of the genes at a cellular level. Adeno-associated viral (AAV) vectors possess unique features that make them attractive for delivering foreign DNA to cells for a transgene to be expressed, e.g., for use in gene therapy. AAV is a replication-deficient parvovirus, and its natural, single-stranded DNA genome is about 4.7 kilo bases (kb) in length including two 145-base nucleotide inverted terminal repeat (ITRs). Thus, AAV vectors usually have a packaging capacity of about 4.7 kb, ideally not more than 5 kb. And with promoters and other regulatory elements, the size of the transgene that can be accommodated in an AAV vector is usually limited to about 4.2 kb. However, some of the therapeutic proteins to be expressed to treat heart diseases can be quite large, for example, comprise at least about 1000 or more amino acids. Accordingly, there exists a need for novel, compact promoters that can drive cardiacspecific expression of proteins, including large proteins, with comparable, equivalent, or better efficacy to natural cardiac-specific promoters. The present technology addresses that need.

SUMMARY

[0005] The present technology provides compositions and methods for gene therapy and/or cell therapy for treatment of heart diseases including, for example, cardiac-specific promoters and vectors for expressing transgenes (e.g., large transgenes) in cardiac cells, for example, with the AAV system.

[0006] In some aspects, provided is a cardiac-specific chimeric promoter comprising a transcription factor (TF) binding rich region having one or more fragments derived from the promoter regions of one or more cardiac-specific genes. In some embodiments, the TF binding rich region comprises binding sites for one or more transcription factors selected from the group consisting of GATA4, MEF2C, TBX5, NKS2.5, HAND2, and SRF. In some embodiments, the TF binding rich region comprises binding sites for transcription factors GATA4, MEF2C, TBX5, NKS2.5, and HAND2.

[0007] In some embodiments, the TF binding rich region comprises two or more fragments derived from the promoter regions of two or more cardiac-specific genes. In some embodiments, the two or more cardiac-specific genes are selected from the group consisting of TNNT2, MYBPC3, MYH6, MYH7, MYL2, and MYL3. In some embodiments, the two or more fragments are each about 50 bp in length.

[0008] In some embodiments, the TF binding rich region comprises five fragments derived from the promoter regions of two or more cardiac-specific genes. In some embodiments, the chimeric promoter is about 250 bp in length. In some embodiments, the two or more cardiac-specific genes comprise TNNT2 and MYBPC3. In some embodiments, the two or more cardiac-specific genes are TNNT2 and MYBPC3. In some embodiments, the chimeric promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the chimeric promoter further comprises a binding site for SRF. In some embodiments, the chimeric promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2.

[0009] In some embodiments, the chimeric promoter has at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, or at least 100% activity compared to a wild-type human TNNT2 promoter of SEQ ID NO: 6.

[0010] In some aspects, provided is a cardiac-specific synthetic promoter comprising: (i) a transcription factor (TF) binding rich region comprising one or more fragments derived from the promoter regions of one or more cardiac-specific genes; and (ii) a core promoter comprising a TATA box and a transcription start site (TSS).

[0011] In some embodiments, the core promoter is a CMV core promoter. In some embodiments, the CMV core promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13.

[0012] In some embodiments, the TF binding rich region is less than 300 bp, less than 250 bp, or less than 200 bp in length. In some embodiments, the TF binding rich region comprises binding sites for one or more transcription factors selected from the group consisting of GATA4, MEF2C, TBX5, NKS2.5, HAND2, and SRF. In some embodiments, the TF binding rich region comprises binding sites for transcription factors GATA4, MEF2C, TBX5, and SRF.

[0013] In some embodiments, the TF binding rich region comprises two or more fragments derived from the promoter regions of two or more cardiac-specific genes.

[0014] In some embodiments, the two or more cardiac-specific genes are selected from the group consisting of TNNT2, MYBPC3, MYH6, MYH7, MYL2, and MYL3. In some embodiments, the two or more cardiac-specific genes comprise TNNT2 and MYBPC3. In some embodiments, the two or more cardiac-specific genes are TNNT2 and MYBPC3. In some embodiments, the TF binding rich region comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3. In some embodiments, the synthetic promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14. [0015] In some embodiments, the TF binding rich region is derived from the promoter region of a cardiac-specific gene. In some embodiments, the cardiac-specific gene is selected from the group consisting of human TNNT2, mouse TNNT2, and chicken TNNT2. In some embodiments, the cardiac-specific gene is human TNNT2. In some embodiments, the TF binding rich region comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8. In some embodiments, the synthetic promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15. In some embodiments, the cardiac-specific gene is mouse TNNT2. In some embodiments, the TF binding rich region comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10. In some embodiments, the synthetic promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16. In some embodiments, the cardiac-specific gene is chicken TNNT2. In some embodiments, the TF binding rich region comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12. In some embodiments, the synthetic promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17.

[0016] In some embodiments, the synthetic promoter has at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, or at least 100% activity compared to a wild-type human TNNT2 promoter of SEQ ID NO: 6.

[0017] In some aspects, provided is a vector comprising a cardiac-specific chimeric promoter or a cardiac-specific synthetic promoter according to various embodiments disclosed herein. In some embodiments, the vector further comprises a transgene. In some embodiments, the vector is a viral vector, for example, an adeno-associated virus (AAV) vector (e.g., an AAV9 vector). [0018] In some aspects, provided is a recombinant AAV (rAAV) virus or virion comprising a vector and a capsid protein according to various embodiments disclosed herein.

[0019] In some aspects, provided is a cell comprising a vector or an rAAV virus or virion according to various embodiments disclosed herein.

[0020] In some aspects, provided is a pharmaceutical composition comprising (i) a vector, an rAAV virus or virion, or a cell according to various embodiments disclosed herein; and (ii) a pharmaceutically acceptable excipient.

[0021] In some aspects, provided is a method of treating a condition or disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to various embodiments disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a diagram showing the generation and testing of cardiac-specific (e.g., cardiomyocyte (CM)-specific) chimeric promoters. The promoter sequences of five human cardiomyocyte-specific genes (MYBPC3, MYH6, MYH7, MYL3, and TNNT2) were randomly cut into 50 bp fragments and fused to generate a library of 250 bp-long chimeric promoters. These chimeric promoters were closed in AAV vectors carrying a GFP reporter gene and tested in an in vitro screening. High expressers were selected for further testing in vivo.

[0023] FIG. 2A is a diagram showing the testing of twelve high-expression chimeric promoter constructs selected from the in vitro screening in non-human primates (NHP) and mice to measure the promoter strength. A 400 bp natural promoter sequence from the human TNNT2 gene (HuTNNT2 400bp; see Table 2, SEQ ID NO: 6) was used as a benchmark to compare the strengths of the promoter constructs. HuTNNT2 400 bp and each of the twelve chimeric promoter constructs were cloned into AAV9 vectors and given at 4x10¹¹ genome copies (gc)/kg intravenously. Four weeks after administration, heart and liver tissue were dissected and analyzed by next generation sequencing (NGS). FIG. 2B shows that the chimeric promoter 6C2 had cardiac-specific gene expression in NHP and mice.

[0024] FIG. 3 shows a schematic of the chimeric promoter 6C2, which is a 250 bp promoter composed of four 50 bp human TNNT2 promoter fragments and one 50 bp MYBPC3 promoter fragment, arranged randomly. The fragment from MYBPC3 promoter contains a TATA box and a transcription start site (TSS).

[0025] FIG. 4 transcription factor (TF) binding site profiles and numbers of the 6C2 promoter and the natural human (HsTNNT2), mouse (MsTNNT2), and chicken (ChTNNT2) TNNT2 promoters. The 6C2 promoter does not have a binding site for transcription factor SRF.

[0026] FIG. 5A shows modifications of the 6C2 promoter by converting the 12 nucleotides between positions 99-112 of 6C2 to an SRF transcription factor binding site, to generate the 6C2SRF promoter. FIG. 5B show that insertion of an SRF binding site at the indicated site improved promoter strength of 6C2.

[0027] FIG. 6A shows modifications of the 6C2SRF promoter fusing a 65 bp CMV core promoter sequence that carries a TATA and a TSS to the 3’ end of the first 154 bp of 6C2SRF, to generate the 6C2SRF_CMV core (also referred to as 6C2SRF219 promoter. FIG. 6B shows that insertion of an SRF binding site in the 6C2 promoter increased the expression to about 50% compared with HuTNNT2, and fusion of the 65 bp CMV core promoter to the first 154 bp of 6C2SRF further increased expression by 2.3-fold compared to that of HuTNNT2.

[0028] FIGS. 7A-7B show development of mini cardiomyocyte specific promoters. FIG. 7A shows the comparison of 400 bp sequences from human (HsTNNT2), mouse (MsTNNT2), and chicken (ChTNNT2) TNNT2 promoters. The transcription factor (TF) binding rich regions are circled. FIG. 7B shows creation of synthetic mini promoters by fusing the circled TF binding rich regions independently with a 65 bp CMV core promoter carrying a TATA box and a TSS, and testing of the generated mini promoters in comparison to their corresponding wild-type promoters.

DETAILED DESCRIPTION

[0029] The present technology provides compositions and methods for gene therapy and/or cell therapy for treatment of heart diseases. In particular, the present technology provides cardiac-specific promoters and vectors for expressing transgenes (e.g., large transgenes) in cardiac cells, for example, with the AAV system. The present technology also provides expression cassettes, recombinant AAV (rAAV) viral genomes, rAAV viruses or virions, pharmaceutical compositions, and methods of use in treatment or prevention of diseases (e.g., heart disease, such as cardiomyopathy).

[0030] While the present disclosure is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present disclosure is to be considered as an exemplification of the invention and is not intended to limit the invention to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the invention in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

[0031] The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word "about." It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about.” It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios, such as about 2, about 3, and about 4, and sub-ranges, such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0032] All publications disclosed herein are incorporated by reference in their entirety. To the extent any materials incorporated by reference conflict with the present disclosure, the present disclosure controls.

Definitions

[0033] Generally, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, each of the following terms has the meaning set forth in this section.

[0034] The indefinite articles “a” and “an” denote at least one of the associated nouns and are used interchangeably with the terms “at least one” and “one or more.” For example, the phrase “a module” means at least one module, or one or more modules.

[0035] The conjunctions “or” and “and/or” are used interchangeably.

[0036] The term “about,” as used herein when referring to a measurable value, such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount.

[0037] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises,” “comprising”, or equivalents such as “have,” “has,” “having,” “contain,” “contains,” “containing,” “include,” “includes,” or “including” will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0038] The term “AAV” is an abbreviation for adeno-associated virus. The term covers all subtypes of AAV, except where a subtype is indicated, and to both naturally occurring and recombinant forms. The abbreviation “rAAV” refers to recombinant adeno- associated virus. “AAV5” refers to AAV subtype 5. “AAV9” refers to AAV subtype 9. The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits may be found in the literature or in public databases such as GenBank. An “AAV vector” or “rAAV vector” is used in the art to refer either to the DNA packaged into in the rAAV virion or to the rAAV virion itself, depending on context. As used herein, unless otherwise apparent from context, rAAV vector refers to a nucleic acid (typically a plasmid) comprising a polynucleotide sequence capable of being packaged into an rAAV virion, but with the capsid or other proteins of the rAAV virion. Generally, an rAAV vector comprises a heterologous polynucleotide sequence (/.e., a polynucleotide not of AAV origin) and one or two AAV ITRs flanking the heterologous polynucleotide sequence. An “AAV particle” refers to an extracellular viral particle including at least one viral capsid protein (e.g., VP1 ) and an encapsidated AAV vector (or fragment thereof), including the capsid proteins. [0039] The term “administering” to a subject is a procedure by which one or more delivery agents, together or separately, are introduced into or applied onto a subject such that target cells which are present in the subject are eventually contacted with the agent.

[0040] For brevity and clarity, the disclosure refers to “capsid protein” or “capsid proteins” of AAV. Those skilled in the art understand that such references refer to VP1 , VP2, or VP3, or combinations thereof. As in wild-type AAV and most recombinant expression systems VP1 , VP2, and VP3 are expressed from the same open reading frame, engineering of the sequence that encodes VP3 inevitably alters the sequences of the C-terminal domain of VP1 and VP2. One may also express the capsid proteins from different open reading frames, in which case the capsid of the resulting rAAV virion could contain a mixture of wild-type and engineered capsid proteins, and mixtures of different engineered capsid proteins.

[0041] The term “cardiomyopathy” refers to the deterioration of the function of the myocardium (/.e., the actual heart muscle) for any reason. Subjects with cardiomyopathy are often at risk of arrhythmia or sudden cardiac death or both. The term “hypertrophic cardiomyopathy” refers to a disease of the heart and myocardium in which a portion of the myocardium is hypertrophied. The term “familial hypertrophic cardiomyopathy” refers to a genetic disorder characterized by increased growth (/.e., hypertrophy) in thickness of the wall of the left ventricle.

[0042] A “clinically effective amount,” “clinically effective concentration,” or “clinically effective dose” refers to a concentration or dose of a peptide, composition, or pharmaceutical composition that is shown to be effective in clinical trials or is predicted to be effective based on early phase or pre-clinical trials. In some embodiments, a “clinically effective amount” is the same as a “therapeutically effective amount.” In some embodiments, a “clinically effective amount” is higher or lower than a “therapeutically effective amount.” Further, the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and seventy of the condition may require an increase or decrease in the actual effective amount administered.

[0043] The term “construct” refers to any polynucleotide that contains a recombinant nucleic acid molecule. A construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A “vector” is a nucleic acid molecule that is capable of introducing a specific nucleic acid sequence into a cell or into another nucleic acid sequence, or as a means of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, an RNA vector, or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).

[0044] The term “delivery”, which is used interchangeably with “transduction,” refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Delivery of nucleic acids is a distinct process from expression of nucleic acids.

[0045] The term “expression” refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

[0046] The term “expression cassette” or “expression construct” refers to a DNA polynucleotide sequence operably linked to a promoter.

[0047] The term “gene therapy” involves the transfer of heterologous DNA to cells of a mammal, particularly a human, with a disorder or conditions for which therapy or diagnosis is sought. The DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed, and a therapeutic product encoded thereby is produced. Alternatively, the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product; it may encode a product, such as a peptide or RNA that in some manner mediates, directly or indirectly, expression of a therapeutic product. Gene therapy may also be used to deliver nucleic acid encoding a gene product to replace a defective gene or supplement a gene product produced by the mammal or the cell in which it is introduced. The introduced nucleic acid may encode a therapeutic gene product that is not normally produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time. The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host to enhance or otherwise alter the product or expression thereof.

[0048] The term “host cell” as used herein refers to a cell or microorganism targeted for genetic modification by introduction of a construct or vector carrying a nucleotide sequence for expression of a protein or polypeptide of interest.

[0049] The term “modified” refers to a substance or compound (e.g., a cell, a polynucleotide sequence, and/or a polypeptide sequence) that has been altered or changed as compared to the corresponding unmodified substance or compound.

[0050] The term “nucleic acid” or “polynucleotide” refers to a polymeric compound including covalently linked nucleotides comprising natural subunits (e.g., purine or pyrimidine bases). Purine bases include adenine and guanine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single- or double-stranded. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence.

[0051] The term “operably linked” or “operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a polynucleotide sequence if the promoter affects the transcription or expression of the polynucleotide sequence.

[0052] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length, though a number of amino acid residues may be specified. Polypeptides 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 embodiments, the polypeptides 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.

[0053] The term “promoter” as used herein refers a polynucleotide sequence that has one or more recognition site(s) to which an RNA polymerase binds, such that in a host or target cell, an RNA polymerase may initiate and transcribe a polynucleotide sequence “downstream” of the promoter into an RNA. Similarly stated, a “promoter” is operably linked or operatively linked to a polynucleotide sequence if in a host or target cell in which the promoter is active, an RNA polymerase initiates transcription of the polynucleotide at a transcription state site. Promoters operative in mammalian cells generally comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.

[0054] 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, or that the polynucleotide is assembled from synthetic oligonucleotides. A “recombinant” protein is a protein produced from a recombinant polypeptide. A recombinant virion is a virion that comprises a recombinant polynucleotide and/or a recombinant protein, e.g., a recombinant capsid protein.

[0055] The term “sequence identity” or “identity” when referring to a polynucleotide or polypeptide sequence refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. Methods of sequence alignment for comparison and determination of percent sequence identity is well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology (2003)), by use of algorithms know in the art including the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977); and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In some embodiments, the determination of the percentage of sequence identity may take place after a local alignment. Such alignments are well known in the art, for instance, the service EMBOSS Matcher identifies local similarities between two sequences using an algorithm based on the LALIGN application, version 2.0u4. In an example, the identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g., matrix (DNAfull), gap open (16), gap extend (4), alternative matches (1 ).

[0056] The term “subject” refers to a mammalian subject, preferably a human. A “subject in need thereof” refers to a subject who has been diagnosed with a cardiac disease (e.g., cardiomyopathy) or is at an elevated risk of developing the disease. The phrases “subject” and “patient” are used interchangeably herein.

[0057] A “therapeutically effective amount” as used herein is an amount that produces a desired effect in a subject for an indication, condition, disease, or disorder. In certain embodiments, the therapeutically effective amount is an amount that yields maximum therapeutic effect. In other embodiments, the therapeutically effective amount yields a therapeutic effect that is less than the maximum therapeutic effect. For example, a therapeutically effective amount may be an amount that produces a therapeutic effect while avoiding one or more side effects associated with a dosage that yields maximum therapeutic effect. A therapeutically effective amount for a particular composition will vary based on a variety of factors, including, but not limited to, the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability); the physiological condition of the subject (e.g., age, body weight, sex, disease type and stage, medical history, general physical condition, responsiveness to a given dosage, and other present medications); the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition; and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely, by monitoring a subject’s response to administration of the therapeutic composition and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st Edition, Univ, of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0058] The term “transgene” refers to a nucleic acid sequence encoding a protein or RNA (e.g., a therapeutic protein), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal’s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

[0059] The terms “treat,” “treating,” and “treatment,” as used herein with regard to cancer, refers to alleviating the cancer partially or entirely, inhibiting cancer cell growth, reducing the number of cancer cells, preventing the cancer, decreasing the likelihood of occurrence or recurrence of the cancer, slowing the progression or development of the cancer, or eliminating, reducing, or slowing the development of one or more symptoms associated with the cancer. For example, “treating” may refer to preventing or slowing the existing tumor from growing larger, preventing or slowing the formation or metastasis of cancer, and/or slowing the development of certain symptoms of the cancer. In some embodiments, the term “treat,” “treating,” or “treatment” means that the subject has a reduced number or size of tumor compared to a subject not being administered the treatment. In some embodiments, the term “treat,” “treating,” or “treatment” means that one or more symptoms of the cancer are alleviated in a subject receiving the pharmaceutical compositions as disclosed and described herein, compared to a subject who does not receive such treatment.

