WO2025024039A1

WO2025024039A1 - Methods and compositions for restoring gene expression

Info

Publication number: WO2025024039A1
Application number: PCT/US2024/030774
Authority: WO
Inventors: Zhihong Jane YANG
Original assignee: Tenaya Therapeutics Inc
Current assignee: Tenaya Therapeutics Inc
Priority date: 2023-07-21
Filing date: 2024-05-23
Publication date: 2025-01-30
Anticipated expiration: 2026-01-21

Abstract

Provided herein are methods for restoring gene expression of a plurality of genes in an individual having a heart disease or disorder. In some cases, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, thereby restoring gene expression of the plurality of genes, wherein the plurality of genes comprise one or more of Cacna1c, Ryr2, Asph, Atp2a2, Ank2, Casq2, Scn5a, Des, Jup, Dsp, Dsg2, Dsc2, Mapre1, Myh7, Mybpc3, Myoz2, Actn2, Tpm1, Myl3, Tnnc1, Myh6, Pln, Tnni3, Myl2, Tfn, Tnnt2, Trdn, Ttn, Gja1, Echs1, Cpt1b, Acadsb, Mpc1/2, L2hgdh, Pdp2, or Acss1, TGFβ1, P4HA1, Col1a1, Col3a1, Mmp2, Postn, or Timp1.

Description

METHODS AND COMPOSITIONS FOR RESTORING GENE EXPRESSION

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/515,043, filed July 21, 2023, and U.S. Provisional Application No. 63/606,807, filed December 6, 2023, each of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Mutations in the desmosome gene Plakophillin-2. PKP2, are the most frequent cause of genetic arrhythmogenic right ventricular cardiomyopathy (ARVC) and account for approximately 43% of ARVC cases. Mutations in the PKP2 gene are heterozygous in patients and lead to haploinsufficiency in PKP2 protein levels.

SUMMARY

[0003] In an aspect, provided herein are methods for restoring gene expression of a plurality of genes in an individual having a heart disease or disorder. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, thereby restoring gene expression of the plurality of genes, wherein the plurality of genes comprise one or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Pin, Ryr2, Scn5a, Tfin, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno -associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[0004] In another aspect, provided herein are methods of reducing gene expression of a plurality of genes in an individual having a heart disease or disorder. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, thereby reducing gene expression of the plurality of genes, wherein the plurality of genes comprises one or more of Collal, Col3al, Mmp2, Nppa, Nppb, P4HA1, Postn, TGFpi, or Timpl. In some embodiments, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH polyA) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[0005] In another aspect, provided herein are methods for monitoring efficacy of a treatment for a heart disease or disorder in an individual. In some embodiments, the method comprises: (a) administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) operatively linked to a promoter; and (b) measuring expression of one or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfin, TGF[31, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH polyA) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the method further comprises measuring one or more of fibrofatty tissue replacement; myocardial atrophy; ventricular dilation; ventricular arrhythmias; sudden cardiac death; exercise-triggered cardiac events; right ventricular cardiomyopathy, dilation, or heart failure; left ventricular cardiomyopathy, dilation, or heart failure; atrial arrhythmias; syncope; palpitations; shortness of breath; or chest pain. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof. In some embodiments, (b) comprises RT-qPCR, RNAseq, array hybridization, or probe hybridization.

[0006] In another aspect, provided herein are methods for monitoring efficacy of a treatment for a heart disease or disorder in an individual. In some embodiments, the method comprises: (a) administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) operatively linked to a promoter; and (b) measuring expression of one or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acatl, Acly, Acot3, Acsll, Acssl, Actn2, Ank2, Asph, Atp2a2, Bcatl, Bcat2, Cacnalc, Casq2, Cd36, Cldea, Collal, Col3al, Cptlb, Cpt2, Crat, Cs, Cth, Des, Dgatl, Dgat2, Dsc2, Dsg2, Dsp, Echsl, Elovll, Elovl5, Eno3, Fabp3, Fabp3, Fads2, G0s2, Gjal, Gotl, Gpt2, Hadh, Hadha, Hadhb, Hk2, Idh2, Jup, L2hgdh, Lpl, Maprel, Mgll, Mmp2, Mpcl/2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, Oxctl, P4HA1, Pdk4, Pdp2, Pfkm, Plin2, Pin, Polr21, Postn, Ryr2, Scn5a, Sdha, Sdhd, Suclg2, Tfin, TGF[31, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, Ttn, or Txn2. In some embodiments, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno -associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the method further comprises measuring one or more of fibrofatty tissue replacement; myocardial atrophy; ventricular dilation; ventricular arrhythmias; sudden cardiac death; exercise-triggered cardiac events; right ventricular cardiomyopathy, dilation, or heart failure; left ventricular cardiomyopathy, dilation, or heart failure; atrial arrhythmias; syncope; palpitations; shortness of breath; or chest pain. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof. In some embodiments, (b) comprises RT-qPCR, RNAseq, array hybridization, or probe hybridization.

