Compositions and methods for treating a heart disease

Abstract

The present disclosure provides methods for treating a heart disease using a vector comprising a p21 polynucleotide and compositions for the same.

Description

Docket No.10504-106WO1 ^ ^ COMPOSITIONS AND METHODS FOR TREATING A HEART DISEASE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 663,811, ^^ filed June 25, 2024, the entirety of which is hereby incorporated by reference herein for all purposes. REFERNCE TO SEQUENCE LISTING

[0002] The sequence listing submitted on June 24, 2025, as an .XML entitled “10504- 106WO1.xml” created on June 24, 2025, and having a file size of 40,985 bytes is hereby ^^^ incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant numbers HL136824, HL160890, HL167955, and HL169784 awarded by the National Institutes of ^^^ Health. The government has certain rights in this invention. BACKGROUND OF THE INVENTION

[0004] In the last 30 years, there has been a dramatic increase in the understanding of the genetic and non-genetic mechanisms that contribute to the development of cardiomyopathy in humans. Despite this increased mechanistic understanding, the risk of ^^^ heart failure and sudden death remains high in this patient population. While multiple small molecule agents reduce the morbidity and mortality of patients with cardiomyopathy, many patients still progress to end stage disease. In addition, these small molecule agents act in a non-cell type specific manner and their utilization is often limited by hypotension and other systemic side effects. Likewise, pharmacological ^^^ therapy to prevent sudden death in individuals with cardiomyopathy remains lacking, and many patients still require an implantable cardiac defibrillator (ICD). Therefore, new strategies are required to reduce disease progression and sudden death in patients with cardiomyopathy.

[0005] Accordingly, what are needed are improved compositions and methods for ^^^ treating a heart disease.

[0006] Provided herein are compositions and methods that solve the problems or inadequacies of the prior art. ^Docket No.10504-106WO1 ^ ^ SUMMARY OF THE INVENTION

[0007] In some aspects, disclosed herein is a method of treating a heart disease in a subject comprising administering to the subject a therapeutically effective amount of a vector comprising a polynucleotide encoding a p21 polynucleotide sequence. ^^

[0008] In some embodiments, the p21 polynucleotide has at least 70% identity to SEQ ID NO:4. In some embodiments, the p21 polynucleotide has at least 80% identity to SEQ ID NO:4. In some embodiments, the p21 polynucleotide has at least 90% identity to SEQ ID NO:4. In some embodiments, the vector comprises SEQ ID NO:4.

[0009] In some embodiments, the p21 polynucleotide has at least 70% identity to SEQ ^^^ ID NO:8. In some embodiments, the p21 polynucleotide has at least 80% identity to SEQ ID NO:8. In some embodiments, the p21 polynucleotide has at least 90% identity to SEQ ID NO:8. In some embodiments, the vector comprises SEQ ID NO:8.

[0010] In some embodiments, the p21 polynucleotide has at least 70% identity to SEQ ID NO:16. In some embodiments, the p21 polynucleotide has at least 80% identity to ^^^ SEQ ID NO:16. In some embodiments, the p21 polynucleotide has at least 90% identity to SEQ ID NO:16. In some embodiments, the vector comprises SEQ ID NO:16.

[0011] In some embodiments, the p21 polypeptide has at least 80% identity to SEQ ID NO:7 or SEQ ID NO:20. In some embodiments, the p21 polypeptide has at least 90% identity to SEQ ID NO:7 or SEQ ID NO:20. In some embodiments, the p21 polypeptide ^^^ has at least 95% identity to SEQ ID NO:7 or SEQ ID NO:20. In some embodiments, the p21 polypeptide comprises SEQ ID NO:7, SEQ ID NO:20 or a functional fragment thereof.

[0012] In some embodiments, the p21 polypeptide has at least 80% identity to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25. In some ^^^ embodiments, the p21 polypeptide has at least 90% identity to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25. In some embodiments, the p21 polypeptide has at least 95% identity to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25. In some embodiments, the p21 polypeptide comprises SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID ^^^ NO:25 or a functional fragment thereof.

[0013] In some embodiments, the vector further comprises a cardiomyocyte-specific promoter polynucleotide. In some embodiments, the cardiomyocyte-specific promoter polynucleotide comprises a cardiac troponin T (cTnT) promoter. In some embodiments, ^Docket No.10504-106WO1 ^ ^ the cardiomyocyte-specific promoter polynucleotide comprises SEQ ID NO:3, or a functional fragment thereof.

[0014] In some embodiments, the vector further comprises at least one internal terminal repeat (ITR) sequence. In some embodiments, the at least one ITR sequence comprises ^^ SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:18, or functional fragments thereof. In some embodiments, the vector comprises a polyadenylated (polyA) terminator sequence. In some embodiments, the polyA terminator sequence comprises a simian virus 40 (SV40) polyA terminator sequence. In some embodiments, the polyA terminator sequence comprises SEQ ID NO:5, SEQ ID NO:17, or a functional fragment ^^^ thereof.