[0060] The term “upstream” refers to a portion of a polynucleotide that is, with reference to a transcription start site (TSS), 5’ to the TSS on the sense strand (or coding strand) of the polynucleotide; and 3’ to the TSS on the antisense strand of the polynucleotide. The term “downstream” refers to a portion of a polynucleotide that is, with reference to a TSS, 3’ to TSS on the sense strand (or coding strand) of the polynucleotide; and 5’ to the TSS on the antisense strand of the polynucleotide. Thus, a deletion from the upstream end of a promoter is a deletion of one or more base pairs in the non-transcribed region of the polynucleotide, 5’ to the TSS on the sense strand (or equivalently, 3’ to the TSS on the antisense strand). A deletion from the downstream end of a promoter is a deletion of one or more base pairs in the transcribed region of the polynucleotide, 3’ to the TSS on the sense strand (or equivalently, 5’ to the TSS on the antisense strand).

[0061] The term “variant” refers to a protein or nucleic acid having one or more genetic changes (e.g., insertions, deletions, substitutions, or the like) that returns all or substantially all of the functions of the reference protein or nucleic acid. For example, a variant of a therapeutic protein retains the same or substantially the same activity and/or provides the same or substantially the same therapeutic benefit to a subject in need thereof. A variant of a promoter sequence retains the ability to initiate transcription at the same or substantially the same level as the reference promoter, and retains the same or substantially the same cell type specificity. In particular embodiments, polynucleotides variants have at least or about 50%, 55%, 60%, 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence. In particular embodiments, protein variants have at least or about 50%, 55%, 60%, 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.

[0062] A “vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. [0063] The term “wild-type” or “WT” refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo in a normal or healthy subject.

Cardiac-specific Promoters, Expression Cassettes, Vectors, Virions, and Compositions

[0064] In some aspects, provided are cardiac-specific promoters for expressing transgenes, for example, in cardiac cells. The cardiac-specific promoters disclosed herein can be compact in size to accommodate large transgenes in vectors with a limited packaging capacity, for example, AAV vectors. The cardiac-specific promoters disclosed herein can be chimeric promoters or synthetic promoters. A “cardio-specific promoter”, as used herein, specifies a promoter whose activity in a cardiac cell type (such as cardiomyocyte) is at least 2-fold higher than in any other non-cardiac cell type. Preferably, a cardiac-specific promoter of the present disclosure has an activity in a cardiac cell type which is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher compared to its activity in a non-cardiac cell type. The transgene whose expression is driven by the cardiac-specific promoter may encode a therapeutic gene product, e.g., a wild-type, functional protein or a cardioprotective protein, for use to treat heart diseases, e.g., cardiomyopathy.

Cardiac-specific chimeric promoters

[0065] In some embodiments, the cardiac-specific promoter is a chimeric promoter. In some embodiments, the cardiac-specific chimeric promoter comprises a transcription factor (TF) binding rich region comprising one or more fragments derived from the promoter region of one or more cardiac-specific genes. A “cardio-specific gene” as used herein specifies a gene whose RNA or protein expression in a cardiac cell type is at least 2-fold higher than in any other non-cardiac cell type. Preferably, a cardiac-specific gene suitable for providing fragments for use in the chimeric promoters of the present technology has an RNA or protein expression level in a cardiac cell type (such as cardiomyocytes) which is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher compared to its activity in a non-cardiac cell type. Examples of cardiac-specific genes include, but are not limited to, cardiac troponin T (TNNT2), myosin heavy chain (e.g., MYH6 or MYH7), myosin binding protein C (MYBPC3), myosin light chain 2 (MYL2), myosin light chain 3 (MYL3), actin, alpha cardiac muscle 1 (ACTC1 ), tropomyosin 1 (TPM1 ), 5’-AMP-activated protein kinase subunit gamma-2 (PRKAG2), troponin I type 3 (TNNI3), titin (TTN), potassium voltagegated channel, KQT-like subfamily member 1 (KCNQ1 ), myocyte enhancer factor 2c (MEF2C), and cardiac LIM protein (CSRP3).

[0066] In some embodiments, the TF binding rich region is less than 600 base pairs, less than 550 base pairs, less than 500 base pairs, less than 450 base pairs, less than 400 base pairs, less than 350 base pairs, less than 300 base pairs, less than 250 base pairs, or less than 200 base pairs in length.

[0067] In some embodiments, the TF binding rich region comprises binding sites for one or more transcription factors that promote the expression of cardiac-specific genes or gene expression in cardiac-cells. In some embodiments, the TF binding rich region comprises one or more binding sites for at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different TFs. In some embodiments, the TFs are selected from GATA4, MEF2C, TBX5, NKS2.5, HAND2, and SRF. In some embodiments, the TF binding rich region comprises binding sites for GATA4, MEF2C, TBX5, NKS2.5, HAND2, and SRF. In some embodiments, the TF binding rich region comprises binding sites for GATA4, MEF2C, TBX5, NKS2.5, and HAND2.

[0068] In some embodiments, the TF binding rich region comprises more two or more (e.g., 2, 3, 4, 5, or more) fragments derived from the promoter regions of two or more (e.g., 2, 3, 4, 5, or more) cardiac-specific genes. Each fragment may be about 50 bp in length, and thus, for example, for a chimeric promoter comprising 5 such fragments, the total length of the promoter is around 250 bp. A chimeric promoter that is shorter in length may enable expression of transgenes of larger sizes using the vectors of the present disclosure. In some embodiments, each fragment is about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, or about 80 bp in length.

[0069] In some embodiments, the TF binding rich region comprises two or more (e.g., 2, 3, 4, 5, or more) fragments derived from the promoter regions of two or more (e.g., 2, 3, 4, 5, or more) cardiac-specific genes selected from TNNT2, MYBPC3, MYH6, MYH7, MYL2, and MYL3. In some embodiments, the two or more cardiac-specific genes comprise TNNT2 and MYBPC3. In some embodiments, the two or more cardiac-specific genes are TNNT2 and MYBPC3. Exemplary cardiac-specific chimeric promoters, including 6C2, and their sequences are provided in Table 1 below (also see FIG. 3).

Table 1. Exemplary cardiac-specific chimeric promoters

[0070] In some embodiments, the cardiac-specific chimeric promoter comprises or consists of a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1.

[0071] In some embodiments, the TF binding rich region of the cardiac-specific chimeric promoter further comprises and is modified to comprise a binding site for transcription factor SRF, which may further enhance expression of the transgene in cardiac cells. For example, the 6C2 promoter, which does not have an SRF binding site (see FIG. 4), may be further modified to include one by converting the 12 nucleotides sequence between positions 99-112 of SEQ ID NO: 1 to a SRF transcription factor binding site, resulting in the 6C2SRF promoter which has improved promoter strength (see FIGS. 5A-5B). In some embodiments, the cardiac-specific chimeric promoter comprises or consists of a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2.

[0072] In some embodiments, the cardiac-specific chimeric promoter is further modified by insertion of one or more polynucleotides. A modification may include one, two, three, or more internal insertions. Each insertion may be an insertion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to an exemplary chimeric promoter disclosed herein (e.g., 6C2 or 6C2SRF promoter). In some embodiments, the cardiac-specific chimeric promoter is modified by the insertion of polynucleotides from the downstream end of the chimeric promoter. In some embodiments, the cardiac-specific chimeric promoter is modified by the insertion of polynucleotides from the upstream end of the chimeric promoter.

[0073] In some embodiments, the cardiac-specific chimeric promoter is further modified by deletion of one or more polynucleotides. A modification may include one, two, three, or more internal deletions. Each deletion may be a deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to an exemplary chimeric promoter disclosed herein (e.g., 6C2 or 6C2SRF promoter). In some embodiments, the cardiac-specific chimeric promoter is modified by the deletion of polynucleotides from the downstream end of the chimeric promoter. In some embodiments, the cardiac-specific chimeric promoter is modified by the deletion of polynucleotides from the upstream end of the chimeric promoter.

[0074] In some embodiments, the cardiac-specific chimeric promoter is further modified by substitution of one or more polynucleotides. A modification may include one, two, three or more internal substitutions. Each substitution may include the substitution of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to an exemplary chimeric promoter disclosed herein (e.g., 6C2 or 6C2SRF promoter). In some embodiments, the cardiac-specific chimeric promoter is modified by the substitution of polynucleotides from the downstream end of the chimeric promoter. In some embodiments, the cardiac-specific chimeric promoter is modified by the substitution of polynucleotides from the upstream end of the chimeric promoter.

Cardiac-specific synthetic promoters

[0075] In some embodiments, the cardiac-specific promoter is a synthetic promoter. In some embodiments, the cardiac-specific synthetic promoter comprises (i) a TF binding rich region comprising one or more fragments derived from the promoter region of one or more cardiac-specific genes; and (ii) a core promoter comprising a TATA box and a transcription start site (TSS). A “core promoter” as used herein refers to a region within a promoter sequence that harbors an RNA polymerase complex binding site (e.g., canonically a TATAA sequence or other non-canonical sequences) and a TSS.

[0076] In some embodiments, the TF binding rich region is less than 600 base pairs, less than 550 base pairs, less than 500 base pairs, less than 450 base pairs, less than 400 base pairs, less than 350 base pairs, less than 300 base pairs, less than 250 base pairs, or less than 200 base pairs in length.

[0077] In some embodiments, the TF binding rich region comprises binding sites for one or more transcription factors that promote the expression of cardiac-specific genes or gene expression in cardiac-cells. In some embodiments, the TF binding rich region comprises one or more binding sites for at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different TFs. In some embodiments, the TFs are selected from GATA4, MEF2C, TBX5, NKS2.5, HAND2, and SRF. In some embodiments, the TF binding rich region comprises binding sites for GATA4, MEF2C, TBX5, and SRF.

[0078] In some embodiments, the TF binding rich region comprises two or more (e.g., 2, 3, 4, 5, or more) fragments derived from the promoter regions of two or more (e.g., 2, 3, 4, 5, or more) cardiac-specific genes selected from TNNT2, MYBPC3, MYH6, MYH7, MYL2, and MYL3. In some embodiments, the two or more cardiac-specific genes comprise TNNT2 and MYBPC3. In some embodiments, the two or more cardiac-specific genes are TNNT2 and MYBPC3. For example, the 6C2SRF promoter described above can be further reduced to the first 154 bp fragment at the 5’ end (see Table 1, SEQ ID NO: 3), which contains the engineered SRF binding site, that is then combined with a core promoter, e.g., a CMV core promoter, to generate a synthetic promoter for use in the present technology (see FIGS. 6A-6B). In some embodiments, the TF binding rich region comprises or consists of a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3.

[0079] In some embodiments, the TF binding rich region comprises one or more (e.g., 1 , 2, 3, 4, 5, or more) fragments derived the promoter region of a cardiac-specific gene selected from TNNT2, MYBPC3, MYH6, MYH7, MYL2, and MYL3. In some embodiments, the cardiac-specific gene is TNNT2, for example, human TNNT2, mouse TNNT2, or chicken TNNT2. In some embodiments, the cardiac-specific gene is human TNNT2. In certain of these embodiments, the TF binding rich region comprises a fragment of the promoter of a TNNT2 gene (e.g., human TNNT2, mouse TNNT2, or chicken TNNT2), which can also be referred to as a TNNT2 mini promoter. Exemplary TNNT2 promoters and fragments/mini promoters derived therefrom for use in the TF binding rich region of the present technology are provided in Table 2 below. The TSS of the wild-type TNNT2 promoters is bolded and underlined. In some embodiments, the TF binding rich region comprises or consists of a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8, 10, or 12.

Table 2. Exemplary TNNT2 promoters and fragments derived therefrom

[0080] In some embodiments, the core promoter is a CMV core promoter. Exemplary CMV core promoter sequences are provided in Table 3 below. In some embodiments, the CMV core promoter comprises or consists of a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13.

Table 3. Exemplary core promoters

[0081] In some embodiments, the cardiac-specific synthetic promoter comprises (i) a TF binding rich region as any described herein; and (ii) a core promoter as any described herein. Exemplary cardiac-specific synthetic promoters are provided in Table 4 below. The exemplary cardiac-specific synthetic promoters in Table 4 comprise a TF binding rich region derived from the promoter regions of one or more cardiac-specific genes as described, combined with a CMV core promoter of SEQ ID NO: 13. The CMV core promoter portion is underlined.

Table 4. Exemplary cardiac-specific synthetic promoters

[0082] In some embodiments, the cardiac-specific synthetic promoter comprises or consists of a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14. In some embodiments, the cardiac-specific synthetic promoter comprises or consists of a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15. In some embodiments, the cardiac-specific synthetic promoter comprises or consists of a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16. In some embodiments, the cardiac-specific synthetic promoter comprises or consists of a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17.

[0083] In some embodiments, the cardiac-specific synthetic promoter is further modified by insertion of one or more polynucleotides. A modification may include one, two, three, or more internal insertions. Each insertion may be an insertion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to an exemplary synthetic promoter disclosed herein. In some embodiments, the cardiac-specific synthetic promoter is modified by the insertion of polynucleotides from the downstream end of the synthetic promoter. In some embodiments, the cardiac-specific synthetic promoter is modified by the insertion of polynucleotides from the upstream end of the synthetic promoter.

[0084] In some embodiments, the cardiac-specific synthetic promoter is further modified by deletion of one or more polynucleotides. A modification may include one, two, three, or more internal deletions. Each deletion may be a deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to an exemplary synthetic promoter disclosed herein. In some embodiments, the cardiac-specific synthetic promoter is modified by the deletion of polynucleotides from the downstream end of the synthetic promoter. In some embodiments, the cardiac-specific synthetic promoter is modified by the deletion of polynucleotides from the upstream end of the synthetic promoter.

[0085] In some embodiments, the cardiac-specific synthetic promoter is further modified by substitution of one or more polynucleotides. A modification may include one, two, three or more internal substitutions. Each substitution may include the substitution of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to an exemplary synthetic promoter disclosed herein. In some embodiments, the cardiac-specific synthetic promoter is modified by the substitution of polynucleotides from the downstream end of the synthetic promoter. In some embodiments, the cardiac-specific synthetic promoter is modified by the substitution of polynucleotides from the upstream end of the synthetic promoter.

[0086] In some embodiments, the cardiac-specific promoter, including any chimeric or synthetic promoter disclosed herein, has an activity in cardiac cells (e.g., cardiomyocytes) that is at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher compared to its activity in a noncardiac cell type when assessed by gene expression in vitro (e.g., in cardiomyocytes or induced cardiomyocytes) or in vivo (e.g., in mice, monkeys, or human)..

[0087] In some embodiments, the cardiac-specific promoter, including any chimeric or synthetic promoter disclosed herein, has 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%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% activity compared to a reference promoter when assessed by gene expression in vitro (e.g., in cardiomyocytes or induced cardiomyocytes) or in vivo (e.g., in mice, monkeys, or human). The reference promoter may be a natural or a wild-type cardiac-specific promoter, for example, a TNNT2 promoter (e.g., a human TNNT2 promoter of SEQ ID NO: 6).

Expression cassettes and/or vectors

[0088] In some embodiments, the cardiac-specific promoter according to various embodiments disclosed herein is placed in an expression cassette and/or vector for use in delivering and/or expressing transgenes, for example, in cardiac cells. The cardiacspecific promoters disclosed herein, due to their compact size, would be especially useful for delivering and/or expressing large transgenes in vectors with a limited packaging capacity, for example, AAV vectors.

[0089] The expression cassettes and/or vectors contemplated herein may be combined with other sequences, such as promoters, enhancers, untranslated regions (UTRs), introns, signal sequences, Kozak sequences, polyadenylation (poly(A)) signals, post-transcriptional regulatory elements, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, polynucleotides encoding self-cleaving polypeptides, epitope tags, and/or any other regulatory elements as disclosed elsewhere herein or as known in the art. In some embodiments, the polynucleotides, expression cassettes, and/or vectors described herein may also contain a ribosome binding site for translation initiation, a transcription terminator, and/or polynucleotide sequences for amplifying expression. The expression cassette may be flanked by one or more inverted terminal repeats (ITRs). The ITRs in an expression cassette serve as markers used for viral packaging of the expression cassette. The expression cassette can be integrated into the host cell genome, thereby expressing the transgene within a host cell.

[0090] As used herein, the term “regulatory element” refers to those non-translated regions of the vector (e.g., origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Kozak sequence), introns, poly(A) sequences, 5' and 3' untranslated regions) which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. The transcriptional regulatory element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a polynucleotide sequence described herein is operably linked to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells.

Enhancers

[0091] The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and/or other enhancer elements. The term “enhancer” further refers to a DNA sequence that directs the binding of transcriptional regulatory proteins (e.g., transcriptional machinery) and RNA polymerase, and thereby promotes RNA synthesis. An enhancer may overlap with a promoter or be upstream or downstream of the promoter.

[0092] In some embodiments, the expression cassette and/or vector further comprises one or more enhancers. The one or more enhancers can be operably linked to the cardiac-specific promoter and modulate the expression of a transgene operably linked to the promoter. The presence of an enhancer can modulate transgene expression by, for example, increasing expression or decreasing expression. An enhancer can modulate transgene expression by, for example, increasing expression levels in a desired cell type, for example, a cardiac cell. An enhancer can modulate transgene expression by, for example, decreasing expression levels in an “off-target” cell type, or a cell type in which expression is not desired. For example, a ACTC1 cardiac enhancer can be linked to the cardiac-specific promoter. In some embodiments, the expression cassette and/or vector comprises an enhancer that is operably linked to another enhancer. For example, a ACTC1 cardiac enhancer can be operably linked to an aMHC enhancer. In some embodiments, the expression cassette and/or vector comprises an enhancer that is operably linked to a promoter and operably linked to another enhancer. Exemplary enhancers are provided in Table 5A below. Table 5A. Exemplary enhancer sequences

[0093] In some embodiments, the enhancer comprises an ACTC1 cardiac enhancer (ACTCIe). In some embodiments, the enhancer comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 18, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18.