[0007] In another aspect, provided herein are methods of restoring lipid metabolism and/or regulating energy homeostasis in cardiomyocytes of an individual in need thereof. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector restores activity of genes associated with lipid metabolism and/or energy homeostasis in the cardiomyocytes of the individual. In some embodiments, the genes associated with lipid metabolism and/or energy homeostasis comprise one or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acly, Acot3, Acsll, Acssl, Cd36, Cldea, Cptlb, Cpt2, Crat, Dgatl, Dgat2, Echsl, Elovll, Elovl5, Fabp3, Fabp3, Fads2, G0s2, Hadh, Hadha, Hadhb, L2hgdh, Lpl, Mgll, Mpcl/2, Pdp2, or Plin2. In some embodiments, the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno- associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[0008] In another aspect, provided herein are methods of restoring reducing collagen synthesis and/or fibrosis in cardiomyocytes of an individual in need thereof. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector reduces activity of genes associated with collagen synthesis and/or fibrosis in the cardiomyocytes of the individual. In some embodiments, the genes associated with collagen synthesis and/or fibrosis in cardiomyocytes comprise one or more of Collal, Col3al, Mmp2, P4HA1, Postn, TGF[31, or Timpl. In some embodiments, the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3 ’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[0009] In another aspect, provided herein are methods of modulating amino acid homeostasis in cardiomyocytes of an individual in need thereof. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter. In some embodiments, administration of the viral vector modulates activity of genes associated with amino acid homeostasis in the cardiomyocytes of the individual. In some embodiments, the genes associated with modulating amino acid homeostasis in cardiomyocytes comprise one or more of Beat 1, Bcat2, Cth, Gotl, or Gpt2. In some embodiments, the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno- associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof. In some embodiments, amino acid homeostasis is modulated for at least 12 months.

[0010] In another aspect, provided herein are methods of modulating ketone homeostasis in cardiomyocytes of an individual in need thereof. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter. In some embodiments, administration of the viral vector modulates activity of genes associated with ketone homeostasis in the cardiomyocytes of the individual. In some embodiments, the genes associated with modulating ketone homeostasis in cardiomyocytes comprise one or more of Acatl or Oxctl. In some embodiments, the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno -associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof. In some embodiments, ketone homeostasis is modulated for at least 12 months.

[0011] In another aspect, provided herein are methods of modulating glucose homeostasis and glycolysis in cardiomyocytes of an individual in need thereof. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter. In some embodiments, administration of the viral vector modulates activity of genes associated with glucose homeostasis and glycolysis in the cardiomyocytes of the individual. In some embodiments, the genes associated with modulating glucose homeostasis and glycolysis in cardiomyocytes comprise one or more of Cs, Eno3, Hk2, Idh2, Pdk4, Pfkm, Sdha, Sdhd, or Suclg2. In some embodiments, the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof. In some embodiments, glucose homeostasis and glycolysis are modulated for at least 12 months.

[0012] In another aspect, provided herein are methods of modulating nucleic acid homeostasis in cardiomyocytes of an individual in need thereof. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter. In some embodiments, administration of the viral vector modulates activity of genes associated with nucleic acid homeostasis in the cardiomyocytes of the individual. In some embodiments, the genes associated with modulating nucleic acid homeostasis in cardiomyocytes comprise one or more of Polr21 or Txn2. In some embodiments, the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alphamyosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno- associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof. In some embodiments, modulating nucleic acid homeostasis is modulated for at least 12 months. [0013] In another aspect, provided herein are methods of restoring calcium handling in cardiomyocytes of an individual in need thereof. In some cases, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter. In some embodiments, administration of the viral vector increases activity of genes associated with calcium handling in the cardiomyocytes of the individual. In some embodiments, the genes associated with calcium handling in cardiomyocytes comprise one or more of Ank2, Asph, Atp2a2, Cacnalc, Pin, Ryr2, or Trdn. In some embodiments, the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some embodiments, the individual has a genome with a mutation in a PKP2 gene. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the viral vector further comprises a cardiac specific enhancer. In some embodiments, the viral vector is selected from the group consisting of an adeno -associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some embodiments, the viral vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9. In some embodiments, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof. In some embodiments, calcium handling is restored for at least 12 months.

[0014] In another aspect, provided herein are viral vectors comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, for use in any method provided herein.

INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [0017] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0018] FIG. 1A shows a heatmap of RNA sequencing analyses (n = 3) from iPSC-CMs harvested 6 days after treatment with siRNAs against PKP2 (siPKP2) or negative control siRNAs (siNeg) highlighting effects on genes encoding components of the desmosome, sarcomere, and ion channels. [0019] FIG. IB shows PKP2 silencing led to reduction in protein expression of DSP, JUP, DES, and MyBPC3 in response to reduced PKP2 protein (Western blot on the left panel) and reduction in SCN5A mRNA in response to reduced PKP2 mRNA (RT-qPCR on the right panel).

[0020] FIG. 1C shows PKP2 silencing resulted in disappearance of PKP2 and DSP protein from the cellular membrane and cell disarray in patterned iPSC-CMs.

[0021] FIG. ID shows PKP2 silencing led to defective contraction as quantified by contraction velocity. [0022] FIG. IE shows upon PKP2 silencing, reductions in gene expression of ion channels led to depressed beat period, amplitude, and propagation of electrical signal detected as an extracellular field potential from the cardiac monolayers.

[0023] FIG. 2A shows a schematic representation of the 1st generation and the 2nd generation AAV expression cassette of PKP2a.

[0024] FIG. 2B illustrates a Western blot analysis showed that the second generation of AAV:hPKP2 is expressed in iPSC-CMs in a dose-dependent fashion by applying viruses at different multiplicity of infection (MOI).

[0025] FIG. 2C shows day 10 of PKP2 silencing and day 8 of AAV transduction, GFP expression of the first generation of PKP2 expression cassette was used to label AAV transduced iPSC-CMs.

[0026] FIG. 2D shows day 10 of PKP2 silencing and day 8 of AAV transduction, the left bar graph summarized the percentage of cells without GFP (n=12 technical replicates) and with GFP (n=36 technical replicates). The right graph shows restored DSP protein expression quantified by total intensity of immunofluorescence signal post PKP2 silencing in the absence (n=6 technical replicates) or the presence (n=12-18 technical replicates) of AAV:hPKP2 transgene.

[0027] FIG. 2E shows AAV:hPKP2 showed rescue of contraction velocity post PKP2 silencing in iPSC-CMs (n=18-27 technical replicates).

[0028] FIG. 3A shows Pkp2-cKO ARVC mice (aMyHC-Cre-ER(T2), Pkp2^fl/fl) at ~3 months of age that were injected with tamoxifen to induce cardiac knock-out of the Pkp2 gene. Representative immunoblots showed reduction of desmosome proteins PKP2, DSP, PKG, and GJ protein, Cx43.

[0029] FIG. 3B shows that Pkp2-cKO mice developed spontaneous PVCs as observed during 30 minutes of continuous recording of EKG.

[0030] FIG. 3C shows that Pkp2-cKO mice started to develop biventricular dilatation at 2 weeks post tamoxifen induction.

[0031] FIG. 3D shows that LV performance measured by % ejection fraction sharply declined at 2 weeks post tamoxifen induction. [0032] FIG. 3E shows a Kaplan-Meier survival curve showing a sharp decline of survival of Pkp2 cKO mice beginning 3 weeks post tamoxifen induction.

[0033] FIG. 4A shows the study design to evaluate AAV9:hPKP2 or AAV9:mPkp2 efficacy using Pkp2-cKO ARVC mouse model.

[0034] FIG. 4B shows raw EKG traces showing a significant contrast in spontaneous arrhythmias in Pkp2-cKO mice in the absence and the presence of AAV9:mPKP2 treatment.

[0035] FIG. 4C shows AAV9:PKP2 treatment of Pkp2-cKO mice demonstrated efficacy in reducing RV dilation as estimated by RV area normalized to body weight.

[0036] FIG. 4D shows AAV9:PKP2 treatment of Pkp2-cKO mice demonstrated efficacy in maintaining left ventricular ejection fraction at 4 weeks post gene deletion.

[0037] FIG. 4E shows a Kaplan-Meier survival curve showing that AAV9:hPKP2 extended life span of Pkp2-cKO mice after 72 weeks post gene deletion.

[0038] FIG. 5A illustrates the study design to evaluate dose-dependent efficacy of AAV9:hPKP2 using Pkp2-cKO ARVC mouse model.

[0039] FIG. 5B illustrates AAV9:PKP2 showing a dose-dependent response at 3 weeks in preventing RV dilation as estimated by RV area normalized to body weight, preventing decline of % LV ejection fraction, and trending improvement in arrhythmia scores.

[0040] FIG. 5C shows semi-quantitative Western blot analyses showed restoration of PKP2, JUP, and DSP protein at 3 weeks post AAV treatment.

[0041] FIG. 5D shows immuno-histochemistry for the gap junction protein, Connexin-43 (Cx43), in heart tissue sections showed restoration of Cx43 expression at intercalated discs (ID) at 3 weeks post AAV treatment (top panels). Red arrows indicate ID. Trichrome staining showed a significant reduction of fibrosis, muscle (red) and fibrosis (blue), in heart sections at 3 weeks post AAV treatment (bottom panel). Yellow arrows highlight areas with fibrosis in Pkp2-cKO mouse heart. The percentage of collagen-positive tissue was quantified and shown in the right graph.