[0015] In some embodiments, the vector comprises an adeno-associated vector (AAV). In some embodiments, the AAV is an AAV1, AAV2, AAV6, AAV7, AAV9 or AAVrh10. In some embodiments, the AAV is an AAV9 vector.

[0016] In some embodiments, the method selectively increases p21 expression in ^^^ cardiomyocytes in the subject relative to an untreated control. In some embodiments, the method increases a level of p21 polypeptide in a nucleus of a cardiomyocyte in the subject relative to an untreated control.

[0017] In some embodiments, the heart disease comprises a cardiomyopathy. In some embodiments, the cardiomyopathy comprises a dilated cardiomyopathy, a hypertrophic ^^^ cardiomyopathy, an arrhythmogenic cardiomyopathy, a restrictive cardiomyopathy, or a combination thereof. In some embodiments, the cardiomyopathy comprises a hypertrophic cardiomyopathy.

[0018] In some embodiments, the cardiomyopathy comprises a genetically-linked cardiomyopathy. In some embodiments, the genetically-linked cardiomyopathy ^^^ comprises a mutation in one or more genes selected from myosin heavy chain 6 (MYH6), ^-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), ^-tropomyosin (TPM1), cardiac actin (ACTC1), essential myosin light chain 3 (MYL3), regulatory myosin light chain (MYL2), ribosomal binding protein 20 (RBM20), titan (TTN), and lamin A (LMNA). In ^^^ some embodiments, the genetically-linked cardiomyopathy comprises a mutation in MYH6.

[0019] In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS ^Docket No.10504-106WO1 ^ ^

[0020] Figure 1 shows a schematic of the vector map of pAAV-cTnT-mCdkn1a also referred to as AAV9-cTnT-mCdkn1a.

[0021] Figure 2 shows the in vivo injection of AAV9-cTnT-mCdkn1a increases left ventricular p21 protein levels. One day old Cdkn1a- / -(p21 null) mice were injected with ^^ either AAV9-cTnT-GFP or AAV9-cTnT-Cdkn1a virus (2x1011v.g. / per mouse). At 25 days of age left ventricular myocardial tissue lysates were obtained and immunoblots were performed for p21 and ^-actin protein in both groups of mice.

[0022] Figure 3(A-C) shows the in vivo injection of AAV9-cTnT-mCdkn1a reduces left ventricular hypertrophy in a murine model of hypertrophic cardiomyopathy. One day old ^^^ Myh6R404Q / WTmice were injected with either AAV9-cTnT-GFP or AAV9-cTnT-Cdkn1a virus (2x1011v.g. / per mouse). Echocardiograms were performed at 90 days of age in Ctl (wild type mice) or Myh6R404Q / WTmice injected with AAV9-cTnT-GFP or AAV9-cTnT- Cdkn1a. The following parameters were measured: Figure 3A shows the interventricular septal thickness at end diastole (IVSd), Figure 3B shows the left ventricular posterior ^^^ wall thickness at end diastole (LVPWd), and Figure 3C shows the left ventricular internal diameter at end diastole (LVIDd). N=5-6 / group. Mean + / - SEM.

[0023] Figure 4(A-D) shows that the in vivo injection of AAV9-cTnT-mCdkn1a improves left ventricular diastolic function in a murine model of hypertrophic cardiomyopathy. One day old Myh6R404Q / WTmice were injected with either AAV9-cTnT- ^^^ GFP or AAV9-cTnT-Cdkn1a virus (2x1011v.g. / per mouse). Echocardiograms were performed at 90 days of age in Ctl (wild type mice) or Myh6R404Q / WTmice injected with AAV9-cTnT-GFP or AAV9-cTnT-Cdkn1a. The following parameters were measured: Figure 4A shows the ratio of peak mitral inflow velocity from left ventricular relaxation in early diastole (E wave) to peak mitral inflow velocity in late diastole caused by atrial ^^^ contraction (A wave). Figure 4B shows the ratio of peak mitral inflow velocity from left ventricular relaxation in early diastole (E wave) to mitral annular early diastolic velocity measured by tissue doppler (e’ wave). Figure 4C shows the isovolumic relaxation time (IVRT). Figure 4D shows the left ventricular systolic fractional shortening (FS). N=5- 6 / group. Mean + / - SEM. ^^^

[0024] Figure 5(A-F) shows that in vitro knockdown of CDKN1A in human cardiomyocytes causes increased cardiomyocyte hypertrophy after serum stimulation. (A) Knockdown of p21 in hiPSC-CMs using siRNA targeting CDKN1A or scrambled control siRNA (Ctl). Western blots were then performed for p21 (SCBT, sc-6246) and GAPDH (n=2-3). (B) Representative immunofluorescence staining and (C) ^Docket No.10504-106WO1 ^ ^ quantification of Ki67 from control and CDKN1A siRNA knockdown hiPSC-CMs stimulated with or without serum (n=4). Scale bars, 50 µm. Minimum 100 nuclei / sample. (D) Representative WGA staining with nuclei labeled by DAPI from control and CDKN1A siRNA knockdown hiPSC-CMs stimulated with or without serum. Scale bars, ^^ 70^m. (E) Relative quantification of cardiomyocyte DNA content (relative to Ctl siRNA without serum) and (F) quantification of cardiomyocyte area in control and CDKN1A siRNA knockdown hiPSC-CMs stimulated with or without serum (n=4). Minimum 100 CM / sample. All results are shown as mean±SEM.