[0094] In some embodiments, the enhancer comprises an aMHC enhancer (aMHCe). In some embodiments, the enhancer comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 19, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19.

Introns

[0095] In some embodiments, the expression cassette and/or vector further comprises one or more intron sequences, for example, a synthetic or chimeric intron sequence. The intron sequence can be used to adjust the length (/.e., size) of the expression cassette for improving recombinant AAV packaging. The intron sequence can also be used to improve the efficiency of transgene expression (/.e., mRNA production or transcription) in a host cell containing the expression cassette from the vector. Exemplary intron sequences are provided in Table 5B below. Table 5B. Exemplary intron sequences

[0096] In some embodiments, the intron comprises an CMV intron (CMVint). In some embodiments, the intron comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 20, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 20.

[0097] In some embodiments, the intron comprises a chimeric intron (Chimint). In some embodiments, the intron comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 21 , or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 21 .

WPRE sequences and other post-transcriptional elements

[0098] In some embodiments, the expression cassette and/or vector further comprises one or more post-transcriptional regulatory elements, for example, a woodchuck hepatitis virus post-transcriptional element (WPRE). The WPRE sequence can be inserted, for example, proximal to on the 3’ end of a transgene in a viral vector to, for example, optimize gene expression in a viral vector. In some embodiments, the WPRE comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 22, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 22.

Table 5C. Exemplary WPRE sequences

Poly(A) sequences

[0099] In some embodiments, the expression cassette and/or vector further comprises one or more poly(A) sequences. The term “poly(A) sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by addition of a poly(A) tail to the 3' end of the coding sequence and thus, contribute to increased translational efficiency. Cleavage and polyadenylation are directed by a poly(A) sequence in the RNA. The core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site. Typically, an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or Gil residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5’ cleavage product. In someembodiments, the core poly(A) sequence is an ideal poly(A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). Non-limiting examples of poly(A) sequences include SV40 poly(A) sequence, bovine growth hormone (BGH) poly(A) sequence, rabbit [3-globin poly(A) sequence (r|3gpA), variants thereof, and other suitable heterologous or endogenous poly(A) sequences known in the art. Exemplary poly(A) sequences are provided in Table 5D below.

Table 5D. Exemplary poly(A) sequences

[0100] In some embodiments, the poly(A) sequence comprises a synthetic poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 23, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 23.

[0101] In some embodiments, the poly(A) sequence comprises a BGH poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 24, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 24.

[0102] In some embodiments, the poly(A) sequence comprises a SV40 poly(A) sequence. In some embodiments, the poly(A) sequence comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 25, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 25.

Additional elements

[0103] In some embodiments, the expression cassette and/or vector further comprises one or more additional elements, for example, a Kozak sequence and a nuclear localization sequence (NLS). Exemplary additional regulatory sequences are provided in Table 5E below.

Table 5E. Exemplary additional regulatory sequences

[0104] In some embodiments, the expression cassette and/or vector comprises a Kozak sequence comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 26, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 26.

[0105] In some embodiments, the expression cassette and/or vector comprises a SV40 NLS comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 27, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 27.

[0106] In some embodiments, the expression cassette and/or vector comprises a nucleoplasmin NLS comprising, consisting of, or consisting essentially of a nucleotide sequence set forth in SEQ ID NO: 28, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 28.

[0107] In some embodiments, the expression cassette and/or vector further comprises a transcription termination signal. Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal.

ITRs

[0108] In some embodiments, the expression cassette is flanked by AAV inverted terminal repeats (ITRs) at the 5’ and 3’ ends. ITRs function as recognition sites for replication and markers used for viral packaging of the expression cassette. ITRs form T-shaped secondary structures by two adjacent inverted repeats separated by an unpaired nucleotide. ITRs are required for packaging the expression cassette into an rAAV virion, which provide the function of expressing the transgene after a host cell is targeted by the rAAV virion. The ITRs contain tetranucleotide repeat motifs called Repbinding elements (RBE) that act as contact points for the Rep68/78 proteins encoded by the rep gene. The ITRs also contain a packaging signal for genome encapsidation, which directs 3’ genomic transport into preassembled capsids by Rep proteins. Any naturally occurring or synthetically derived ITRs described herein or known in the art can be used. [0109] In some embodiments, the ITRs flanking the expression cassette are ITRs of the same AAV serotype as the Rep protein used in making the virions described herein. For example, where a Rep protein from AAV9 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV9 as well. In another example, where a Rep protein from AAV2 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV2 as well. In another example, where a Rep protein from AAV5 is used, the transgene expression cassette used in the expression system comprises ITRs from AAV5 as well. The ITRs may be of the same or different serotype as the capsid protein used in packaging the virion described herein.

[0110] In some embodiments, the ITRs comprise, consist of, or consist essentially of a nucleotide sequence set forth in SEQ ID NO: 29 or 30, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 29 or 30, as shown in Table 5F below.

Table 5F. Exemplary ITR sequences

[0111] In some embodiments, the expression cassette is flanked by one or both of a 5’ ITR and a 3’ ITR. In some embodiments, the 5’ ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 29, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 29. In some embodiments, the 3’ ITR comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 30, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 30.

Transgenes [0112] In some embodiments, the expression cassette and/or vector comprising the cardiac-specific promoter according to various embodiments disclosed herein further comprises one or more transgenes driven by the cardiac-specific promoter to be delivered to cells for expression of one or more gene products encoded by the transgenes. The transgenes and gene products described herein are non-limiting. Any transgene encoding any gene product may be used in association with the capsid=specific promoters described herein.

[0113] A transgene can be a gene or nucleotide sequence that encodes a product, or a functional fragment thereof. A product can be, for example, a polypeptide or a noncoding nucleotide. By non-coding nucleotide, it is meant that the sequence transcribed from the transgene or nucleotide sequence is not translated into a polypeptide. In some embodiments, the product encoded by the transgene or nucleotide operably linked to an enhancer described herein is a non-coding polynucleotide. A non-coding polynucleotide can be an RNA, such as for example a microRNA (miRNA or mIR), short hairpin RNA (shRNA), long non-coding RNA (InRNA), and/or a short interfering RNA (siRNA). In some embodiments, the transgene encodes a product natively expressed by a cardiac cell, e.g., a cardiomyocyte.

[0114] In some embodiments, the transgene comprises a nucleotide sequence encoding a human protein. In some embodiments, the transgene comprises a human nucleotide sequence (a human DNA sequence). In some embodiments, the transgene comprises a DNA sequence that has been codon-optimized. In some embodiments, the transgene comprises a nucleotide sequence encoding a wild-type protein, or a functionally active fragment thereof. In some embodiments, the transgene comprises a nucleotide sequence encoding a variant of a wild-type protein, such as a functionally active variant thereof.

[0115] In some embodiments, the transgene comprises a sequence encoding a product selected from vascular endothelial growth factor (VEGF), a VEGF isoform, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-DdNdC, VEGF-A116A, VEGF-A165, VEGF-A121 , VEGF-2, placenta growth factor (PIGF), fibroblast growth factor 4 (FGF-4), human growth factor (HGF), human granulocyte colony-stimulating factor (hGCSF), and hypoxia inducible factor 1a (HIF-1a). [0116] In some embodiments, the transgene comprises a sequence encoding a product selected from SERCA2a, stromal cell-derived factor-1 (SDF-1 ), adenylyl cyclase type 6, S100A1 , miRNA-17-92, miR-302-367, anti-miR-29a, anti-miR-30a, antimiR-141 , cyclin A2, cyclin-dependent kinase 2, Tbx20, miRNA-590, miRNA-199, anti-sense oligonucleotide against Lp(a), interfering RNA against PCSK9, anti-sense oligonucleotide against apolipoprotein C-lll, lipoprotein lipaseS447X, anti-sense oligonucleotide against apolipoprotein B, anti-sense oligonucleotide against c-myc, and E2F oligonucleotide decoy.

[0117] In some embodiments, the transgene encodes a gene product whose expression complements a defect in a gene responsible for a genetic disorder. In some embodiments, the disclosure provides, without limitation, polynucleotides encoding one or more of the following — e.g., for use, without limitation, in the disorder indicated in parentheses, or for other disorders caused by each: TAZ (Barth syndrome); FXN (Freidrich’s Ataxia); CASQ2 (CPVT); FBN1 (Marfan); RAF1 and SOS1 s (Noonan); SCN5A (Brugada); KCNQ1 and KCNH2s (Long QT Syndrome); DMPK (Myotonic Dystrophy 1 ); LMNA (Limb Girdle Dystrophy Type 1 B); JUP (Naxos); TGFBR2 (Loeys- Dietz); EMD (X-Linked EDMD); and ELN (SV Aortic Stenosis). In some embodiments, a polynucleotide encodes one or more of: cardiac troponin T (TNNT2); BAG family molecular chaperone regulator 3 (BAG3); myosin heavy chain (MYH7); tropomyosin 1 (TPM1 ); myosin binding protein C (MYBPC3); 5’-AMP-activated protein kinase subunit gamma-2 (PRKAG2); troponin I type 3 (TNNI3); titin (TTN); myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1 ); potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1 ); myocyte enhancer factor 2c (MEF2C); and cardiac LIM protein (CSRP3).

[0118] In some embodiments, the transgene comprises a nucleotide sequence encoding a protein selected from DWORF, junctophilin (e.g., JPH2), BAG family molecular chaperone regulator s (BAG3), phospholamban (PLN), alpha-crystallin B chain (CRYAB), LMNA (such as Lamin A and Lamin C isoforms), troponin I type 3 (TNNI3), lysosomal-associated membrane protein 2 (LAMP2, such as LAMP2a, LAMP2b and LAMP2c isoforms), desmoplakin (DSP, such as DPI and DPII isoforms), desmoglein 2 (DSG2), junction plakoglobin (JUP), and plakophilin-2 (PKP2). In some embodiments, the transgene comprises a nucleotide sequence encoding a matrix metallopeptidase 11 (MMP11 ) protein, a synaptopodin 2 like (SYNPO2L) protein (e.g., SYNPO2LA or SYNP02LA), or an RNA binding motif protein 20 (RBM20). In some embodiments, the transgene comprises a nucleotide sequence encoding an inhibitory oligonucleotide targeting metastasis suppressor protein 1 (MTSS1 ).

[0119] In some embodiments, the transgene comprises a nucleotide sequence encoding a protein selected from DWORF, JPH2, BAG3, CRYAB, LMNA (e.g., Lamin A isoform of LMNA, or Lamin C isoform of LMNA), TNNI3, PLN, LAMP2 (e.g., LAMP2a, LAMP2b, or LAMP2c), DSP (e.g., DPI isoform of DSP or DPII isoform of DSP), DSG2 and JUP.

[0120] In some embodiments, the transgene comprises a polynucleotide sequence encoding a MYBPC3 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human MYBPC3 polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding MYBPC3, e.g., human MYBPC3. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 31 , or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the MYBPC3 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 32.

[0121] In some embodiments, the transgene comprises a polynucleotide sequence encoding a MYBPC3-delC3 variant polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding MYBPC3-delC3. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 33, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 33. In some embodiments, the MYBPC3-delC3 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 34. [0122] In some embodiments, the transgene comprises a polynucleotide sequence encoding a MYBPC3-delC4 variant polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding MYBPC3-delC4. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 35, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 35. In some embodiments, the MYBPC3-delC4 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 36.

[0123] In some embodiments, the transgene comprises a polynucleotide sequence encoding a MYBPC3-delC4b variant polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding MYBPC3-delC4b. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 37, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 37. In some embodiments, the MYBPC3-delC4b polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 38, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 38.

[0124] In some embodiments, the transgene comprises a polynucleotide sequence encoding a DWORF polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human DWORF polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding DWORF, e.g., human DWORF. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 39 or 40, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 39 or 40. In some embodiments, the DWORF polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 41 , or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to

SEQ ID NO: 41.

[0125] In some embodiments, the transgene comprises a polynucleotide sequence encoding a junctophilin 2 (JPH2) polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a full-length JPH2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human JPH2 polypeptide. In some embodiments, the polynucleotide sequence is a codon- optimized sequence encoding JPH2, e.g., human JPH2. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 42, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 42. In some embodiments, the JPH2 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 43, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 43.

[0126] In some embodiments, the transgene comprises a polynucleotide sequence encoding an N-terminal fragment of the JPH2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding an N-terminal fragment of the JPH2 polypeptide, which retains the JPH2 activity. In some embodiments, athe polynucleotide sequence is a codon-optimized sequence encoding N-terminal fragment of JPH2. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 44, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 44. In some embodiments, the JPH2 N-terminal fragment comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 45, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 45.

[0127] In some embodiments, the transgene comprises a polynucleotide sequence encoding a BAG3 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human BAG3 polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding BAG3, e.g., human BAG3. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 46, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 46. In some embodiments, the BAG3 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 47, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 47.

[0128] In some embodiments, the transgene comprises a polynucleotide sequence encoding a C151 R mutant form of BAG3 polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding a C151 R mutant form of BAG3 polypeptide. In some embodiments, the BAG3 C151 R polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO:

48, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 48.

[0129] In some embodiments, the transgene comprises a polynucleotide sequence encoding a CRYAB polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human CRYAB polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding CRYAB, e.g., human CRYAB. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 49, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:

49. In some embodiments, the CRYAB polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 50, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 50.

[0130] In some embodiments, the transgene comprises a polynucleotide sequence encoding a LMNA polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human LMNA polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding the LaminA isoform of LMNA. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding LaminA isoform of LMNA, e.g., human. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 51 , or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 51. In some embodiments, the LaminA isoform of LMNA polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 52, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 52.

[0131] In some embodiments, the transgene comprises a polynucleotide sequence encoding the LaminC isoform of LMNA. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding LaminC isoform of LMNA, e.g., human. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 53, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 53. In some embodiments, the LaminC isoform of LMNA polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 54.

[0132] In some embodiments, the transgene comprises a polynucleotide sequence encoding a TNNI3 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human TNNI3 polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding TNNI3, e.g., human TNNI3. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 55, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 55. In some embodiments, the TNNI3 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 56, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 56.

[0133] In some embodiments, the transgene comprises a polynucleotide sequence encoding a PLN polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human PLN polypeptide. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding PLN, e.g., human PLN. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 57, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 57. In some embodiments, the PLN polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 58, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 58. In some embodiments, the transgene comprises a polynucleotide sequence encoding a guide RNA targeting a mutant PLN gene (such as a deletious mutant of PLN, e.g., PLN-R14Del).

[0134] In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human LAMP2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding the LAMP2a isoform. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding LAMP2a, e.g., human LAMP2a. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 59, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 59. In some embodiments, the LAMP2a polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 60, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 60.

[0135] In some embodiments, the transgene comprises a polynucleotide sequence encoding the LAMP2b isoform. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding LAMP2b, e.g., human LAMP2b. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 61 , or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 61. In some embodiments, the LAMP2b polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 62, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 62.

[0136] In some embodiments, the transgene comprises a polynucleotide sequence encoding the LAMP2c isoform. In some embodiments, the polynucleotide sequence is a codon-optimized sequence encoding LAMP2c, e.g., human LAMP2c. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 63, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 63. In some embodiments, the LAMP2c polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 64, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 64.

[0137] In some embodiments, the transgene comprises a polynucleotide sequence encoding a DSP polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human DSP polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding the DPI isoform of DSP. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding DPI isoform of DSP, e.g., human. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 65, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 65. In some embodiments, the DPI isoform of DSP polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 66, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 66.

[0138] In some embodiments, the transgene comprises a polynucleotide sequence encoding the DPII isoform of DSP. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding DPII isoform of DSP, e.g., human. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 67, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 67. In some embodiments, the DPII isoform of DSP polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 68, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 68.

[0139] In some embodiments, the transgene comprises a polynucleotide sequence encoding a DSG2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human DSG2 polypeptide. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding DSG2, e.g., human DSG2. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 69, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 69. In some embodiments, the DSG2 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 70, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 70.

[0140] In some embodiments, the transgene comprises a polynucleotide sequence encoding a JUP polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human JUP polypeptide. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding JUP, e.g., human JUP. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 71 , or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 71. In some embodiments, the JUP polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 72.

[0141] In some embodiments, the transgene comprises a polynucleotide sequence encoding MMP11. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human MMP11 polypeptide. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding MMP11 , e.g., human MMP11. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 73, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 73. In some embodiments, the MMP11 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 74, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 74.

[0142] In some embodiments, the transgene comprises a polynucleotide sequence encoding SYNPO2L (e.g., SYNPO2LA or SYNPO2LB). In some embodiments, the transgene comprises a polynucleotide sequence encoding a human SYNPO2L (e.g., SYNPO2LA or SYNPO2LB). In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding SYNPO2LA, e.g., human SYNPO2LA. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 75, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 75. In some embodiments, the SYNPO2LA polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 76. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding SYNPO2LB, e.g., human SYNPO2LB. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 77, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 77. In some embodiments, the SYNPO2LB polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 78, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 78.

[0143] In some embodiments, the transgene comprises a polynucleotide sequence encoding an inhibitory oligonucleotide (e.g., siRNA) targeting MTSS1. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 79, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 79.

[0144] In some embodiments, the transgene comprises a polynucleotide sequence encoding a Cas nuclease, for example, a Cas9 nuclease (e.g., saCas9). In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding saCas9. In some embodiments, the transgene comprises, consists of, or consists essentially of a nucleotide sequence set forth in SEQ ID NO: 80, or a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 80. In some embodiments, the saCas9 polypeptide comprises, consists of, or consists essentially of an amino acid sequence set forth in SEQ ID NO: 81 , or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 81 .