[0025] Figure 6(A-B) shows in vitro overexpression of CDKN1A in human ^^^ cardiomyocytes causes reduced cardiomyocyte hypertrophy after serum stimulation. (A) Representative WGA images and (B) quantification of cardiomyocyte area from GFP and CDKN1A-Flag plasmid transfected hiPSC-CMs stimulated with serum for 24 hours (n=4). Minimum 50 CM / sample. CDKN1A overexpression was confirmed with Flag immunostaining. GFP plasmid was used as control. Scale bars, 50 ^m. ^^^ DETAILED DESCRIPTION OF THE INVENTION

[0026] Provided herein are methods of treating a heart disease in a subject that include administering to the subject a therapeutically effective amount of a vector comprising a polynucleotide encoding a p21 polypeptide. It is a surprising finding disclosed herein ^^^ that administration of a vector encoding a p21 polypeptide, and thereby increasing expression of the p21 polypeptide results in improved cardiac function and / or reduced pathologic remodeling in subjects with a heart disease.

[0027] Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicant desires that the ^^^ following terms be given the particular definition as defined below. Terminology

[0028] As used in the specification and claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. ^^^

[0029] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%. ^Docket No.10504-106WO1 ^ ^

[0030] The term “administering” refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The ^^ term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

[0031] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting ^^^ essentially of" when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. ^^^ "Consisting of" shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.

[0032] A "control" is an alternative subject or sample used in an experiment for ^^^ comparison purpose. A control can be "positive" or "negative."

[0033] As used herein, the term “expression” refers to either or both “gene expression” and “protein expression.” “Gene expression” refers to the process by which polynucleotides are transcribed into mRNA and “protein expression” refers to the process by which mRNA is translated into peptides, polypeptides, or proteins. If the ^^^ polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. “Gene overexpression” refers to the overproduction of the mRNA transcribed from the gene, at a level that is at least about 2.5 times higher, at least about 5 times higher, or at least about 10 times higher than the expression level detected in a control sample. “Protein overexpression” includes the overproduction of the protein ^^^ product encoded by a gene at a level that is at least about 2.5 times higher, at least about 5 times higher, or at least about 10 times higher than the expression level detected in a control sample.

[0034] The term "identity" shall be construed to mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical with the bases or ^Docket No.10504-106WO1 ^ ^ residues of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed ^^ as reducing identity. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) that has a certain percentage (for example, 80%, 85%, 90%, or 95%) of " identity" to another sequence means that, when aligned over their full lengths, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the sequence identity can be determined using software programs known ^^^ in the art. In one embodiment, default parameters are used for alignment. In one embodiment a BLAST program is used with default parameters. In one embodiment, BLAST programs BLASTN and BLASTP are used with the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; ^^^ Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR.

[0035] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. ^^^

[0036] A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ^^^ ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g., enhancers and coding sequences) do not have to be contiguous. Linking is accomplished by ligation at ^^^ convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In embodiments, a promoter is operably linked with a coding sequence when it is capable of affecting (e.g., modulating relative to the absence of the promoter) the expression of a protein from that ^Docket No.10504-106WO1 ^ ^ coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

[0037] The terms “pharmaceutically effective amount”, “therapeutically effective amount” and “therapeutically effective dose” refer to the amount of a compound such as ^^ a vector encoding a p21 polypeptide that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. In some embodiments, a desired response is a heart function improvement. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject ^^^ over a period of days, weeks, or years. The terms “pharmaceutically effective amount”, “therapeutically effective amount” or “therapeutically effective dose” include that amount of a compound such as a vector encoding a p21 polypeptide that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated. The therapeutically ^^^ effective amount will vary depending on the compound such as a vector encoding a p21 polypeptide, the disorder or conditions and its severity, the route of administration, time of administration, rate of excretion, drug combination, judgment of the treating physician, dosage form, and the age, weight, general health, sex and / or diet of the subject to be treated. In the context of the present method, a pharmaceutically or therapeutically ^^^ effective amount or dose of a vector encoding a p21 polypeptide includes an amount that is sufficient to improve a heart function, reduce a thickening of heart muscle, reduce progression of a thickening of heart muscle, reduce myofiber disarray, reduce progression of myofiber disarray, reduce atrial fibrillation, reduce progression of atrial fibrillation, reduce blockage of blood flow leaving the heart, reduce mitral valve disease, ^^^ reduce progression of a mitral valve disease, reduce progression of heart failure, reduce dilated cardiomyopathy, reduce progression of dilated cardiomyopathy.

[0038] As used herein, the terms “reduce,” “reduced” and “reducing” mean to decrease by a statistically significant amount. In some embodiments, the reduction is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70, about 80%, about ^^^ 90%, or about 95%.

[0039] The term “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human. ^Docket No.10504-106WO1 ^ ^

[0040] The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and / or alleviating, mitigating or impeding one or more causes of a disorder or condition. ^^ Treatments according to the invention may be applied preventively, prophylactically, palliatively, or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of heart disease), during early onset (e.g., upon initial signs and symptoms of heart disease), or after an established development of heart disease. Prophylactic administration can occur for several days to years prior to the manifestation ^^^ of symptoms of an infection.

[0041] In some instances, the terms “treat”, “treating”, “treatment” and grammatical variations thereof, include one or more of reducing a thickening of heart muscle, reducing progression of a thickening of heart muscle, reducing myofiber disarray, reducing progression of myofiber disarray, reducing atrial fibrillation, reducing ^^^ progression of atrial fibrillation, reducing blockage of blood flow leaving the heart, reducing mitral valve disease, reducing progression of a mitral valve disease, reducing progression of heart failure, reducing dilated cardiomyopathy, reducing progression of dilated cardiomyopathy, improving diastolic function, and improving a heart condition as compared with prior to treatment of the subject or as compared with the incidence of ^^^ such symptom in a general or study population. In some embodiments, “treating” comprises improving diastolic function, including, but not limited to, improving left ventricular diastolic function. In some embodiments, “treating” comprises reducing progression of a thickening of heart muscle. In some embodiments, “treating” comprises reducing left ventricular hypertrophy. Methods for measuring diastolic function and ^^^ thickening of heart muscle are known in the art.

[0042] The word “vector” refers to any vehicle that carries a polynucleotide into a cell for the expression of the polynucleotide in the cell. The vector may be, for example, a plasmid, a phage particle, or a nanoparticle. 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. In some embodiments, the vector is a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of affecting the expression of the DNA in a suitable host cell. Such control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome ^Docket No.10504-106WO1 ^ ^ binding sites, and sequences which control the termination of transcription and translation. In other embodiments, the vector is a lipid nanoparticle. Lipid nanoparticles can be used to deliver mRNA to a host cell for expression of the mRNA in the host cell. Compositions and Methods ^^

[0043] In some aspects, disclosed herein is a method of treating a heart disease in a subject comprising administering to the subject a therapeutically effective amount of a vector comprising a polynucleotide encoding a p21 polypeptide.

[0044] As used herein, “p21” refers herein to a polypeptide that, in humans, is encoded by the CDKN1A gene or a corresponding cDNA, or a modification or fragment thereof as ^^^ described herein. The p21 polypeptide is a cyclin-dependent kinase inhibitor. Thus, it should be noted that p21 and Cdkn1a can be used interchangeably. For example, the p21 polypeptide can also be referred to as a Cdkn1a polypeptide, and the CDKN1A gene can be referred to as a P21 gene. In some embodiments, the p21 polypeptide is as identified in the following database: UniProtKB / SwissProt: P38936 or P39689. In some ^^^ embodiments, the p21 polypeptide comprises SEQ ID NO:7, or a functional fragment thereof. In some embodiments, the p21 polypeptide comprises a sequence having at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:7, or a polypeptide comprising a fragment of SEQ ID NO:7, wherein the p21 polypeptide retains the function of SEQ ID NO:7. In some ^^^ embodiments, the p21 polypeptide comprises SEQ ID NO: 9. In some embodiments, the p21 polypeptide comprises a polypeptide sequence having at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:9, or a polypeptide comprising a fragment of SEQ ID NO:9, wherein the p21 polypeptide retains the function of SEQ ID NO:9. In some embodiments, the p21 ^^^ polypeptide comprises a polypeptide sequence having at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO:20, or a polypeptide comprising a fragment of SEQ ID NO:20, wherein the p21 polypeptide retains the function of SEQ ID NO:20.

[0045] SEQ ID NO:20 ^^^ MSEPAGDVRQNPCGSKACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNFDF VTETPLEGDFAWERVRGLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGTAE EDHVDLSLSCTLVPRSGEQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFSKR KP ^Docket No.10504-106WO1 ^ ^

[0046] In some embodiments, the p21 polypeptide comprises a modification at an end. In some embodiments, the N-terminal modification increases p21 protein levels by the reduction of p21 protein degradation. In some embodiments, the polypeptide sequence comprises SEQ ID NO:21, or a functional fragment thereof. In some embodiments, the ^^ p21 polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:21. In some embodiments, the polynucleotide sequence comprises SEQ ID NO:22, or a functional fragment thereof. In some embodiments, p21 polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, ^^^ about 95%, about 98%, or about 99% identity with SEQ ID NO:22. In some embodiments, the polynucleotide sequence comprises SEQ ID NO:23, or a functional fragment thereof. In some embodiments, p21 polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:23. In some embodiments, the ^^^ polynucleotide sequence comprises SEQ ID NO:24, or a functional fragment thereof. In some embodiments, p21 polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:24. In some embodiments, the polynucleotide sequence comprises SEQ ID NO:25, or a functional fragment thereof. In some embodiments, p21 ^^^ polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:25.