[0145] Exemplary polynucleotide and amino acid sequences of the transgenes and gene products as described are provided in Table 6 below.

Table 6. Exemplary transgenes and gene products

[0146] In some embodiments, the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from MYBPC3, KCNH2, TRPM4, DSG2, ATP2A2, CACNA1 C, DMD, DMPK, EPG5, EVC, EVC2, FBN1 , NF1 , SCN5A, SOS1 , NPR1 , ERBB4, VIP, MYH6, MYH7, or a mutant, variant, or fragment thereof. In some embodiments, the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from TGFBR2, TGFBR1 , EMD, KCNQ1 , TAZ, COL3A1 , JUP, CASQ2, MLRP44, DNAJC19, LMNA, TNNI3, DSP, DSG2, RAF1 , SOS1 , FBN1 , LAMP2, FXN, RAF1 , BAG3, KCNQ1 , MYLK3, CRYAB, ALPK3, and ACTN2. In some embodiments the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from MYBPC3, DWORF, JPH2, BAG3, CRYAB, Lamin A isoform of LMNA, Lamin C isoform of LMNA, TNNI3, PLN, LAMP2a, LAMP2b, LAMP2c, DPI isoform of DSP, DPII isoform of DSP, DSG2, MYH6, MYH7, RBM20, and JUP.

[0147] In some embodiments, the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from ASCL1 , MYOCD, MEF2C, and TBX5. In some embodiments, the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from ASCL1 , MYOCD, MEF2C, AND TBX5, CCNB1 , CCND1 , CDK1 , CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1 , MESP2, NKX2.5, SRF, TBX20, ZFPM2, and MIR- 133.

[0148] In some embodiments, the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from MYBPC3, DWORF, KCNH2, TRPM4, DSG2, and ATP2A2.

[0149] In some embodiments, the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from TGFBR2, TGFBR1 , EMD, KCNQ1 , TAZ, COL3A1 , JUP, CASQ2, MLRP44, DNAJC19, LMNA, TNNI3, DSP, DSG2, RAF1 , SOS1 , FBN1 , LAMP2, FXN, RAF1 , BAG3, KCNQ1 , MYLK3, CRYAB, ALP K3, and ACTN2.

[0150] In some embodiments, the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from CACNA1 C, DMD, DMPK, EPG5, EVC, EVC2, FBN1 , NF1 , SCN5A, SOS1 , NPR1 , ERBB4, VIP, MYH6, MYH7, and Cas9. In some embodiments, the transgene comprises a polynucleotide sequence that encodes saCas9.

[0151] In some embodiments, the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from MYOCD, ASCL1 , GATA4, MEF2C, TBX5, miR-133, and MESP1.

[0152] In some embodiments, the transgene comprises a polynucleotide sequence that encodes one or more gene products selected from MMP11 , SYNPO2L (e.g., SYNPO2LA or SYNPO2LA), and an inhibitory oligonucleotide targeting MTSS1 .

[0153] In some embodiments, the transgene comprises a polynucleotide sequence that encodes any of the above-identified gene products.

Vectors

[0154] In some embodiments, the cardiac-specific promoter, one or more transgenes, and/or one or more regulatory elements according to various embodiments disclosed herein are in the form of a vector. The vector can be any viral vector or non- viral vector known in the art or described herein. In some embodiments, the vector is a viral vector. In some embodiments the viral vector is an adeno-associated virus vector (AAV), an adenoviral vector, a lenti viral vector, a retroviral vector, a herpes simplex virus vector (HSV), or a poxvirus vector.

[0155] As used herein, the term “retrovirus” or “retroviral” refers an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Retrovirus vectors are a common tool for gene delivery. Once the virus is integrated into the host genome, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoded by the virus. In some embodiments, a retroviral vector is altered so that it does not integrate into the host cell genome. Illustrative retroviruses include, but are not limited to, (1 ) genus gammaretrovirus, such as, Moloney murine leukemia virus (M-MuLV or MMLV), Moloney murine sarcoma virus (MoMSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), and feline leukemia virus (FLV); (2) genus spumavirus, such as, simian foamy virus; and (3) genus lentivirus, such as, human immunodeficiency virus-1 and simian immunodeficiency virus. [0156] As used herein, the term “ lentiviral” or “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include but are not limited to, human immunodeficiency virus (HIV), including HIV type 1 , and HIV type 2; visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0157] In some embodiments, the viral vector is an adenoviral vector. The genetic organization of adenovirus includes an approximate 36 kb, linear, double-stranded DNA virus, which allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb.

[0158] In some embodiments, the viral vector is an AW vector, such as an AAV vector selected from the group consisting of serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, rh.10, rh.20, rh.74, and a variant or chimeric AAV derived thereof. In some embodiments, the AAV expression vector is pseudotyped to enhance targeting. A pseudotyping strategy can promote gene transfer and sustain expression in a target cell type. For example, the AAV2 genome can be packaged into the capsid of another AAV serotype such as AAV5, AAV7, or AAV8, producing pseudotyped vectors such as AAV2/5, AAV2/7, and AAV2/8 respectively, as described in Balaji et al., J. Surg. Res. Sep. (2013) 184(1 ):691 -698. In some embodiments, an AAV9 may be used to target expression in myofibroblast-like lineages, as described in Piras et al., Gene Therapy (2016) 23:469- 478. In some embodiments, AAV1 , AAV6, or AAV9 is used, and in some embodiments, the AAV is engineered, as described in Asokari et al., Hum. Gene Then Nov. (2013) 24(11 ):906-913; Pozsgai et al., Mol. Then (2017) 25(4): 855-869; Kotterman, M.A. and D.V. Schaffer, Nature Reviews Genetics (2014) 15:445-451 ; and US20160340393A1 to Schaffer et al. In some embodiments, the viral vector is AAV engineered to increase target cell infectivity as described in US20180066285A1 . In some embodiments, the vector is an AAV9 vector.

[0159] In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a naked DNA (e.g., a DNA plasmid). In some embodiments, the non-viral vector is a plasmid. In some embodiments, the non-viral vector is a liposome or lipid vector comprising plasmid DNA and a lipid solution.

[0160] In some embodiments, the vector is a recombinant vector. [0161] In some aspects of the disclosure, a vector is used to deliver the expression cassette described herein to cardiac cells of a subject, e.g., to treat a heart disease, e.g., cardiomyopathy.

[0162] In some embodiments, the viral vectors described herein are replication incompetent, in that it cannot independently further replicate and package its genome. For example, when a cardiac cell is targeted with a virion, the transgene is expressed in the targeted cardiac cell, however, since the targeted cardiac cell lacks packaging and accessory function genes, the virion is not able to replicate. In some embodiments, the viral vectors described herein are replication competent.

[0163] In some embodiments, the vectors described herein are capable of being delivered to both dividing and non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to dividing cells.

[0164] In some embodiments, the vectors comprising the expression cassettes described herein lead to cardiac cell-specific expression of the coding sequence(s). In some embodiments, the vectors comprising the expression cassettes described herein lead to cardiomyocyte-specific expression of the coding sequence(s). In some embodiments, the vectors comprising the expression cassettes described herein allow high expression of the coding sequence(s) in a cardiac cell (e.g., a cardiomyocyte) and low or no expression in other cells (e.g., low or no expression in liver cells, low or no expression in muscle cells except for muscle cells of the heart, low or no expression in cardiac fibroblasts). In some embodiments, the vectors comprising the expression cassettes described herein allow high expression of the coding sequence(s) in heart tissue of a subject (e.g., in human heart). In some embodiments, the vectors comprising the expression cassettes described herein allow no or low expression of the coding sequence(s) in tissues of a subject other than the heart (e.g., in liver or in muscles except those of the heart). “High” and “low” can be relative to each other, for example, the expression of a transgene in cardiac cells (e.g., cardiomyocytes) and/or heart tissue can be at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 150-fold, or 200-fold higher than its expression in other cells and tissues (e.g., liver, muscle except for the heart). [0165] In some embodiments, the vector genome has a size of less than 6 kilobases. In some embodiments, the vector genome has a size of less than 5.6 kilobases. In some embodiments, the vector genome has a size of about, at most or less than 4.0 kilobases, 4.5 kilobases, 4.6 kilobases, 4.7 kilobases, 4.8 kilobases, 4.9 kilobases, 5 kilobases, 5.1 kilobases, 5.2 kilobases, 5.3 kilobases, 5.4 kilobases, or 5.5 kilobases. In some embodiments, the vector genome has a size of 4 kilobases to 5.2 kilobases. In some embodiments, the vector genome has a size of 4 kilobases to 5 kilobases. In some embodiments, the vector genome has a size of 4 kilobases to 4.8 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.9 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.8 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.7 kilobases. In some of these embodiments, the vector is an AAV vector, e.g., an AAV9 vector.

[0166] In some of these embodiments, the vector is an AAV vector or a variant thereof. In some of these embodiments, the vector is an AAV9 vector or a variant thereof. In some of these embodiments, the vector is an AAV5 vector or a variant thereof. In some of these embodiments, the vector is an AAV2 vector or a variant thereof.

[0167] The capsid proteins of AAV largely determine the immunogenicity and tropism of AAV vectors. In some embodiments, the AAV is an AAV subtype 9 (AAV9). In some embodiments, AAV9 is a preferred AAV vector due to its ability to transduce the heart following systemic delivery. While AAV9 can achieve moderate transduction of the heart, the majority of vector traffics to the liver. Moreover, in order to achieve therapeutic levels of transduction in the heart, relatively high systemic doses are required, potentially leading to systemic inflammation and in turn, toxicity.

[0168] Methods of introducing polynucleotides into a host cell are known in the art, and any known method can be used to introduce the polynucleotides described herein into a cell. Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, microfluidics delivery methods, and the like. Recombinant AA V virions

[0169] In some embodiments, the cardiac-specific promoter, one or more transgenes, and/or one or more regulatory elements described herein are in the form of an AAV vector or a recombinant AAV (rAAV) virus or virion, for example, to deliver the expression cassettes described herein to cardiac cells.

[0170] In some embodiments, the AAV is any AAV known in the art or described herein. In some embodiments, the AAV is an AAV selected from the group consisting of serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, rh.1O, rh.20, rh.74, or a chimeric or variant AAV derived therefrom. In some embodiments, the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAVrh.10, AAVrh.20, AAVrh.74, or a variant thereof.

[0171] In some embodiments, the rAAV virus or virion comprises an AAV capsid protein and an expression cassette as described herein. Capsid proteins are structural proteins that make up the assembled icosahedral packaging of the rAAV virion that contains the expression cassette. Capsid proteins are classified by the serotype. Wildtype capsid serotypes in rAAV virions can be, for example, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAVrh.10, AAVrh.20, or AAVrh.74. Engineered capsid types include chimeric capsids and mosaic capsids. Capsids are selected for rAAV virions based on their ability to transduce specific tissue or cell types.

[0172] Any capsid protein that can facilitate rAAV virion transduction into cardiac cells for delivery of a transgene, as described herein, can be used. Capsid proteins used in rAAV virions for transgene delivery to cardiac cells that result in high expression can be, for example, AAV4, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the AAV capsid protein described herein is a wild-type AAV capsid protein from AAV serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, rh.1O, rh.20, rh.74, or a variant thereof. In some embodiments, the AAV is AAV9 or a variant thereof. In some embodiments, the AAV is AAV5 or a variant thereof. In some embodiments, the AAV is AAV2 or a variant thereof.

[0173] Artificial capsids, such as chimeric capsids generated through combinatorial libraries, can also be used for transgene delivery to cardiac cells that results in high expression. Other capsid proteins with various features can also be used in the rAAV virions of the disclosure. AAV vectors and capsids are provided in U.S. Pat. Pub. Nos. US10011640B2; US7892809B2, US8632764B2, US8889641 B2, US9475845B2, US10889833B2, US10480011 B2, and US10894949B2, the entire contents of each of which are incorporated by reference herein; and Int’l Pat. Pub. Nos. WO2020198737A1 , W02019028306A2, WO2016054554A1 , WO2018152333A1 , WQ2017106236A1 , WQ2008124724A1 , W02017212019A1 , W02020117898A1 , WQ2017192750A1 , W02020191300A1 , and W02017100671A1 , the entire contents of each of which are incorporated by reference herein.

[0174] In some embodiments, the rAAV virus or virion comprises a wild-type AAV9 capsid protein or a variant thereof. Wild-type AAV9 VP1 has the amino acid sequence of SEQ ID NO: 82; wild-type AAV9 VP2 has the amino acid sequence of SEQ ID NO: 83; wild-type AAV9 VP3 has the amino acid sequence of SEQ ID NO: 84, as shown below and provided in Table 7A. The N-terminal residue of VP1 , VP2, and VP3, the VR sites (VR-I, VR-II, VR-IV, VR-V, VR-VII, and VR-VIII), as well as the last 35 amino acid positions, are indicated (in bold, and underlined) in the sequence of full-length VP1 (SEQ ID NO: 82). In some embodiments, the capsid protein comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 82-84.

VPl — > ( SEQ ID NO : 82 )

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEP VNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEP VP2 — > ( SEQ ID NO : 83 )

LGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPP VPS — > ( SEQ ID NO : 84 )

AAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHL YKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNI QVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLND GSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALN GRNSLMNPGPAMASHKEGEDRFFPLSGSLI FGKQGTGRDNVDADKVMITNEEEIKTTNPVATES YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGM KHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSN

YYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL [0175] As labeled in AAV9 VP1 (SEQ ID NO: 82) above, the VR-I site is between amino acids 262 and 269 in the parental sequence (“NSTSGGSS”, SEQ ID NO: 155); the VR-I I site is between amino acids 327 and 332 in the parental sequence (“DNNGVK”, SEQ ID NO: 156); the VR-IV site is between amino acids 452 and 458 in the parental sequence (“NGSGQNQ”, SEQ ID NO: 85); the VR-V site is between amino acids 497 and 502 in the parental sequence (“NNSEFA”, SEQ ID NO: 86); the VR-VII site is between amino acids 549 and 553 in the parental sequence (“GRDNV”, SEQ ID NO: 87); the VR-VII I site is between amino acids 581 and 594 in the parental sequence (“ATNHQSAQAQAQTG”, SEQ ID NO: 88); the last 35 amino acid positions are between amino acids 702 to 736 in the parental sequence (“TSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL”, SEQ ID NO: 157.

[0176] In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 82, excluding the VR-I, VR-I I, VR-IV, VR-V, VR-VII, VR- VIII, and/or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 82, excluding the VR-IV and/or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 82, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 82, excluding the VR-VIII site.

[0177] In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 83, excluding the VR-I, VR-I I, VR-IV, VR-V, VR-VII, VR- VIII site, and/or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 83, excluding the VR-IV and/or VR-VIII site. In some embodiments, the engineered AAV9 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 83, excluding the VR-II site and/or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 83, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 83, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 83, excluding the VR-II site and/or the last 35 amino acid positions.

[0178] In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 84, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR- VIII, and/or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 84, excluding the VR-IV and/or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 84, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 84, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 83, excluding the VR-II site and/or the last 35 amino acid positions.

[0179] In some embodiments, the rAAV virus or virion comprises a wild-type AAV5 capsid protein or a variant thereof. Wild-type AAV5 VP1 has the amino acid sequence of SEQ ID NO: 89; wild-type AAV5 VP2 has the amino acid sequence of SEQ ID NO: 90; wild-type AAV5 VP3 has the amino acid sequence of SEQ ID NO: 91 , as shown below and provided in Table 7A. The N-terminal residue of VP1 , VP2, and VP3 VR sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, as well as the last 35 amino acid positions), are indicated (in bold, and underlined) in the sequence of full-length VP1 (SEQ ID NO: 89). In some embodiments, the capsid protein comprises a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to any one of SEQ ID NOs: 89-91 .

VP1 — > ( SEQ ID NO : 89 )

MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPV NRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPF VP2 — > ( SEQ ID NO : 90 )

GLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADT VPS — > ( SEQ ID NO : 91 ) MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRWTKSTRTWVLPSYNNHQYREIKSGSVD GSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKI FNIQVKEVTVQDST TTIANNLTSTVQVFTDDDYQLPYWGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSS FFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQ FNKNLAGRYANTYKNW FPGPMGRT QGWNLGSGVNRASVSAFAT TNRME LE GAS YQVP PQPNGMT NNLQGSNTYALENTMI FNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSS TTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKN TPVPGNI TS FSDVPVS S FI TQYS TGQVTVEMEWELKKENSKRWNPE I QYTNNYNDPQFVDFAPD STGEYRTTRPIGTRYLTRPL

[0180] As labeled in AAV5 VP1 (SEQ ID NO: 89) above, the VR-I site is between amino acids 252 and 256 in the parental sequence (“SGSVD”, SEQ ID NO: 158); the VR- II site is between amino acids 316 and 321 in the parental sequence (“VQDSTT”, SEQ ID NO: 159); the VR-IV site is between amino acids 437 and 461 in the parental sequence (“RFVSTNNTGGVQFNKNLAGRYANTY”, SEQ ID NO: 92); the VR-V site is between amino acids 477 and 490 in the parental sequence (“LGSGVNRASVSAFA”, SEQ ID NO: 93); the VR-VII site is between amino acids 533 and 546 in the parental sequence (“PANPGTTATYLEGN”, SEQ ID NO: 94); the VR-VIII site is between amino acids 570 and 584 in the parental sequence (“ATNNQSSTTAPATGT”, SEQ ID NO: 95) ; the last 35 amino acid positions are between amino acids 690 and 724 in the parental sequence (“TNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL”, SEQ ID NO: 160).

[0181] In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 89, excluding the VR-I, VR-I I , VR-IV, VR-V, VR-VII, VR- VIII, and/or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 89, excluding the VR-IV and/or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 89, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 89, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 89, excluding the VR-II site and/or the last 35 amino acid positions.

[0182] In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 90, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR- VIII, and/or the last 35 amino acid position. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 90, excluding the VR-IV and/or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 90, excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 90, excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 90, excluding the VR-II site and/or the last 35 amino acid positions.

[0183] In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 91 , excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR- VIII, and/or the last 35 amino acid positions. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 91 , excluding the VR-IV and/or VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 91 , excluding the VR-IV site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 91 , excluding the VR-VIII site. In some embodiments, the capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) identity to SEQ ID NO: 91 , excluding the VR-II site and/or the last 35 amino acid positions.

Table 7A. Exemplary wild-type AAV capsid protein sequences

[0184] In some embodiments, the engineered capsid protein is an engineered AAVrh.10 capsid protein comprising one or more amino acid substitutions and/or insertions compared to the wild-type AAVrh.10 capsid protein described herein. In some embodiments, the engineered capsid protein is an engineered AAVrh.10 capsid protein comprising an insertion peptide sequence or insertion motif compared to the wild-type AAVrh.10 capsid protein.

[0185] The wild-type AAVrh.10 VP1 has the amino acid sequence of SEQ ID NO: 161 ; the wild-type AAVrh.10 VP2 has the amino acid sequence of SEQ ID NO: 162; the wild-type AAVrh.10 VP3 has the amino acid sequence of SEQ ID NO: 163, as shown below and provided in Table 7 A. The N-terminal residue of VP1 , VP2, and VP3, the variable region (VR) sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII and VR-VIII), as well as the last 35 amino acid positions, are indicated in bold and underlined in the sequence of full-length VP1 (SEQ ID NO: 161 ). In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 161. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 162. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 163.

VPl — > ( SEQ ID NO : 161 )

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEP VNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEP

VP2 — > ( SEQ ID NO : 162 )

LGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEP VPS — > ( SEQ ID NO : 163 )

PAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNH LYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFN IQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLN NGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYQFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYL SRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYH LNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVAT EQYGWADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT SNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNL

[0186] As labeled in AAVrh.10 VP1 (SEQ ID NO: 161 ) above, the VR-I site is between amino acids 263 and 267 in the parental sequence (“NGTSG”, SEQ ID NO: 164); the VR-II site is between amino acids 328 and 333 in the parental sequence (“QNEGTK”, SEQ ID NO: 165); the VR-IV site is between amino acids 449 and 464 in the parental sequence (“SRTQSTGGTAGTQQLL”, SEQ ID NO: 166); the VR-V site is between amino acids 493 and 506 in the parental sequence (“TTLSQNNNSNFAWT”, SEQ ID NO: 167); the VR-VII site is between amino acids 549 and 559 in the parental sequence (“GAGKDNVDYSS”, SEQ ID NO: 168); the VR-VIII site is between amino acids 583 and 597 in the parental sequence (“ADNLQQQNAAPIVGA”, SEQ ID NO: 169); the last 35 amino acid positions are between amino acids 704 and 738 in the parental sequence (“TSNYYKSTNVDFAVNTDGTYSEPRPIGTRYLTRNL”, SEQ ID NO: 170).

[0187] In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 161 , excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and/or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 161 , excluding the VR-IV and/or VR-VIII site. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 161 , excluding the VR-II site and/or the last 35 amino acid positions.

[0188] In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 162, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and/or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 162, excluding the VR-VI and/or VR-VIII site. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 162, excluding the VR-II site and/or the last 35 amino acid positions.

[0189] In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 163, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and/or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 163, excluding the VR-IV and/or VR-VIII site. In some embodiments, the engineered AAVrh.10 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 163, excluding the VR-II site and/or the last 35 amino acid positions.

[0190] In some embodiments, the engineered capsid protein is an engineered AAVrh.74 capsid protein comprising one or more amino acid substitutions and/or insertions compared to the wild-type AAVrh.74 capsid protein described herein. In some embodiments, the engineered capsid protein is an engineered AAVrh.74 capsid protein comprising an insertion peptide sequence or insertion motif compared to the wild-type AAVrh.74 capsid protein. The wild-type AAVrh.74 VP1 has the amino acid sequence of SEQ ID NO: 171 ; the wild-type AAVrh.74 VP2 has the amino acid sequence of SEQ ID NO: 172; the wild-type AAVrh.74 VP3 has the amino acid sequence of SEQ ID NO: 173, as shown below and provided in Table 7A. The N-terminal residue of VP1 , VP2, and VP3, the variable region (VR) sites (e.g., VR-I, VR-II, VR-IV, VR-V, VR-VII and VR-VIII), as well as the last 35 amino acid positions, are indicated in bold and underlined in the sequence of full-length VP1 (SEQ ID NO: 171 ). In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 171 . In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 172. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 173.

VPl — > ( SEQ ID NO : 171 )

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEP VNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEP

VP2 — > ( SEQ ID NO : 172 )

LGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEP VPS — > ( SEQ ID NO : 173 )

PAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNH LYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFN IQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLN NGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYL SRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYH LNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVAT EQYGWADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT SNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0191] As labeled in AAVrh.74 VP1 (SEQ ID NO: 171 ) above, the VR-I site is between amino acids 263 and 267 in the parental sequence (“NGTSG”, SEQ ID NO: 164); the VR-II site is between amino acids 328 and 333 in the parental sequence (“QNEGTK”, SEQ ID NO: 165); the VR-IV site is between amino acids 449 and 464 in the parental sequence (“SRTQSTGGTAGTQQLL”, SEQ ID NO: 166); the VR-V site is between amino acids 493 and 506 in the parental sequence (“TTLSQNNNSNFAWT”, SEQ ID NO: 167); the VR-VII site is between amino acids 549 and 559 in the parental sequence (“GAGKDNVDYSS”, SEQ ID NO: 168); the VR-VIII site is between amino acids 583 and 597 in the parental sequence (“ADNLQQQNAAPIVGA”, SEQ ID NO: 169); the last 35 amino acid positions are between amino acids 704 and 738 in the parental sequence (“TSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL”, SEQ ID NO: 174).

[0192] In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 171 , excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and/or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 172, excluding the VR-IV and/or VR-VIII site. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 173, excluding the VR-II site and/or the last 35 amino acid positions.

[0193] In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 172, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and/or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 172, excluding the VR-IV and/or VR-VIII site. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 172, excluding the VR-II and/or the last 35 amino acid positions.

[0194] In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 173, excluding the VR-I, VR-II, VR-IV, VR-V, VR-VII, VR-VIII, and/or the last 35 amino acid positions. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 173, excluding the VR-IV and/or VR-VIII site. In some embodiments, the engineered AAVrh.74 capsid protein comprises a sequence that shares at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 173, excluding the VR-II site and/or the last 35 amino acid positions.

[0195] In some embodiments, the rAAV virus or virion comprises a chimeric capsid protein, for example, an AAV5/AAV9 chimeric capsid protein. In some embodiments, the AAV5/AAV9 chimeric capsid protein comprises at least 1 , 2, 3, 4, 5 or more polypeptide segments each that are derived from AAV9 capsid protein and from AAV5 capsid protein. In some embodiments, at least one polypeptide segment is derived from the AAV5 capsid protein and at least one polypeptide segment is derived from the AAV9 capsid protein.

[0196] In some embodiments, the rAAV virus or virion comprises a combinatory capsid protein. As used herein, “combinatory capsid protein” refers to a AAV5/AAV9 chimeric capsid protein, which further comprises amino acid variations with respect to the chimeric parental sequence at one or more sites. In some embodiments, the one or more sites of the chimeric parental sequence are selected from those equivalent to the VR-IV site, the VR-V site, the VR-VII site, and the VR-VIII site of the corresponding wildtype capsid protein.

[0197] In some embodiments, the rAAV virus or virion comprises an engineered capsid protein. Engineered capsid proteins can be derived from a parental, e.g., wildtype, capsid and include, for example, a variant polypeptide sequence with respect to a parental capsid sequence at one or more sites. For example, variant polypeptide sequences of the parental capsid can occur at the VR-IV site, VR-V site, VR-VII site and/or VR-VIII site. In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-IV region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-V region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-VII region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-VIII region of the capsid protein (e.g., of wild-type AAV9). In some embodiments, the engineered capsid protein has a substitution or insertion at the VR-IV region and the VR-VIII region of the capsid protein (e.g., of wildtype AAV9). Exemplary variant AAV9 capsid proteins (e.g., comprising one or more substitutions or insertions) are described in WO2021/163357, WO2021/216456, and U.S. Patent No. 11 ,129,908, the entire contents of each of which are incorporated by reference herein. In some embodiments, the present disclosure provides a rAAV capsid protein disclosed in WO2021/163357 as CR9-01 , and this disclosure is specifically incorporated by reference herein in its entirety.

[0198] In some embodiments, the rAAV virus or virion comprises an engineered AAV9 capsid protein comprising a variant polypeptide sequence. In some embodiments, the engineered AAV9 capsid protein comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least six substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least eight substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site.

[0199] In some embodiments, the parental sequence is the AAV9 VP1 parental sequence (SEQ ID NO: 82). In some embodiments, the parental sequence is the AAV5 VP1 parental sequence (SEQ ID NO: 89). In some embodiments, the parental sequence is the AAVrh.10 VP1 parental sequence (SEQ ID NO: 161 ). In some embodiments, the parental sequence is the AAVrh.74 VP1 parental sequence (SEQ ID NO: 171 ). In some embodiments, the variant polypeptide sequence occurs only at the VR-VIII site.

[0200] In some embodiments, the engineered capsid protein comprises a variant polypeptide in the VR-VIII site. In some embodiments, the engineered capsid protein comprises a variant polypeptide at positions 581-594 of the capsid protein, wherein the amino acid numbering is according to the AAV9 VP1 sequence of SEQ ID NO: 82. A person of skill in the art will recognize the equivalent positions in other AAV serotype capsid proteins, e.g., positions 570-583 of the AAV5 VP1 capsid sequence of SEQ ID NO: 89, positions 583-596 of the AAVrh.10 VP1 capsid sequence of SEQ ID NO: 161 , and positions 583-596 of the AAVrh.74 VP1 capsid sequence of SEQ ID NO: 171. The variant polypeptide may comprise substitutions across the entire VR-VIII site (e.g., between positions 581 -594) or may comprise substitutions in a portion of the VR-VIII site (e.g., between positions 582-591 ).

[0201] In some embodiments, the engineered capsid protein comprises the variant amino acid sequence of X1X2X3X4X5K at a specified site. In some embodiments, the engineered capsid protein comprises a variant amino acid sequence in the VR-II site. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3X4X5K in a VR-II site of an AAV VP capsid polypeptide sequence. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3X4X5K at amino acid positions 327 to 332 according to the amino acid numbering of SEQ ID NO: 82 (WT AAV9 VP1 protein). In some embodiments, the site is between amino acids 316 and 333 according to an AAV VP1 amino acid sequence. In some embodiments, the site is at positions 327 to 332, wherein the amino acid numbering is according to SEQ ID NO: 89. In some embodiments, the site is at positions 328 to 333, wherein the amino acid numbering is according to SEQ ID NO: 161. In some embodiments, the site is at positions 328 to 333, wherein the amino acid numbering is according to SEQ ID NO: 171 .

[0202] In some embodiments, the variant amino acid sequence X1X2X3X4X5K is QTDGVK (SEQ ID NO: 179) In some embodiments, the variant amino acid sequence X1X2X3X4X5K is QQDGTK (SEQ ID NO: 180).

[0203] In some embodiments, the engineered capsid protein comprises a variant amino acid sequence within the last 35 amino acid positions of the capsid protein. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3GX4 within the last 35 amino acid positions of an AAV VP capsid polypeptide sequence. In some embodiments, the engineered capsid protein comprises the variant amino acid sequence X1X2X3GX4 at amino acid positions 716 to 720 according to the amino acid numbering of SEQ ID NO: 82.

[0204] In some embodiments, the capsid protein comprises the variant amino acid sequence of X1X2X3GX4 at a specified site. In some embodiments, the site is at amino acid positions 716 to 720 wherein the amino acid numbering is according to SEQ ID NO: 82. In some embodiments, the site is at positions 718 to 722, wherein the amino acid numbering is according to SEQ ID NO: 89. In some embodiments, the site is at positions 718 to 722, wherein the amino acid numbering is according to SEQ ID NO: 161. In some embodiments, the site is at positions 704 to 708, wherein the amino acid numbering is according to SEQ ID NO: 171 .

[0205] In some embodiments, the variant amino acid sequence X1X2X3GX4 is selected from the group consisting of: NQYGV (SEQ ID NO: 181 ), NVHGV (SEQ ID NO: 182), NTHGV (SEQ ID NO: 183), and NTRGE (SEQ ID NO: 184). In some embodiments, the variant amino acid sequence X1X2X3GX4 is NQYGV (SEQ ID NO: 181 ). In some embodiments, the variant amino acid sequence X1X2X3GX4 is NTRGE (SEQ ID NO: 184).

[0206] In some embodiments, the variant polypeptide comprises one or more substitutions (e.g., a substitution motif). In some embodiments, the non-naturally occurring amino acid motif comprises a substitution motif and does not comprise an insertion motif.

[0207] In some embodiments, the engineered AAV9 capsid protein: (i) is cardiotrophic, (ii) exhibits increased transduction efficiency in cardiac cells compared to the parental sequence, (iii) exhibits decreased transduction efficiency in liver cells compared to the parental sequence, and/or (iv) exhibits increased selectivity for the cardiac cells over liver cells compared to the parental sequence. These characteristics may be assessed in cells (e.g., iPSC-derived cardiac cells or cardiomyocytes) in vitro, or in mice or primates in vivo, by any methods known in the art.

[0208] In some embodiments, the engineered AAV9 capsid protein comprises an amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 (SEQ ID NO: 96) at the VR-VIII site (e.g., of a wild-type AAV9 capsid protein or a variant thereof), wherein each of Xi , X2, X3, X4, Xs, Xe, X7, and Xs is any amino acid.

[0209] In some embodiments, Xi is alanine (A). [0210] In some embodiments, X2 is glutamine (Q).

[0211] In some embodiments, X7 is threonine (T).

[0212] In some embodiments, X5 is alanine (A) or proline (P).

[0213] In some embodiments, Xs is glutamine (Q) or glutamic acid (E).

[0214] In some embodiments, X4 is glutamine (Q), glycine (G), arginine (R), asparagine (N), histidine (H), methionine (M), proline (P), or serine (S).

[0215] In some embodiments, Xs is glutamic acid (E), methionine (M), glutamine (Q), aspartic acid (D), leucine (L), alanine (A), cysteine (C), histidine (H), phenylalanine (F), tyrosine (Y), threonine (T), valine (V), isoleucine (I), serine (S), or asparagine (N). In some embodiments, Xs is glutamic acid (E).

[0216] In some embodiments, X3 is leucine (L), histidine (H), valine (V), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), methionine (M), phenylalanine (F), proline (P), threonine (T), or tyrosine (Y).

[0217] In some embodiments, Xi is A, X2 is Q, X7 is T, and/or the capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4X5X6TX8 (SEQ ID NO: 97), wherein each of X3, X4, X5, Xs, and Xs is any amino acid.

[0218] In some embodiments, X5 is A or P, and Xs is Q or E, and/or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1 DVQX2X3PGFX4AQX7X8 (SEQ ID NO: 98), X1 DVQX2X3PGFX4AEX7X8 (SEQ ID NO: 99), X1 DVQX2X3PGFX4PQX7X8 (SEQ ID NO: 100), X1DVQX2X3PGFX4PEX7X8 (SEQ ID NO: 101 ), ADVQQX3PGFX4AQTX8 (SEQ ID NO: 102), ADVQQX3PGFX4AETX8 (SEQ ID NO: 103), ADVQQX3PGFX4PQTX8 (SEQ ID NO: 104), or ADVQQX3PGFX4PETX8 (SEQ ID NO: 105), wherein each of Xi , X2, X3, X4, X7, and Xs is any amino acid.

[0219] In some embodiments, X3 is selected from L, H, V, C, Q, G, I, M, F, P, T, or Y, X4 is selected from Q, G, R, N, H, M, P, or S, and Xs is selected from E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments, X3 is L and Xs is E.

[0220] In some embodiments, X4 is Q, X5 is A, and Xs is Q, and/or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1DVQX2X3PGFQAQX7X8 (SEQ ID NO: 106) or ADVQQXsPGFQAQTXs (SEQ ID NO: 107), wherein each of Xi, X2, X₃, X7, and Xs is any amino acid. [0221] In some embodiments, X3 is L, and Xs is E, and/or the capsid protein comprises in the VR-VIII site an amino acid sequence ofXi DVQX2LPGFX4X5X6X7E (SEQ ID NO: 108) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 109), wherein each of Xi, X2, X₄, Xs, Xs, and X7 is any amino acid. In some embodiments, X4 is Q.

[0222] In some embodiments, X4 is Q, and/or the capsid protein comprises in the VR-VIII site an amino acid sequence of X1 DVQX2X3PGFQX5X6X7X8 (SEQ ID NO: 110) or ADVQQXsPGFQXsXeTXs (SEQ ID NO: 111 ), wherein each of Xi , X₂, X₃, X₅, X₆, X₇, and Xs is any amino acid.

[0223] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of X1 DVQX2X3PGFX4AX6X7X8, wherein each of Xi , X2, X3, X4, Xs, X7, and Xs is any amino acid (SEQ ID NO: 185).

[0224] In some embodiments, the engineered AAV capsid comprises in the VR-VIII site an amino acid sequence of X1 DVQX2X3PGFX4X5QX7X8, wherein each of Xi, X2, X3, X4, Xs, X7, and Xs is any amino acid (SEQ ID NO: 186).

[0225] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4PETX8, wherein each of X3, X4 and/or Xs is any amino acid (SEQ ID NO: 187). In some embodiments, Xs is L, H, S, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4 is Q, G, R, N, H, M, P, or S. In some embodiments, Xs is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0226] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4AQTX8, wherein each of X3, X4 and/or Xs is any amino acid (SEQ ID NO: 188). In some embodiments, Xs is L, H, S, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4 is Q, G, R, N, H, M, P, or S. In some embodiments, Xs is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0227] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFQAQTE, wherein X3 is any amino acid (SEQ ID NO: 189).

[0228] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4X5X6X7X8, wherein each of X3, X4, Xs, Xs, X7, and Xs is any amino acid (SEQ ID NO: 190). [0229] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQX3PGFX4X5X6X7E, wherein each of, X3, X4, Xs, Xe, and X7 is any amino acid (SEQ ID NO: 191 ).