[0047] SEQ ID NO:21:^^^ DKSEPAGDVRQNPCGSKACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNFD FVTETPLEGDFAWERVRGLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGTA EEDHVDLSLSCTLVPRSGEQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFSK RKP

[0048] SEQ ID NO:22 ^^^ MEQKLISEEDLEQKLISEEDLEQKLISEEDLEQKLISEEDLEQKLISEEDLYPYDVP DYASEPAGDVRQNPCGSKACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNF DFVTETPLEGDFAWERVRGLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGT AEEDHVDLSLSCTLVPRSGEQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFS KRKP ^Docket No.10504-106WO1 ^ ^

[0049] SEQ ID NO:23 MEQKLISEEDLEQKLISEEDLEQKLISEEDLEQKLISEEDLEQKLISEEDLSEPAGD VRQNPCGSKACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNFDFVTETPLEG DFAWERVRGLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGTAEEDHVDLSL ^^ SCTLVPRSGEQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFSKRKP

[0050] SEQ ID NO:24 MEQKLISEEDLEQKLISEEDLEQKLISEEDLEQKLISEEDLEQKLISEEDLEQKLISE EDLSEPAGDVRQNPCGSKACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNF DFVTETPLEGDFAWERVRGLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGT ^^^ AEEDHVDLSLSCTLVPRSGEQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFS KRKP

[0051] SEQ ID NO:25 MEQKLISEEDLEQKLISEEDLEQKLISEEDLEQKLISEEDLSEPAGDVRQNPCGSK ACRRLFGPVDSEQLSRDCDALMAGCIQEARERWNFDFVTETPLEGDFAWERVR ^^^ GLGLPKLYLPTGPRRGRDELGGGRRPGTSPALLQGTAEEDHVDLSLSCTLVPRSG EQAEGSPGGPGDSQGRKRRQTSMTDFYHSKRRLIFSKRKP

[0052] In some embodiments, the p21 polynucleotide sequence is identified in one or more publicly available databases as follows: HGNC: 1784, NCBI Gene: 1026, Ensembl: ENSG00000124762, and OMIM: 116899. In some embodiments, the p21 polynucleotide ^^^ sequence comprises SEQ ID NO:4, or a functional fragment thereof. In some embodiments, the p21 polynucleotide sequence comprises SEQ ID NO:8, or a functional fragment thereof. In some embodiments, the p21 polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:8. In some embodiments, the p21 ^^^ polynucleotide sequence is identified by the NCBI Reference identifier: NM_000389.5. In some embodiments, the p21 polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:4.

[0053] In some embodiments, the p21 polynucleotide sequence comprises a modification ^^^ at the 5’ end. In some embodiments, the p21 polypeptide sequence comprises a modification at the amino (N) terminal. In some embodiments, the N-terminal modification increases p21 protein levels by the reduction of p21 protein degradation. In some embodiments, the polynucleotide sequence comprises SEQ ID NO:10, or a functional fragment thereof. In some embodiments, the p21 polynucleotide sequence ^Docket No.10504-106WO1 ^ ^ comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:10. In some embodiments, the polynucleotide sequence comprises SEQ ID NO:11, or a functional fragment thereof. In some embodiments, p21 polynucleotide sequence comprises at or ^^ greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:11. In some embodiments, the polynucleotide sequence comprises SEQ ID NO:12, or a functional fragment thereof. In some embodiments, p21 polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about ^^^ 99% identity with SEQ ID NO:12. In some embodiments, the polynucleotide sequence comprises SEQ ID NO:13, or a functional fragment thereof. In some embodiments, p21 polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:13. In some embodiments, the polynucleotide sequence comprises SEQ ID NO:14, ^^^ or a functional fragment thereof. In some embodiments, p21 polynucleotide sequence comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with SEQ ID NO:14.

[0054] A non-limiting example of a vector disclosed herein is an adeno-associated virus (AAV) vector. There are multiple AAV serotypes including AAV1, AAV2, AAV3, ^^^ AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV2 / 9n, AAVrh10 and AAV helper. In some embodiments, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV7 and AAVrh10. In some embodiments, the AAV vector is an AAV1 vector. In some embodiments, the AAV vector is an AA26 vector. In some embodiments, the AAV vector is an AAV7 vector. In some ^^^ embodiments, the AAV vector is an AAV2 / 9n vector (RepCap Addgene 112865). In some embodiments, the AAV vector is an AAVrh10 vector. In some embodiments, the AAV vector is selected from the group consisting of AAV6, AAV8 and AAV9. In some embodiments, the AAV vector is an AAV6 vector (RepCap, Addgene 110770). In some embodiments, an AAV helper vector is also administered the cell. In some ^^^ embodiments, the AAV helper vector is an AAV helper (Addgene 112867).

[0055] In some embodiments, the AAV vector is an AAV9 vector. In some embodiments the AAV vector further comprises one or more elements shown in Figure 1. In some embodiments, the vector comprises at or greater than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity with ^Docket No.10504-106WO1 ^ ^ SEQ ID NO:1 or SEQ ID NO:19. In some embodiments, the vector comprises SEQ ID NO:1 or SEQ ID NO:19. In some embodiments, the vector comprises one or more elements shown in Figure 1 and wherein the mouse Cdkn1a polynucleotide is replaced with a human Cdkn1a polynucleotide. ^^

[0056] An AAV vector can, in certain aspects, contain a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene, such as a CDKN1A gene. “Heterologous” in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus. Typically, the AAV and B19 coding regions have^^^ been deleted. The AAV ITRs, or modifications thereof, confer infectivity and site- specific integration, and the promoter directs cell-specific expression. United States Patent No.6,261,834 is herein incorporated by reference for material related to the AAV vector.

[0057] Accordingly, in some embodiments, the vector comprises one or more regulatory ^^^ components (such as, for example, an internal terminal repeat (ITR) sequence, or a polyadenylate (polyA) tail) necessary for AAV replication, packaging, and / or vector persistence. In some embodiments, the vector comprises at least one ITR sequence. In some embodiments, the at least one ITR sequence comprises SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:18, or functional fragments thereof. In some ^^^ embodiments, the vector comprises a polyadenylated (polyA) terminator sequence. In some embodiments, the polyA terminator sequence is a simian virus 40 (SV40) polyA terminator sequence. In some embodiments, the polyA terminator sequence comprises SEQ ID NO:5, SEQ ID NO:17, or a functional fragment thereof.

[0058] In other or further embodiments, the vector comprises a cardiomyocyte-specific ^^^ promoter polynucleotide. The cardiomyocyte-specific promoter can be a cardiac troponin T (cTnT) promoter, an alpha myosin heavy chain (^-MHC / MYH6) promoter, a beta myosin heavy chain (β-MHC / MYH7) promoter an alpha cardiac actin (^-actin) promoter, a myosin light chain-2v (MLC-2v) promoter, an atrial natriuretic peptide (ANP) promoter, or other appropriate promoter. In some aspects, the cardiomyocyte- ^^^ specific promoter is a cTnT promoter, and in some further embodiments, the cardiomyocyte-specific promoter polynucleotide comprises SEQ ID NO:3, or a functional fragment thereof.

[0059] Use of a cardiomyocyte-specific promoter can result in the selective increase in p21 expression in cardiomyocytes of the subject. The term “selectively increased” refers ^Docket No.10504-106WO1 ^ ^ to a statistically significant increase in expression in a limited cell type, the cardiomyocyte, without a same increase in other cell types. In some embodiments, there is an about 0% increase in p21 expression in non-cardiomyocytes. In some embodiments, there is an about 1%, about 2%, about 3%, about 4%, about 5%, or about ^^ 10% increase in p21 expression in non-cardiomyocytes.

[0060] In some embodiments, the method selectively increases p21 expression in cardiomyocytes in the subject relative to an untreated control. In some embodiments, the method selectively increases p21 expression by 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, ^^^ 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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%, 100%, or more in cardiomyocytes in the ^^^ subject relative to an untreated control. In some embodiments, the method increases a level of p21 polypeptide in a nucleus of a cardiomyocyte relative to an untreated control. In some embodiments, the method increases a level of p21 polypeptide in a nucleus of a cardiomyocyte by 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, ^^^ 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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%, 100%, or more in the subject relative to an untreated control. ^^^

[0061] The methods disclosed herein are for treating a heart disease. As used herein, “heart disease” refers to any condition involving a dysfunction in cardiomyocytes. In some embodiments, the heart disease comprises a cardiomyopathy. In some embodiments, the cardiomyopathy comprises a dilated cardiomyopathy, a hypertrophic cardiomyopathy, an arrhythmogenic cardiomyopathy, a restrictive cardiomyopathy, or a ^^^ combination thereof. In some embodiments, the cardiomyopathy comprises a hypertrophic cardiomyopathy. “Hypertrophic cardiomyopathy” refers to a thickening of the heart muscle or tissue. In some embodiments, the hypertrophic cardiomyopathy comprises a thickening of the septal wall of the heart. In other embodiments, the hypertrophic cardiomyopathy comprises a thickening in a right ventricle or a left ^Docket No.10504-106WO1 ^ ^ ventricle. In some embodiments, the hypertrophic cardiomyopathy is obstructive. In other embodiments the hypertrophic cardiomyopathy is nonobstructive. In some embodiments, the heart disease is preclinical, including, but not limited to preclinical hypertrophic cardiomyopathy. ^^

[0062] The cardiomyopathy treated according to the present methods can be genetically- linked or non-genetically linked. In some embodiments, the cardiomyopathy comprises a genetically-linked cardiomyopathy. As used herein, “genetically-linked” means a disease that is caused or worsened at least in part by a change or mutation in a gene as compared to a general or study population. A genetically-linked cardiomyopathy can include a ^^^ cardiomyopathy caused, at least in part, by a mutation in one or more genes selected from, but not limited to, myosin heavy chain 6 (MYH6), ^-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), ^-tropomyosin (TPM1), cardiac actin (ACTC1), essential myosin light chain 3 (MYL3), regulatory myosin light chain (MYL2), ribosomal binding protein 20^^^ (RBM20), titan (TTN), and lamin A (LMNA). In some embodiments, the genetically- linked cardiomyopathy can include a cardiomyopathy caused, at least in part, by a mutation in MYH6. Using a preclinical mouse model with mutation in MYH6, it was determined that p21 could be a potential target for cardiomyopathy progression. However, both genetic and non-genetic cardiomyopathies associated with left ventricular ^^^ hypertrophy would be eligible to be treated with this method.