[0230] In some embodiments, the engineered AAV capsid protein comprises in the VR-VIII site an amino acid sequence of ADVQQHPGFX4X5X6TE, wherein each of X4, X5, and Xe is any amino acid (SEQ ID NO: 192).

[0231] In some embodiments, the engineered AAV9 capsid protein comprises, consists essentially of, or consists of a sequence having at least about 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%) identity to any one of the following sequences at the VR-VIII site (positions 581-595 relative to reference sequence SEQ ID NO: 82), with up to 1 , 2, or 3 substitutions: ADVQQLPGFQAQTEW (SEQ ID NO: 112), ADVQQHPGFQAQTEW (SEQ ID NO: 113), ADVQQVPGFQAQTMW (SEQ ID NO: 114), ADVQQVPGFQAQTQW (SEQ ID NO: 115), ADVQQLPGFGAQTEW (SEQ ID NO: 116), ADVQQLPGFRPETEW (SEQ ID NO: 117), and ADVQQLPGFNAQTEW (SEQ ID NO: 118).

[0232] In some embodiments, the engineered AAV9 capsid protein comprises in the VR-VIII site an amino acid sequence selected from ADVQQLPGFQAQTE (SEQ ID NO: 112), ADVQQHPGFQAQTE (SEQ ID NO: 113), ADVQQVPGFQAQTM (SEQ ID NO: 83), ADVQQVPGFQAQTQ (SEQ ID NO: 114), ADVQQLPGFGAQTE (SEQ ID NO: 116), ADVQQLPGFRPETE (SEQ ID NO: 117), and ADVQQLPGFNAQTE (SEQ ID NO: 118).

[0233] In some embodiments, the engineered AAV9 capsid protein comprises in the VR-VIII site an amino acid sequence selected from ADVQQLPGFQAQTE (SEQ ID NO: 112) and ADVQQHPGFQAQTE (SEQ ID NO: 113).

[0234] In some embodiments, the engineered AAV9 capsid protein comprises one or more amino acid substitutions selected from the group consisting of A581 R, A581 N, A581 D, A581 C, A581 Q, A581 E, A581 G, A581 H, A5811, A581 L, A581 K, A581 M, A581 F, A581 P, A581 O, A581 S, A581T, A581W, A581Y, A581V, T582D, N583V, H584Q, Q585T, Q585C, Q585V, Q585L, Q585N, Q585S, Q585P, Q585A, Q585M, Q585E, Q585Y, Q585G, Q585H, Q585I, Q585R, Q585D, Q585K, Q585F, Q585O, Q585, S586D, S586T, S586G, S586K, S586M, S586N, S586I, S586Q, S586L, S586P, S586F, S586R, S586A, S586C, S586E, S586H, S586O, S586W, S586Y, S586V, A587P, A587S, A587N, Q588G, Q588R, Q588V, A589F, A589T, Q590I, Q590S, Q590N, Q590G, Q590D,

Q590R, Q590H, Q590T, Q590M, Q590F, Q590Y, Q590L, Q590A, Q590C, Q590E,

Q590K, Q590P, Q590O, Q590W, Q590V, A591 I, A591 R, A591 N, A591 D, A591 C,

A591 Q, A591 E, A591 G, A591 H, A591 L, A591 K, A591 M, A591 F, A591 P, A5910, A591 S, A591T, A591W, A591Y, A591V, Q592I, Q592R, Q592N, Q592D, Q592C, Q592A,

Q592E, Q592G, Q592H, Q592 L, Q592K, Q592M, Q592F, Q592P, Q592O, Q592S,

Q592T, Q592W, Q592Y, Q592V, T593I, T593R, T593N, T593D, T593C, T593A, T593E, T593G, T593H, T593L, T593K, T593M, T593F, T593P, T593O, T593S, T593Q, T593W, T593Y, T593V, G594I, G594R, G594N, G594D, G594C, G594A, G594E, G594Q, G594H, G594 L, G594K, G594M, G594F, G594P, G594O, G594S, G594T, G594W, G594Y, and G594V.

[0235] In some embodiments, the engineered capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected from the group consisting of S586L, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 82.

[0236] In some embodiments, the engineered capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected from the group consisting of S586H, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 82.

[0237] In some embodiments, the engineered capsid protein comprises one or more amino acid substitutions selected from the group consisting of: D327Q, D327V, N328T, N328Q, N328S, N329D, G330N, and V331T. In some embodiments, engineered the capsid protein comprises the amino acid substitutions D327Q, N328T, and N329D. In some embodiments, the engineered capsid protein comprises the amino acid substitutions D327Q, N328Q, N329D, V331T.

[0238] In some embodiments, the engineered capsid protein comprises one or more amino acid substitutions selected from the group consisting of: N716D, T717Q, T717M, T717V, T717A, T717P, T717I, E718Y, E718R, E718H, E718N, E718M, E718Q, V720E, V720Q, V720I, and V720A. In some embodiments, the capsid protein comprises the amino acid substitutions T717Q and E718Y. In some embodiments, the capsid protein comprises the amino acid substitutions E718R and V720E. [0239] In some embodiments, the engineered AAV9 capsid protein comprises any substitution and/or insertion motif described herein. In some embodiments, the engineered capsid protein comprises a substitution motif having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any substitution motif described herein. In some embodiments, the engineered capsid protein comprises an insertion motif having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any insertion motif described herein.

[0240] It should be noted that the above modified VR-VIII motifs are described in the context of AAV9 capsid proteins for illustrative purposes only and are not meant to be limited to AAV9 capsid proteins. Instead, any modified VR-VIII motif described herein can be applied to other AAV capsid proteins of a different serotype (e.g., AAV5, AAVrh.10, or AAVrh.74), for example, by replacing the wild-type sequence at the VR-VIII site of the corresponding capsid protein (e.g., amino acid positions 570 to 583 of wildtype AAV5 VP1 capsid protein sequence according to SEQ ID NO: 89, amino acid positions 583 to 596 of wild-type AAVrh.10 VP1 capsid protein sequence, or amino acid positions 583 to 596 of wild-type AAVrh.74 VP1 capsid protein sequence) with any of the modified VR-VIII motifs described herein to generate a variant of the capsid protein of a particular serotype. In some embodiments, the engineered capsid protein is a variant of an AAV5, AAV9, AAVth.10, or AAVrh.74 capsid protein.

[0241] In some embodiments, the engineered AAV9 capsid protein comprises one, two, three, four, five, or more insertions in the VR-VIII site. In some embodiments, the engineered AAV9 capsid protein comprises, relative to reference SEQ ID NO: 82, one, two, three, four, five, or more insertions at positions from 584 to 590 in the VR-VIII site, or one, two, three, four, five, or more insertions at positions from 585 to 590 in the VR- VIII site.

[0242] In some embodiments, the engineered capsid protein comprises an insertion polypeptide or insertion motif compared to the wild-type or parental capsid protein. In some embodiments, the engineered capsid protein additionally comprises one or more amino acid substitutions in the amino acid sequence of the wild-type or parental capsid protein sequence from which it is derived. In some embodiments, the insertion motif is inserted at a surface loop region of the capsid protein, for example, at a VR-IV, VR-V, VR-VII and/or VR-VIII site, as described.

[0243] In some embodiments, the insertion motif comprises or consists of the amino acid sequence RGDXKGL, wherein X can be any amino acid. In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDAARL” (SEQ ID NO: 119); and/or the engineered capsid protein comprises an amino acid sequence of “RGDAARL” (SEQ ID NO: 119).

[0244] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “SHVRGDL” (SEQ ID NO: 120); and/or the engineered capsid protein comprises an amino acid sequence of “SHVRGDL” (SEQ ID NO: 120).

[0245] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “VVSSGAR” (SEQ ID NO: 121 ); and/or the engineered capsid protein comprises an amino acid sequence of “WSSGAR” (SEQ ID NO: 121 ).

[0246] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “VRGD” (SEQ ID NO: 122); and/or the engineered capsid protein comprises an amino acid sequence of “VRGD” (SEQ ID NO: 122).

[0247] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RTDLKGL” (SEQ ID NO: 123); and/or the engineered capsid protein comprises an amino acid sequence of “RTDLKGL” (SEQ ID NO: 123).

[0248] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDTKGL” (SEQ ID NO: 194); and/or the engineered capsid protein comprises an amino acid sequence of “RGDTKGL” (SEQ ID NO: 194).

[0249] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDAKGL” (SEQ ID NO: 195); and/or the engineered capsid protein comprises an amino acid sequence of “RGDAKGL” (SEQ ID NO: 195).

[0250] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDVKGL” (SEQ ID NO: 196); and/or the engineered capsid protein comprises an amino acid sequence of “RGDVKGL” (SEQ ID NO: 196). [0251] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDVKGL” (SEQ ID NO: 196); and/or the engineered capsid protein comprises an amino acid sequence of “RGDLVST” (SEQ ID NO: 243).

[0252] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDVKGL” (SEQ ID NO: 196); and/or the engineered capsid protein comprises an amino acid sequence of “RGDGGVL” (SEQ ID NO: 244).

[0253] In some embodiments, the insertion motif comprises or consists of an amino acid sequence of “RGDVKGL” (SEQ ID NO: 196); and/or the engineered capsid protein comprises an amino acid sequence of “RGDHASW” (SEQ ID NO: 245).

[0254] The insertion motif can occur (e.g., be inserted) at any position of the capsid protein, for example, at a surface or an exposed region of the capsid protein. In some embodiments, the engineered AAV capsid protein comprises an insertion motif as described herein inserted at a surface loop region of the capsid protein, e.g., the VR-I, VR-II, VR-IV, VR-V, VR-VII, and/or VR-VIII site of the capsid protein. In some embodiments, the engineered capsid protein comprises an insertion motif as described inserted at the VR-IV and/or the VR-VIII site of the capsid protein. In certain of these embodiments, the engineered capsid protein additionally comprises one or more amino acid substitutions in the same VR site as the insertion or at a different location from the insertion.

[0255] In some embodiments, the engineered capsid protein is an engineered AAV9 capsid protein that comprises an insertion polypeptide or insertion motif at the VR-VIII site, e.g., between amino acids 588 (glutamine (Q)) and 589 (alanine (A)) within the VR- VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 82. In some embodiments, the engineered AAV9 capsid protein further comprises one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 587-590 in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 82. In certain of these embodiments, the insertion motif comprises an amino acid sequence of RGDAARL (SEQ ID NO: 119), RTDLKGL (SEQ ID NO: 123), YPSTGSG (SEQ ID NO: 124), FAGSLTRA (SEQ ID NO: 125), DRTLTTR (SEQ ID NO: 126), RIAGRDV (SEQ ID NO: 127), SLGSGVR (SEQ ID NO: 128) , RGDTKGL (SEQ ID NO: 194), RGDAKGL (SEQ ID NO: 164), RGDVKGL (SEQ ID NO: 165), RGDLVST (SEQ ID NO: 243), RGDGGVL (SEQ ID NO: 244), or RGDHASW” (SEQ ID NO: 245).

[0256] In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQSX1X2X3X4AQTGW” (SEQ ID NO: 206) in the VR-VIII site, wherein Xi , X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence). In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQSX1X2X3X4AQTEW’ (SEQ ID NO: 207) wherein Xi, X2, X₃, and X₄ can individually be any amino acid, and an insertion motif is inserted between X2 and Xs (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence). In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQLX1X2X3X4AQTGW” (SEQ ID NO: 208) in the VR-VIII site, wherein Xi , X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence). In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence “ATNHQLX1X2X3X4AQTEW” (SEQ ID NO: 209) in the VR-VIII site, wherein Xi , X2, X3, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (for example, between amino acid positions 588 and 589 of wild-type AAV9 VP1 capsid protein sequence).

[0257] In some embodiments, the engineered capsid protein is an engineered AAV9 capsid protein that comprises an insertion polypeptide or insertion motif at the VR-IV site, e.g., between amino acids 453 (glycine (G)) and 454 (serine (S)), and/or between amino acids 456 (glutamine (Q)) and 457 (asparagine (N)), within the VR-IV site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 82. In certain of these embodiments, the insertion motif comprises an amino acid sequence of SHVRGDL (SEQ ID NO: 120), VVSSGAR (SEQ ID NO: 121 ), PQYGRGG (SEQ ID NO: 129), LQVSRVS (SEQ ID NO: 130), VRSYSSN (SEQ ID NO: 131 ), “TMRVGSL” (SEQ ID NO: 132), GAYSRGV (SEQ ID NO: 133), LRGGSLG (SEQ ID NO: 134), or “VYGTGVR” (SEQ ID NO: 135). [0258] In some embodiments, the engineered capsid protein is an engineered AAV5 capsid protein that comprises an insertion polypeptide or insertion motif at the VR-VIII site, e.g., between amino acids 574 (glutamine (Q)) and 575 (serine (S)) within the VR- VIII site in reference to the wild-type full-length AAV5 capsid protein of SEQ ID NO: 89. In certain of these embodiments, the insertion motif comprises an amino acid sequence of DKLIIVS (SEQ ID NO: 136), AEDRTKL (SEQ ID NO: 137), LSASASL (SEQ ID NO: 138), LADQTKL (SEQ ID NO: 139), LLLKLQE (SEQ ID NO: 140), ELPVKTG (SEQ ID NO: 141 ), LDLKWG (SEQ ID NO: 142), or RDAVL (SEQ ID NO: 143).

[0259] In some embodiments, the engineered AAV9 capsid protein comprises an insertion peptide sequence or insertion motif at the VR-VIII site, e.g., between amino acids 588 (glutamine (Q)) and 589 (alanine (A)) within the VR-VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 82. The insertion motif can be any motif as described herein, including those provided in Table 7B below. In some embodiments, the engineered AAV9 capsid protein further comprises one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 587-590 in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 82.

[0260] In some embodiments, the engineered AAV9 capsid protein further comprises one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 586 and/or 594 in reference to the wildtype full-length AAV9 capsid protein of SEQ ID NO: 82. In certain of these embodiments, the engineered AAV9 capsid protein comprises a sequence of “ATNHQSX1X2X3X4AQTEW’ (SEQ ID NO: 207) at the VR-VIII site, where Xi , X2, X₃, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (i.e., between amino acid positions 588 and 589). In certain of these embodiments, the engineered AAV9 capsid protein comprises a sequence of “ATNHQLX1X2X3X4AQTGW’ (SEQ ID NO: 208) at the VR-VIII site, where Xi , X2, X₃, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (i.e., between amino acid positions 588 and 589). In certain of these embodiments, the engineered AAV9 capsid protein comprises a sequence of “ATNHQLX1X2X3X4AQTEW’ (SEQ ID NO: 209) at the VR-VIII site, where Xi , X2, X₃, and X4 can individually be any amino acid, and an insertion motif is inserted between X2 and X3 (i.e., between amino acid positions 588 and 589). Table 7B. Exemplary engineered AAV9 capsid protein VR-VIII sequences with superior performance in nonhuman primates.

[0261] In some embodiments, the engineered AAV9 capsid protein comprises a “substitution + insertion motif”, wherein the substitution + insertion motif comprises: an insertion peptide sequence or insertion motif at the VR-VIII site, e.g., between amino acids 588 (glutamine (Q)) and 589 (alanine (A)) within the VR-VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 82, and (ii) one or more amino acid substitutions within the VR-VIII site, including, for example, at one or more of amino acid positions 586, 587, 588, 590, and 594 in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 82. In some embodiments, the substitution + insertion motif comprises amino acids 586 to 594, including the insertion motif, within the VR-VIII site in reference to the wild-type full-length AAV9 capsid protein of SEQ ID NO: 82. In some embodiments, the substitution + insertion motif can be any disclosed herein, including those provided in Table 7C below.

Table 7C. Exemplary substitution + insertion motifs

[0262] In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 119, 123, 194-196, and 243-245. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 194. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 195. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 243. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 244. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 245.

[0263] In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 119, 123, 194-196, and 243-245, with up to 1 , 2, or 3 amino acid substitutions. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises an insertion motif comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 194-196, and 243-245, with up to 1 , 2, or 3 amino acid substitutions.

[0264] In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 197, or the amino acid sequence set forth SEQ ID NO: 197. In some embodiments, the engineered AAV9 capsid protein, at the VR- VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 199, or the amino acid sequence set forth in SEQ ID NO: 199. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NOs: 246, or the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 247, or the amino acid sequence set forth in SEQ ID NOs: 247. In some embodiments, the engineered AAV9 capsid protein, at the VR-VIII site, comprises, consists essentially of, or consists of an amino acid sequence that shares at least about 70% or 80% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 248, or the amino acid sequence set forth in SEQ ID NO: 248.

[0265] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 84, and comprises the amino acid sequence of ATNHQSSVRGDTKGLAGAQTGW (SEQ ID NO: 197) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 82.

[0266] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 84, and comprises the amino acid sequence of ATNHQSSVRTDLKGLAGAQTGW (SEQ ID NO: 199) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 82.

[0267] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 84, and comprises the amino acid sequence of ATNHQENRRGDLVSTTQAQTGW (SEQ ID NO: 246) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 82.

[0268] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 84, and comprises the amino acid sequence of ATNHQENRRGDGGVLAQAQTGW (SEQ ID NO: 247) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 82.

[0269] In some embodiments, the engineered AAV9 capsid protein comprises at least 90% or at least 95% amino acid sequence identity to AAV9 VP3 SEQ ID NO: 84, and comprises the amino acid sequence of ATNHQSSVRGDHASWAQAQTGW(SEQ ID NO: 248) replacing the natural amino acid sequence at amino acid positions 581 to 595, wherein the amino acid numbering is according to AAV9 VP1 SEQ ID NO: 82. [0270] Exemplary engineered capsid protein sequences are provided in Table 7B below. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence set forth in any one of SEQ ID NOs: 144- 154 and 229-242, or an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 144-154 and 229-242.

[0271] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 144 or SEQ ID NO: 145. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 144. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 145.

[0272] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 229 or 231. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 229. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 231 .

[0273] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 239- 242. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 239. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 240. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 241. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 242.

[0274] In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of an amino acid sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 255-257. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 255. In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 256 In some embodiments, the engineered capsid protein comprises, consists of, or consists essentially of SEQ ID NO: 257.