[0063] The method disclosed herein can be performed any time prior to and / or after the onset of a heart disease of any preceding aspect. In some embodiments, the disclosed methods can be performed 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, ^^^ 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years;12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 months; 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 days; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours prior to the onset of a heart disease of any preceding aspect; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120 minutes; 3, 4, 5, ^^^ 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, 60, 90 or more days; 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, ^Docket No.10504-106WO1 ^ ^ 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 years after the onset of a heart disease of any preceding aspect.

[0064] Dosing frequency for a vector of any preceding aspect, includes, but is not limited to, at least once every 12 months, once every 11 months, once every 10 months, ^^ once every 9 months, once every 8 months, once every 7 months, once every 6 months, once every 5 months, once every 4 months, once every 3 months, once every two months, once every month; or at least once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, or daily. In some embodiment, the interval between each administration is ^^^ less than about 4 months, less than about 3 months, less than about 2 months, less than about a month, less than about 3 weeks, less than about 2 weeks, or less than less than about a week, such as less than about any of 6, 5, 4, 3, 2, or 1 day. In some embodiment, the dosing frequency for the T cells disclosed herein includes, but is not limited to, at least once a day, twice a day, or three times a day. In some embodiment, the interval ^^^ between each administration is less than about 48 hours, 36 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, or 7 hours. In some embodiment, the interval between each administration is less than about 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, or 6 hours. In some embodiments, the interval between each administration is ^^^ constant. For example, the administration can be carried out daily, every two days, every three days, every four days, every five days, or weekly. Administration can also be continuous and adjusted to maintaining a level of the compound within any desired and specified range.

[0065] It should be understood that the foregoing relates to preferred embodiments of the ^^^ present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading ^^^ the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and / or the scope of the appended claims. All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety for all purposes. ^Docket No.10504-106WO1 ^ ^ EXAMPLES Example 1:

[0066] In vivo p21 vector to validate cardiomyocyte transduction:

[0067] p21 null mice (Cdkn1a- / -) were injected with AAV9-cTnT-GFP or AAV9-cTnT- ^^ mCdkn1a (Figure 1) virus (2x1011v.g. / per mouse) at 1 day of age and then euthanized at 25 days of age. Immunoblotting was performed on left ventricle tissue lysate using an anti-p21 antibody followed by a secondary antibody. See Figure 2. Chemiluminescence imaging was performed to visualize the p21 left ventricular protein levels. Data not shown. ^^^

[0068] In vivo p21 vector injection to test if cardiomyocyte p21 overexpression impacts left ventricular hypertrophy in a hypertrophic cardiomyopathy murine model:

[0069] One day old Myh6R404Q / WTmice were injected with either AAV9-cTnT-GFP or AAV9-cTnT-Myc / mCdkn1a virus (2x1011v.g. / per mouse). At 90 days of age the mice had transthoracic echocardiograms performed to measure the following: intraventricular ^^^ septal thickness at end diastole (IVSd) (Figure 3A), left ventricular posterior wall thickness at end diastole (LVPWd) (Figure 3B), left ventricular internal diameter at end diastole (LVIDd) (Figure 3C).

[0070] In vivo p21 vector injection to test if left ventricle diastolic function changes in a hypertrophic cardiomyopathy model: ^^^

[0071] One day old Myh6WT / R404Qmice were injected with either AAV9-cTnT-GFP or AAV9-cTnT-Myc / mCdkn1a virus (2x1011v.g. / per mouse). At 90 days of age the mice had transthoracic echocardiograms performed under isoflurane anesthesia. The following measurements were made: (1) Ratio of peak mitral inflow velocity from left ventricular relaxation in early diastole (E wave) to mitral annular early diastolic velocity measured ^^^ by tissue doppler (e’ wave) (Figure 4B). (2) isovolumic relaxation time (IVRT) (Figure 4C). (3) left ventricular systolic fractional shortening (FS) (Figure 4D).

[0072] Matured hiPSC-CMs were seeded onto an 8-well chamber slide. Pre-designed siRNA for CDKN1A or negative control siRNA to a final concentration of 100 nM was introduced into culture medium using LIPOFECTAMINE® RNAiMAX Transfection ^^^ Reagent. Media was refreshed every 48 hours with a new siRNA-Transfection Reagent mixture. Immunoblotting was performed on cell lysate after 8 days of transfection using an anti-p21 antibody followed by a secondary antibody (Figure 5A). After 8 days of transfection, cells were incubated with fetal bovine serum (10% v / v) for 24 hours followed by immunofluorescence staining using primary antibodies against Ki67 and ^Docket No.10504-106WO1 ^ ^ sarcomeric ^-actinin followed by the Alexa fluor 594 and 488 secondary antibodies (Figure 5B). Quantification of Ki67 (% positive nuclei) was performed from control and CDKN1A siRNA knockdown cells stimulated with or without serum (Figure 5C). For measuring cardiomyocyte size, hiPSC-CMs were fixed and stained with a wheat germ ^^ agglutinin (WGA) Texas red conjugate and mounted with DAPI mounting media (Figure 5D). Cardiomyocyte nuclear DNA contents were quantified by measuring DAPI intensity (Figure 5E) and cell areas were measured with WGA staining (Figure 5E).