Table 7D. Exemplary engineered AAV capsid protein sequences

[0275] In some embodiments, the rAAV virus or virion comprises a wild-type

AAVrh.10 capsid protein or a variant thereof as known in the art. In some embodiments, the rAAV virus or virion comprises a wild-type AAVrh.74 capsid protein or a variant thereof as known in the art. In some embodiments, the rAAV virus or virion comprises an AAV-SLB101 capsid protein or a variant thereof as known in the art or described in, e.g., WO 2021/072197, which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises an AAVmod capsid protein or a variant thereof as known in the art or described in, e.g., WO 2022/173847 or in Olivieri et al. (2021 ) 24th Annual Meeting of the American Society of Gene & Cell Therapy available at https://www.affiniatx.com/pdf/asgct_2021_olivieri.pdf, both of which are incorporated by reference herein in their entireties. In some embodiments, the rAAV virus or virion comprises an AAVmut1 dec1 , AAVdecol , and/or AAVmutl capsid protein or a variant thereof as known in the art or described in, e.g. , WO 2022/173847, which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises an AAVcc.47 capsid protein or a variant thereof as known in the art or described in, e.g., Gonzalez et al. Nature Communications 13:5947 (2022), which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises an AAVHSC16 capsid protein or a variant thereof as known in the art or described in, e.g., Smith et al. Molecular Therapy Methods & Clinical Development 26:224-238 (2022), which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises a MyoAAV capsid protein or variant thereof as known in the art or described in, e.g., Tabebordbar et al. Cell 184(19):4919- 4938. (2021 ), which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises a MyoAAV-4E, MyoAAV-3F, MyoAAV- 4A, or MyoAAV-4D capsid protein or variant thereof as known in the art or described in, e.g., Tabebordbar et al, which is incorporated by reference herein in its entirety. In some embodiments, the rAAV virus or virion comprises a 4D-C102 or C102 capsid protein or a variant thereof as known in the art or described in, e.g., US2021/0380643, which is incorporated by reference herein in its entirety.

[0276] In some embodiments, the rAAV virus or virion comprises a capsid protein (such as any described herein) and a vector genome, and the vector genome comprises an expression cassette flanked by ITRs. In some embodiments, the rAAV virus or virion specifically transduces heart cells and/or cardiomyocytes. In some embodiments, the rAAV virus or virion traffics to the heart. In some embodiments, the rAAV virus or virion traffics to at least one organ other than the liver. [0277] In some embodiments, the rAAV virus or virion exhibits a higher transduction efficiency (e.g., a higher heart transduction efficiency) than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 82. In some embodiments, the rAAV virus or virion exhibits a higher transduction efficiency (e.g., a higher heart transduction efficiency) in a primate or as assessed in a primate than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 82.

[0278] In some embodiments, administration of the rAAV virus or virion to a subject leads to a lower liver viral load than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 82. In some embodiments, administration of the rAAV virus or virion to a subject leads to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower liver viral load than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 82. In some embodiments, administration of the rAAV virus or virion to a subject leads to a lower liver viral load in a primate or as assessed in a primate than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 82. In some embodiments, administration of the rAAV virus or virion to a subject leads to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times lower liver viral load in a primate or as assessed in a primate than administration of an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 82.

[0279] In some embodiments, the rAAV virus or virion exhibits a higher heart-to- liver transduction ratio than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 82. In some embodiments, the rAAV virion exhibits a heart-to- liver transduction ratio which is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 82. In some embodiments, the rAAV virus or virion exhibits a higher heart-to-liver transduction ratio in a primate or as assessed in a primate than an rAAV virus or virion having a wildtype AAV9 VP1 capsid protein of SEQ ID NO: 82. In some embodiments, the rAAV virion exhibits a heart-to-liver transduction ratio which is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher in a primate or as assessed in a primate than an rAAV virus or virion having a wild-type AAV9 VP1 capsid protein of SEQ ID NO: 82.

[0280] In some embodiments, the rAAV virus or virion is replication defective, in that the rAAV virus or virion cannot independently further replicate and package its genome. For example, when a cardiac cell is targeted with rAAV virions, the transgene is expressed in the targeted cardiac cell, however, due to the fact that the targeted cardiac cell lacks AAV rep and cap genes and accessory function genes, the rAAV is not able to replicate.

[0281] In some embodiments, rAAV virus or virion encapsulating the expression cassettes as described herein can be produced using helper-free production. rAAVs are replication-deficient viruses and normally require components from a live helper virus, such as adenovirus, in a host cell for packaging of infectious rAAV virions. rAAV helper- free production systems allow the production of infectious rAAV virions without the use of a live helper virus. In the helper-free system, a host packaging cell line is cotransfected with three plasmids. A first plasmid may contain adenovirus gene products (e.g., E2A, E4, and VA RNA genes) needed for the packaging of rAAV virions. A second plasmid may contain required AAV genes (e.g., REP and CAP genes). A third plasmid contains the polynucleotide sequence encoding the transgene of interest and a promoter flanked by ITRs. A host packaging cell line can be, for example, AAV-293 host cells. Suitable host cells contain additional components required for packaging infectious rAAV virions that are not supplied by the plasmids. In some embodiments, the CAP genes can encode, for example, AAV capsid proteins as described herein.

Compositions

[0282] In some embodiments, an expression cassette and/or vector (e.g., rAAV viruses or virions) comprising the cardiac-specific promoter according to various embodiments disclosed herein is in a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients for parenteral delivery. Pharmaceutically acceptable carriers, diluents, or excipients can include vehicles that are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. Illustrative pharmaceutical forms suitable for injectable use include, e.g., sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

[0283] In some embodiments, the pharmaceutical composition comprises about 1 X10⁸ genome copies per milliliter (GC/mL), about 5X10⁸ GC/mL, about 1 xio⁹ GC/mL, about 5x10⁹ GC/mL, about 1 x ¹⁰ GC/mL, about 5x10¹° GC/mL, about 1 X10^{1 1} GC/mL, about 5x10¹¹ GC/mL, about 1 x ¹² GC/mL, about 5x10¹² GC/mL, about 5x10¹³ GC/mL, about 1 x ¹⁴ GC/mL, or about 5x10¹⁴ GC/mL of the vector (e.g., rAAV virus or virion).

[0284] In some embodiments, the pharmaceutical composition comprises about 1 x ⁸ viral genomes per milliliter (vg/mL), about 5X10⁸ vg/mL, about 1 x ⁹ vg/mL, about 5X10⁹ vg/mL, about 1 xi o¹⁰ vg/mL, about 5xl O¹⁰ vg/mL, about 1 xio¹¹ vg/mL, about 5x10¹¹ vg/mL, about 1 xi o¹² vg/mL, about 5x10¹² vg/mL, about 5x10¹³ vg/mL, about 1 x ¹⁴ vg/mL, or about 5x10¹⁴ vg/mL of the vector (e.g., rAAV virus or virion).

[0285] In some embodiments, the pharmaceutical composition comprises less than about 1 xi o¹⁵ viral genomes per milliliter (vg/mL), less than about 5x10¹⁴ vg/mL, less than about 1 x ¹⁴ vg/mL, less than about 5x10¹³ vg/mL, less than about 1 xi o¹³ vg/mL, less than about 5x10¹² vg/mL, less than about 1 xi o¹² vg/mL, less than about 5x10¹¹ vg/mL, or less than about 1 x1 o¹¹ vg/mL of the vector (e.g., rAAV virus or virion).

[0286] In some embodiments, the pharmaceutical composition comprises less than about 1 xi o¹⁴ viral genomes per milliliter (vg/mL) or less than about 1 xi o¹³ vg/mL of the vector (e.g., rAAV virus or virion).

[0287] In some embodiments, the pharmaceutical composition comprises from about 1 X10¹¹ viral genomes per milliliter (vg/mL) to about 1 xi o¹⁵ vg/mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1 xio¹¹ viral genomes per milliliter (vg/mL) to about 1 xi o¹⁴ vg/mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1 xi o¹² viral genomes per milliliter (vg/mL) to about 1 X10¹⁴ vg/mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1 xi o¹² viral genomes per milliliter (vg/mL) to about 1 xi o¹³ vg/mL of the vector (e.g., rAAV virus or virion). In some embodiments, the pharmaceutical composition comprises from about 1 xio¹² viral genomes per milliliter (vg/mL) to about 6x10¹³ vg/mL of the vector (e.g., rAAV virus or virion). [0288] In some embodiments, the pharmaceutical composition comprises any amount or concentration range of the vector (e.g., rAAV virus or virion) between the values referenced herein.

[0289] In some embodiments, the pharmaceutical composition is administered in a total volume of about 1 mL, about 5 mL, about 10 mL, about 20 mL, about 25mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, about 65 mL, about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about 95 mL, about 100 mL, about 105 mL, about 110 mL, about 115 mL, about 120 mL, about 125 mL, about 130 mL, about 135 mL, about 140 mL, about 145 mL, about 150 mL, about 155 mL, about 160 mL, about 165 mL, about 170 mL, about 175 mL, about 180 mL, about 185 mL, about 190 mL, about 200 mL, about 205 mL, about 210 mL, about 215 mL, or about 220 mL.

[0290] In some embodiments, the pharmaceutical composition can be formulated (e.g., injectable, lyophilized, liquid formulations, or oral formulations) to be compatible with its intended route of administration. Examples of routes of administration include oral administration, extracorporeal administration, parenteral administration, intravenous administration, subcutaneous administration, intralesional administration (e.g., injection into tumors), and by administration into biological spaces infiltrated by tumors (e.g., intraspinal administration, intracerebel lar administration, intraperitoneal administration, intralymphatic administration, intranodal administration, and/or pleural administration). For example, a pharmaceutical composition provided herein can be administered systemically by oral administration or by intravenous administration (e.g., injection or infusion). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. [0291] In some embodiments, the pharmaceutical composition can be coformulated in the same dosage unit or can be individually formulated in separate dosage units. The term “dosage unit” herein refers to a portion of a pharmaceutical composition that contains an amount of a therapeutic agent suitable for a single administration to provide a therapeutic effect. Such dosage units may be administered one to a plurality (e.g., 1 to about 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2) of times per day, or as many times as needed to elicit a therapeutic response.

Kits

[0292] In some embodiments, a pharmaceutical composition according to various embodiments disclosed herein is in a kit.

[0293] The kit can include any of compositions described herein, either mixed together or individually packaged, and in dry or hydrated form. The rAAV virions and/or other agents described herein can be packaged separately into discrete vials, bottles or other containers. Alternatively, any of the rAAV virions and/or agents described herein can be packaged together as a single composition, or as two or more compositions that can be used together or separately. The compounds and/or agents described herein can be packaged in appropriate ratios and/or amounts to facilitate conversion of selected cells across differentiation boundaries to form cardiac progenitor cells and/or cardiomyocytes.

[0294] The kit can include instructions for administering those compositions, compounds and/or agents. Such instructions can provide the information described throughout this application. The rAAV virion or pharmaceutical composition can be provided within any of the kits in the form of a delivery device. Alternatively, a delivery device can be separately included in the kits, and the instructions can describe how to assemble the delivery device prior to administration to a subject.

[0295] Any of the kits can also include syringes, catheters, scalpels, sterile containers for sample or cell collection, diluents, pharmaceutically acceptable carriers, and the like. The kits can provide other factors such as any of the supplementary factors or drugs described herein for the compositions in the preceding section or other parts of the application.

Cells [0296] In some embodiments, an expression cassette and/or vector (e.g., rAAV viruses or virions) comprising the cardiac-specific promoter according to various embodiments disclosed herein is in an isolated cell or a population of cells.

[0297] In some embodiments, the cell is a cardiac cell. As used herein the term “cardiac cell” refers to any cell present in the heart that provides a cardiac function, such as heart contraction or blood supply, or otherwise serves to maintain the structure of the heart. Cardiac cells as used herein encompass cells that exist in the epicardium, myocardium or endocardium of the heart. Cardiac cells also include, for example, cardiac muscle cells or cardiomyocytes, and cells of the cardiac vasculatures, such as cells of a coronary artery or vein. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem or progenitor cells, cardiac conducting cells and cardiac pacemaking cells that constitute the cardiac muscle, blood vessels and cardiac cell supporting structure. Cardiac cells may be derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells.

[0298] In some embodiments, the cell is a cardiomyocyte.

[0299] In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, a cell is an iPSC-derived cardiomyocyte.

[0300] In some embodiments, provided are methods of contacting the cell with any vector (e.g., an rAAV virus or virion) described herein. In some embodiments, the cell is a cardiac cell. In some embodiments, the cell is a cardiomyocyte. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0301] In some embodiments, provided are methods of contacting a tissue with any vector (e.g., an rAAV virus or virion) described herein. In some embodiments, the tissue is cardiac tissue. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0302] In some embodiments, provided are methods of contacting an organ with any vector (e.g., an rAAV virus or virion) described herein. In some embodiments, the organ is heart. In some embodiments, the heart is diseased or at risk of disease. In some embodiments, the heart has borderline or reduced ejection fraction. In some embodiments, the heart has a normal ejection fraction. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. [0303] In some embodiments, provided are cell therapy compositions comprising any cell described herein.

Therapeutic Methods

[0304] In some aspects, provided are methods for preventing and/or treating a disease or condition in a subject in need thereof. Subjects who are suitable for the compositions and/or methods of the present technology include individuals (e.g., mammalian subjects, such as humans, non-human primates, domestic mammals, experimental non-human mammalian subjects such as mice, rats, etc.). In some embodiments, the subject is a human. In some embodiments, the method for preventing and/or treating a disease or condition comprises administering to the subject a clinically effective or a therapeutically effective amount of an expression cassette, vector, virus, or virion comprising the cardiac-specific promoter according to various embodiments disclosed herein, or a pharmaceutical composition containing the same.

[0305] In some embodiments, the disease or condition is a heart disease. In some embodiments, the heart disease is cardiomyopathy, including, for example, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), idiopathic DCM, arrhythmogenic cardiomyopathy (ACM), and arrhythmogenic right ventricular cardiomyopathy (ARVC). In some embodiments, the heart disease is heart failure, including, for example, heart failure with reduced ejection fraction and ischemic heart failure. In some embodiments, the heart disease is arrhythmia, including, for example, atrial and/or ventricular arrhythmia and malignant ventricular arrhythmia. In some embodiments, the heart disease is cardiomyopathy associated with a pulmonary embolus, venous thrombosis, myocardial infarction, transient ischemic attack, peripheral vascular disorder, atherosclerosis, ischemic cardiac disease, other myocardial injury or vascular disease, and/or cardiac diseases associated with myocardial tissue hypercontractility, such as heart failure related to left ventricular hypercontractility.

[0306] In some embodiments, the pharmaceutical composition for use in the method of the present technology can be administered to the subject by systemic application (such as parenteral application), for example, by intravenous (e.g., by IV infusion), intra-arterial, or intraperitoneal delivery. In some embodiments, the pharmaceutical composition (e.g., rAAV vectors, viruses, or virions) can be delivered by direct administration to the heart tissue. [0307] In some embodiments, the pharmaceutical composition for use in the method of the present technology can be delivered by intracoronary administration. In some embodiments, the administration is by antegrade epicardial coronary artery infusion, e.g., a single infusion over a 10-minute period in a cardiac catheterization laboratory after angiography (percutaneous intracoronary delivery without vessel balloon occlusion) with the use of standard 5F or 6F guide or diagnostic catheters.

[0308] In some embodiments, the pharmaceutical composition for use in the method of the present technology can be delivered by direct injection into the heart or cardiac catheterization, or by intracardiac catheter delivery via retrograde coronary sinus infusion (RCSI).

[0309] When direct injection is used, it may be performed either by open-heart surgery or by minimally invasive surgery. In some cases, the pharmaceutical composition for use in the method of the present technology can be delivered to the pericardial space by injection or infusion.

[0310] In some embodiments, the amount, concentration, and volume of the pharmaceutical composition that modulates contractile function in myocardial tissue administered to a subject can be controlled and/or optimized to substantially improve the functional parameters of the heart while mitigating adverse side effects.

[0311] The amount of the composition that modulates contractile function administered to myocardial tissue can also be an amount required to result in the detectable expression of a therapeutic protein or nucleic acid in the heart; preserve and/or improve contractile function; delay the emergence of cardiomyopathy or reverse the pathological course of the disease; increase myocyte viability; improve myofilament function; inhibit left ventricular hypertrophy; cardiac hypertrophy regression, normalize systolic and diastolic function in heart; and restore normal cross-bridge behavior at the myofilament level.

[0312] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of about 1 X10⁸ genome copies per milliliter (GC/mL), about 5x10⁸ GC/mL, about 1 x ⁹ GC/mL, about 5x10⁹ GC/mL, about 1 xio¹⁰ GC/mL, about 5x10¹° GC/mL, about 1 X10^{1 1} GC/mL, about 5x10¹¹ GC/mL, about 1 X10¹² GC/mL, about 5x10¹² GC/mL, about 5x10¹³ GC/mL, about 1 x ¹⁴ GC/mL, or about 5x10¹⁴ GC/mL of the rAAV vector, virus, or virion. [0313] In some embodiments, the method comprises intravenously administering an rAAV vector, virus, or virion at a dose of about 3X10¹² GC/mL, about 3X10¹³ GC/mL, about 1 xi o¹⁴ GC/mL, or about 3x10¹⁴ GC/mL of the rAAV vector, virus, or virion.

[0314] In some embodiments, the method comprises administering, by localized delivery to the heart, an rAAV vector, virus, or virion at a dose of about 3X10^{1 1} GC/mL, about 3x10¹² GC/mL, about 1 xi o¹³ GC/mL, or about 3x10¹³ GC/mL of the rAAV vector, virus, or virion.

[0315] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of about 1 xi o⁸ viral genomes per milliliter (vg/mL), about 5x10⁸ vg/mL, about 1 xio⁹ vg/mL, about 5x10⁹ vg/mL, about 1 xi o¹⁰ vg/mL, about 5x10¹⁰ vg/mL, about 1 X10¹¹ vg/mL, about 5x10¹¹ vg/mL, about 1 xi o¹² vg/mL, about 5x10¹² vg/mL, about 5X10¹³ vg/mL, about 1 xi o¹⁴ vg/mL, or about 5X10¹⁴ vg/mL of the rAAV vector, virus, or virion.