[0073] Matured hiPSC-CMs were seeded in 8-well chamber slides, and 500 ng of CDKN1A-pcDNA3.1-C-(k)DYK and cTnT-GFP plasmid DNA was used with ^^^ Lipofectamine 3000 reagent for the transient transfections. The next day after 24 hours, cell media was changed with fresh media and stimulated with 10% serum. After 24 hours, hiPSCs were fixed, and immunofluorescence analysis was performed using primary antibodies against GFP or Flag followed by Alexa Fluor 594 secondary antibody and then stained with a WGA Texas Red conjugate (Figure 6A). Quantification of cell ^^^ area was performed using WGA staining (Figure 6B).

[0074] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods ^^^ disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. ^

Claims

Docket No.10504-106WO1 ^ ^ CLAIMS What is claimed is:

1. A method of treating a heart disease in a subject comprising administering to the subject a therapeutically effective amount of a vector comprising a p21 polynucleotide encoding a p21 polypeptide sequence. The method of claim 1, wherein the p21 polynucleotide has at least 70% identity to SEQ ID NO:8 or SEQ ID NO:

16.

3. The method of claim 1, wherein the p21 polynucleotide comprises SEQ ID NO:8 or SEQ ID NO:

16.

4. The method of claim 1, wherein the p21 polynucleotide has at least 70% identity to SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:

14.

5. The method of claim 1, wherein the p21 polynucleotide comprises SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:

14.

6. The method of claim 1, wherein the p21 polypeptide has at least 80% identity to SEQ ID NO:7 or SEQ ID NO:

20.

7. The method of claim 1, wherein the p21 polypeptide has at least 90% identity to SEQ ID NO:7 or SEQ ID NO:

20.

8. The method of claim 1, wherein the p21 polypeptide has at least 95% identity to SEQ ID NO:7 or SEQ ID NO:

20.

9. The method of claim 1, wherein the p21 polypeptide comprises SEQ ID NO:7, SEQ ID NO:20, or a functional fragment thereof.

10. The method of claim 1, wherein the p21 polypeptide has at least 80% identity to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:

25.

11. The method of any one of claims 1-10, wherein the vector further comprises a cardiomyocyte-specific promoter polynucleotide. ^Docket No.10504-106WO1 ^ ^ 12. The method of claim 11, wherein the cardiomyocyte-specific promoter polynucleotide comprises a cardiac troponin T (cTnT) promoter.

13. The method of claim 11 or 12, wherein the cardiomyocyte-specific promoter polynucleotide comprises SEQ ID NO:3 or a functional fragment thereof.

14. The method of any one of claims 1-13, wherein the vector further comprises at least one internal terminal repeat (ITR) sequence.

15. The method of claim 14, wherein the at least one ITR sequence comprises SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:15 or SEQ ID NO:18, or a functional fragment thereof.

16. The method of any one of claims 1-15, wherein the vector is an adeno-associated vector (AAV).

17. The method of claim 16, wherein the AAV is an AAV1, AAV2, AAV6, AAV7, AAV9 or AAVrh10.

18. The method of claim 17, wherein the AAV is an AAV9 vector.

19. The method of any one of claims 1-18, wherein the method selectively increases p21 expression in cardiomyocytes in the subject relative to an untreated control.

20. The method of any one of claims 1-19, wherein the method increases a level of p21 polypeptide in a nucleus of cardiomyocytes in the subject relative to an untreated control.

21. The method of any one of claims 1-20, wherein the subject is a human.

22. The method of any one of claims 1-21, wherein the heart disease comprises a cardiomyopathy. ^Docket No.10504-106WO1 ^ ^ 23. The method of claim 22, wherein the cardiomyopathy comprises a dilated cardiomyopathy, a hypertrophic cardiomyopathy, an arrhythmogenic cardiomyopathy, a restrictive cardiomyopathy, or a combination thereof.

24. The method of claim 22 or 23, wherein the cardiomyopathy comprises a hypertrophic cardiomyopathy.

25. The method of any one of claims 22-24, wherein the cardiomyopathy comprises a genetically-linked cardiomyopathy.

26. The method of claim 25, wherein the genetically-linked cardiomyopathy comprises a mutation in one or more genes selected from myosin heavy chain 6 (MYH6), ^-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), ^-tropomyosin (TPM1), cardiac actin (ACTC1), essential myosin light chain 3 (MYL3), regulatory myosin light chain (MYL2), ribosomal binding protein 20 (RBM20), titan (TTN), and lamin A (LMNA). The method of claim 25, wherein the genetically-linked cardiomyopathy comprises a mutation in a myosin heavy chain 6 (MYH6) gene. ^

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