[0316] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of less than about 1 xi o¹⁵ viral genomes per milliliter (vg/mL), less than about 5X10¹⁴ vg/mL, less than about 1 xio¹⁴ vg/mL, less than about 5X10¹³ vg/mL, less than about 1 xi o¹³ vg/mL, less than about 5x10¹² vg/mL, less than about 1 X10¹² vg/mL, less than about 5x10¹¹ vg/mL, or less than about 1 xi o¹¹ vg/mL of the rAAV vector, virus, or virion.

[0317] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of less than about 1 xi o¹⁴ viral genomes per milliliter (vg/mL) or less than about 1 xi o¹³ vg/mL of the rAAV vector, virus, or virion.

[0318] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at a dose of from about 1 xi o¹¹ viral genomes per milliliter (vg/mL) to about 1 xi o¹⁵ vg/mL, from about 1 xi o¹¹ vg/mL to about 1 xl 0¹⁴ vg/mL, from about 1 xl0¹² vg/mL to about 1 xl 0¹⁴ vg/mL, from about 1 xi o¹² vg/mL to about 1 xl0¹³ vg/mL, or from about 1 xi o¹² vg/mL to about 6X10¹³ vg/mL of the rAAV vector, virus, or virion.

[0319] In some embodiments, the method comprises administering an rAAV vector, virus, or virion at any dose or dose range of the disclosure between the values referenced herein. [0320] In some embodiments, the method comprises intravenously administering an rAAV vector, virus, or virion at a dose of about 1 x10¹²vg/mL, about 3xlO¹²vg/mL, about 6x10¹² vg/mL, or about 9x10¹² vg/mL of the rAAV vector, virus, or virion.

[0321] In some embodiments, the method comprises administering, by localized delivery to the heart, an rAAV vector, virus, or virion at a dose of about 1 xl0¹²vg/mL, about 3x10¹²vg/mL, about 6X10¹² vg/mL, or about 9x10¹² vg/mL of the rAAV vector, virus, or virion.

[0322] Genome copies per milliliter can be determined by quantitative polymerase change reaction (qPCR) using a standard curve generated with a reference sample having a known concentration of the polynucleotide genome of the virus. For AAV, the reference sample used is often the transfer plasmid used in generation of the rAAV virion, but other reference samples may be used.

[0323] Alternatively, the concentration of a viral vector can be determined by measuring the titer of the vector on a cell line. Viral titer is typically expressed as viral particles (vp) per unit volume (e.g., vp/mL). In various embodiments, the pharmaceutical composition comprises about 1 xi o⁸ viral particles per milliliter (vp/mL), about 5X10⁸ vp/mL, about 1 xio⁹ vp/mL, about 5x10⁹ vp/mL, about 1 xi o¹⁰ vp/mL, about 5x10¹° vp/mL, about 1 X10¹¹ vp/mL, about 5x10¹¹ vp/mL, about 1 xi o¹² vp/mL, about 5x10¹² vp/mL, about 5X10¹³ vp/mL, about 1 xio¹⁴ vp/mL, or about 5X10¹⁴ of the rAAV vector, virus, or virion.

[0324] The vector, virus, or virion administered to the subject can be traced by a variety of methods. For example, recombinant viruses labeled with or expressing a marker (such as green fluorescent protein, or beta-galactosidase) can readily be detected. The recombinant viruses may be engineered to cause the target cell to express a marker protein, such as a surface-expressed protein or a fluorescent protein. Alternatively, the infection of target cells with recombinant viruses can be detected by their expression of a cell marker that is not expressed by the animal employed for testing (for example, a human-specific antigen when injecting cells into an experimental animal). The presence and phenotype of the target cells can be assessed by fluorescence microscopy (e.g., for green fluorescent protein, or beta-galactosidase), by immunohistochemistry (e.g., using an antibody against a human antigen), by ELISA (using an antibody against a human antigen), or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for RNA indicative of a cardiac phenotype.

[0325] In some embodiments, the expression cassette, vector, virus, or virion, or a pharmaceutical composition containing the same, is administered to the subject once a day, twice a day, three times a day, or four times a day for a period of about 1 day, about

2 days, about 3 days, about 5 days, about 7 days, about 10 days, about 2 weeks, about

3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or more than about 5 years. In some embodiments, the expression cassette, vector, virus, or virion, or a pharmaceutical composition containing the same, is administered every day, every other day, 3 times a week, every third day, weekly, biweekly (/.e., every other week), every third week, monthly, every other month, every third month, every fourth month, every fifth month, every sixth month, every ninth month, every year, every 18 months, every 2 years, every 5 years, every 10 years, or every 20 years. In some embodiments, the dose regimens listed above could be repeated after a period of about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, or more than about 5 years. In some embodiments, the schedule of administration is a hybrid of these periods, for example, the expression cassette, vector, virus, or virion, or a pharmaceutical composition containing the same, is administered a number of times a week and that pattern is repeated a number of times a month every month or every second, third, fourth, fifth, or sixth month, and treatment according to that pattern is continued for part of a year to several years, as set out above. In some embodiments, the expression cassette, vector, virus, or virion, or a pharmaceutical composition containing the same, is administered in a cycle of a number or administrations over a week or two weeks, and the cycle is repeated at spaced intervals over a number of months or years, as set out above. In some embodiments treatment is continued until disease is eliminated, until no further improvement is achieved, or as long as the disease does not progress. In some embodiments, a disease or condition within a subject to be treated can be monitored to evaluate the effectiveness of the treatment using any appropriate method known to a skilled artisan. [0326] In some embodiments, the expression cassette, vector, virus, or virion, or a pharmaceutical composition containing the same, is administered over a predetermined time period. Alternatively, the expression cassette, vector, virus, or virion, or a pharmaceutical composition containing the same, is administered until a particular therapeutic benchmark is reached. In some embodiments, the methods provided herein further include a step of evaluating one or more therapeutic benchmarks in the subject to determine whether to continue administration of the treatment.

EXAMPLES

Example 1 : Generation of cardiac-specific chimeric promoters

[0327] As shown in FIG. 1 , to generate cardiac-specific chimeric promoters, promoter regions (about 500 bp each) of five cardiomyocyte specific genes (MYBPC3, MYH6, MYH7, MYL3, and TNNT2) were fragmented into 50 bp sequences. These short 50 bp-long sequences were randomly assembled to generate a library of 1 ,000 chimeric promoters, each 250 bp in length. Each of the five cardiac-specific genes had about equal representation (approximately 20%) at each promoter position. Each of the 250 bp chimeric promoters was cloned into an adeno-associated virus (AAV) expression cassette carrying enhanced green fluorescence protein (EGFP). Promoter strengths were tested in human induced pluripotent stem cell (iPSC)-derived cardiomyocyte (iPSC- CM) cells by transient transfection. iPSC-CM cells (n = 6 wells/promoter construct) were transfected with the chimeric promoters driving the expression of EGFP. Five days after transfection, EGFP signals in the transfected cells were measured using a BioTEK Cytation5 imaging reader.

Example 2: Chimeric promoters confer cardiac-specific gene expression in nonhuman primates (NHP) and mice

[0328] The twelve chimeric promoter constructs that yielded the highest GFP expression in iPSC-derived cardiomyocytes from Example 1 were further tested in NHP and mice to measure the promoter strength (FIG. 2A). A 400 bp natural promoter sequence from the human TNNT2 gene (HuTNNT2 400bp; see Table 2, SEQ ID NO: 6) was used as a reference and control. HuTNNT2 400 bp and each of the twelve chimeric promoter constructs was DNA barcoded and cloned into an AAV9 vector. Two NHP and four mice were dosed with 4x10¹¹ genome copies (gc)/kg intravenously. Four weeks after administration, heart and liver tissue were dissected from the animals. RNA was isolated from the tissues and the DNA barcode of each construct was used to identify and quantify expression by next generation sequencing (NGS). As shown in FIG. 2B, the chimeric promoter 6C2 showed cardiac-specific gene expression in NHP as well as in mice. In NHP, expression from chimeric promoter 6C2 was comparable to expression from HuTNNT2 400 bp promoter. In mice, expression from 6C2 was about 50% compared to the HuTNNT2 400 bp promoter. A schematic of the chimeric promoter 6C2, which is composed of four 50 bp human TNNT2 promoter fragments and one 50 bp MYBPC3 promoter fragment, is shown in FIG. 3. The fragment from MYBPC3 promoter contains a transcription start site (TSS).

Example 3: Targeted modifications enhance chimeric promoter strength to drive gene expression over two-fold higher compared to human TNNT2 promoter

[0329] To further improve the strength of chimeric promoter 6C2, the transcription factor binding profile of 6C2 was compared to that of a 400 bp natural promoter sequence from the human, mouse, and chicken TNNT2 gene, respectively (HsTNNT2, MsTNNT2, and ChTNNT2). As shown in FIG. 4, 6C2 lacks a binding site for transcription factor SRF, which is present in the human, mouse, and chicken TNNT2 promoters. Therefore, a modified chimeric promoter, 6C2SRF, was generated from 6C2 by converting the 12 nucleotides between positions 99-112 of 6C2 to an SRF transcription factor binding site (FIG. 5A). As shown in FIG. 5B, insertion of an SRF binding site at the indicated site improved promoter strength of 6C2.

[0330] Next, another variation of the 6C2SRF promoter was generated by fusing a 65 bp CMV core promoter sequence that carries a TATA and a TSS to the 3’ end of the first 154 bp of 6C2SRF, giving rise to 6C2SRF_CMV core (also referred to as 6C2SRF219), a shorter chimeric promoter 219 bp in length (FIG. 6A). To compare their promoter strengths, iPSC-CM were transiently transfected with an AAV expression cassette encoding EGFP and each of HuTNNT2, chimeric 6C2, 6C2SRF, and 6C2SRF219. As shown in FIG. 6B, the 6C2 promoter yielded about 35% of expression compared with the 400 bp human TNNT2 promoter (HuTNNT2). Insertion of an SRF binding site in the 6C2SRF promoter increased the expression to about 50% compared with HuTNNT2. Fusion of the 65 bp CMV core promoter to the first 154 bp of 6C2SRF increased expression in iPSC-CM by 2.3-fold compared to that of HuTNNT2. These results show that shorter chimeric promoters can drive appreciable levels of cardiacspecific gene expression and enable expression of larger transgenes. Additionally, when combined with the use of core promoter sequences that carries a TATA and a TSS, chimeric promoters can drive expression to a higher level compared to a native cardiacspecific promoter.

Example 4: Synthetic promoters composed of an enhancer region sequence from a TNNT2 promoter and a CMV core promoter drive cardiac-specific gene expression in iPSC-CM

[0331] In an effort to develop short, synthetic promoters, mini cardiomyocyte specific promoters were designed by fusing a CMV core promoter sequence that carries a TATA and a TSS to the 3’ end of a shortened sequence derived from the transcription factor (TF) binding rich region of each of the wild-type human (HuTNNT2), mouse (MsTNNT2), and chicken (ChTNNT2) TNNT2 promoter (FIGS. 7A-7B). The following synthetic promoters were generated:

1 . Hu203: a 203 bp promoter made by fusing a 138 bp sequence from the TF binding rich region of the HuTNNT2 promoter with a 65 bp CMV core promoter;

2. Ms201 : a 201 bp promoter made by fusing a 136 bp sequence from the TF binding rich region of the MsTNNT2 promoter with a 65 bp CMV core promoter;

3. Ch281 : a 281 bp promoter made by fusing a 216 bp sequence from the TF binding rich region of the ChTNNT2 promoter with a 65 bp CMV core promoter.

[0332] As previously described in Example 3, iPSC-CM were transiently transfected with an AAV expression cassette encoding EGFP and one of the following promoters: (1 ) 400 bp human TNNT2 (HuTNNT2); (2) Hu203; (3) 400 bp mouse TNNT2 (MsTNNT2); (4) Ms201 ; (5) 400 bp chicken TNNT2 (ChTNNT2); and (6) Ch281. As shown in FIG. 7B, the mouse TNNT2 promoter was 1.25-fold stronger than the human counterpart in iPSC-CM, while the chicken TNNT2 promoter was weaker than the human TNNT2, with about 0.75-fold expression compared to the benchmark human TNNT2 promoter. When compared to their respective wild-type promoters, the Hu203 and the Ms201 mini promoters had reduced promoter strengths, while the Ch281 mini promoter had increased promoter activity. Notably, the Ch281 promoter yielded comparable expression to the human TNNT2 promoter. [0333] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMS I/We claim:

1 . A cardiac-specific chimeric promoter comprising a transcription factor (TF) binding rich region having one or more fragments derived from the promoter regions of one or more cardiac-specific genes.

2. The chimeric promoter of claim 1 , wherein the TF binding rich region comprises binding sites for one or more transcription factors selected from the group consisting of GATA4, MEF2C, TBX5, NKS2.5, HAND2, and SRF.

3. The chimeric promoter of claim 1 or 2, wherein the TF binding rich region comprises binding sites for transcription factors GATA4, MEF2C, TBX5, NKS2.5, and HAND2.

4. The chimeric promoter of any one of claims claim 1 -3, wherein the TF binding rich region comprises two or more fragments derived from the promoter regions of two or more cardiac-specific genes.

5. The chimeric promoter of claim 4, wherein the two or more cardiac-specific genes are selected from the group consisting of TNNT2, MYBPC3, MYH6, MYH7, MYL2, and MYL3.

6. The chimeric promoter of claim 4 or 5, wherein the two or more fragments are each about 50 bp in length.

7. The chimeric promoter of any one of claims 4-6, wherein the TF binding rich region comprises five fragments derived from the promoter regions of two or more cardiac-specific genes.

8. The chimeric promoter of claim 7, wherein the chimeric promoter is about 250 bp in length.

9. The chimeric promoter of claim 7 or 8, wherein the two or more cardiac-specific genes comprise TNNT2 and MYBPC3.

10. The chimeric promoter of any one of claims 7-9, wherein the two or more cardiacspecific genes are TNNT2 and MYBPC3.

11 . The chimeric promoter of claim 10, comprising a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1 .

12. The chimeric promoter of any one of claims 7-11 , further comprising a binding site for SRF.

13. The chimeric promoter of claim 12, comprising a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2.

14. The chimeric promoter of any one of claims 1 -13, wherein the chimeric promoter has at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, or at least 100% activity compared to a wild-type human TNNT2 promoter of SEQ ID NO: 6.

15. A cardiac-specific synthetic promoter comprising:

(i) a transcription factor (TF) binding rich region comprising one or more fragments derived from the promoter regions of one or more cardiacspecific genes; and

(ii) a core promoter comprising a TATA box and a transcription start site (TSS).

16. The synthetic promoter of claim 15, wherein the core promoter is a CMV core promoter.

17. The synthetic promoter of claim 16, wherein the CMV core promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13.

18. The synthetic promoter of any one of claims 15-17, wherein the TF binding rich region is less than 300 bp, less than 250 bp, or less than 200 bp in length.

19. The synthetic promoter of any one of claims 15-18, wherein the TF binding rich region comprises binding sites for one or more transcription factors selected from the group consisting of GATA4, MEF2C, TBX5, NKS2.5, HAND2, and SRF.

20. The synthetic promoter of claim 19, wherein the TF binding rich region comprises binding sites for transcription factors GATA4, MEF2C, TBX5, and SRF.

21 . The synthetic promoter of any one of claims claim 15-20, wherein the TF binding rich region comprises two or more fragments derived from the promoter regions of two or more cardiac-specific genes.

22. The synthetic promoter of claim 21 , wherein the two or more cardiac-specific genes are selected from the group consisting of TNNT2, MYBPC3, MYH6, MYH7, MYL2, and MYL3.

23. The synthetic promoter of claim 21 or 22, wherein the two or more cardiac-specific genes comprise TNNT2 and MYBPC3.

24. The synthetic promoter of any one of claims 21 -23, wherein the two or more cardiac-specific genes are TNNT2 and MYBPC3.

25. The synthetic promoter of claim 24, wherein the TF binding rich region comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3.

26. The synthetic promoter of claim 25, wherein the synthetic promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14.

27. The synthetic promoter of any one of claims claim 15-20, wherein the TF binding rich region is derived from the promoter region of a cardiac-specific gene.

28. The synthetic promoter of claim 27, wherein the cardiac-specific gene is selected from the group consisting of human TNNT2, mouse TNNT2, and chicken TNNT2.

29. The synthetic promoter of claim 28, wherein the cardiac-specific gene is human TNNT2.

30. The synthetic promoter of claim 29, wherein the TF binding rich region comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8.

31 . The synthetic promoter of claim 30, wherein the synthetic promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15.

32. The synthetic promoter of claim 28, wherein the cardiac-specific gene is mouse TNNT2.

33. The synthetic promoter of claim 32, wherein the TF binding rich region comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 10.

34. The synthetic promoter of claim 33, wherein the synthetic promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16.

35. The synthetic promoter of claim 28, wherein the cardiac-specific gene is chicken TNNT2.

36. The synthetic promoter of claim 35, wherein the TF binding rich region comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 12.

37. The synthetic promoter of claim 36, wherein the synthetic promoter comprises a nucleotide sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17.

38. The synthetic promoter of any one of claims 28-37, wherein the synthetic promoter has at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, or at least 100% activity compared to a wild-type human TNNT2 promoter of SEQ ID NO: 6.

39. A vector comprising the cardiac-specific chimeric promoter of any one of claims 1 -14 or the cardiac-specific synthetic promoter of any one of claims 14-38.

40. The vector of claim 39, further comprising a transgene.

41 . The vector of claim 39 or 40, wherein the vector is a viral vector.

42. The vector of claim 41 , wherein the viral vector is an adeno-associated virus (AAV) vector.

-I SO-

43. The vector of claim 42, wherein the AAV vector is an AAV9 vector.

44. A recombinant AAV (rAAV) virus or virion, comprising the vector of any one of claims 39-43 and a capsid protein described herein.

45. A cell comprising the vector of any one of claims 39-43 or the rAAV virus or virion of claim 44.

46. A pharmaceutical composition comprising (i) the vector of any one of claims 39- 43, the rAAV virus or virion of claim 44, or the cell of claim 45; and (ii) a pharmaceutically acceptable excipient.

47. A method of treating a condition or disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 46.