[0042] FIG. 5E shows RT-qPCR analyses of RV tissue at 3 weeks post AAV treatment which showed expression of hPKP2 transgene and suppression of heart failure markers (Nppa) (Nppb did not show statistical significance) and fibrosis genes (Collal, Col3al, Timpl).

[0043] FIG. 6A shows a study design to evaluate AAV9:mPkp2 efficacy using Pkp2-cKO ARVC mouse model with delivery at 2.5 weeks after Pkp2 cardiac gene deletion by tamoxifen induction.

[0044] FIG. 6B shows HBSS-treated Pkp2-cKO ARVC mice died within 6 weeks of cardiac Pkp2 gene deletion, in contrast, AAV9:mPkp2 treatment significantly reduced mortality and extended life span of Pkp2-cKO mice to 51 weeks.

[0045] FIG. 6C shows treatment improved left ventricle ejection fraction by 36%.

[0046] FIG. 6D shows treatment reversed right ventricle enlargement by 31% and restored RV size similar to that of WT animals (RV size was normalized to body weight, mm2/g).

[0047] FIG. 6E shows treatment prevented further worsening of arrhythmias by 33.3%, all three readouts relative to HBSS treated Pkp2-cKO ARVC mice. [0048] FIG 6F shows a top bar graph showing EF% at 4 weeks post gene deletion and 1.5 weeks post treatment and a bottom bar graph showing multiple comparisons between treatment groups at different time points.

[0049] FIG. 6G shows a top bar graph showing RV size at 4 weeks post gene deletion and 1.5 weeks post treatment and a bottom bar graph showing multiple comparisons between treatment groups at different time points.

[0050] FIG 6H shows a top bar graph showing arrhythmia score at 4 weeks post gene deletion and 1.5 weeks post treatment and a bottom bar graph showing multiple comparisons between treatment groups at different time points.

[0051] FIG. 7A shows the study design to evaluate AAV9:hPKP2 dose-dependent efficacy at week 4 and 9 post tamoxifen induction of Pkp2 cardiac gene deletion in Pkp2-cKO ARVC mouse model.

[0052] FIG. 7B shows human PKP2 transgene mRNA levels at two doses (Low (L), 3E13 vg/kg; High (H), 6E13 vg/kg) quantified in copy number per ng of total LV RNA (top panel). Human PKP2 transgene protein levels at two doses were compared to the levels of endogenous mouse Pkp2 in WT and in Pkp2-cKO mouse post cardiac gene deletion by semi-quantitative Western blot (WB) (bottom panel). [0053] FIG. 7C shows TN-401 treatment at 3E13 or 6E13 vg/kg at week 9 post gene deletion showed comparable efficacy in EF%, RV area (mm2/g, normalized to body weight), LV mass (mg/g, normalized to body weight), and arrhythmia scores.

[0054] FIG. 7D shows a heatmap of gene expression analyses sorted by heart chambers (LV vs RV) clustered gene classes in response to treatment groups.

[0055] FIG. 7E shows volcano plots from differential gene expression analysis showing changes in gene expression between treatment groups of WT vs HBSS (top graph) and AAV9:hPKP2 high dose vs HBSS treated animals (bottom graph).

[0056] FIG. 7F shows boxplots showed group-wise gene expression for each representative gene of the selected gene classes.

[0057] FIG. 8A shows the study design to evaluate AAV9:mPkp2 efficacy in reducing mortality at 51 weeks post tamoxifen induction of Pkp2 deletion in Pkp2-cKO ARVC mouse model.

[0058] FIG. 8B shows Kaplan-Meier curve showing percent survival for each mode of treatment for 51 weeks post Pkp2 deletion.

[0059] FIG. 8C shows Principal Component Analysis showing clusters of gene transcripts from WT (animals taken down at 51 weeks post induction), untreated Pkp2-cKO animals (animals taken down at 4 weeks post induction), and AAV9:mPkp2 treated animals (animals taken down at 51 weeks post induction).

[0060] FIG. 8D shows volcano plots highlighting numbers of down-regulated genes in blue and numbers of up-regulated genes in red between pair-wise comparisons of untreated vs WT, preventive vs WT, and therapeutic vs WT.

[0061] FIG. 8E shows Gene Set Enrichment Analysis (GSEA) showing top 10 positively and negatively enriched cardiac gene sets in WT vs Pkp2-cKO. [0062] FIG. 8F shows a heatmap of RNA sequencing results showing relative expression of selected genes categorized in treatment groups, cardiac RV and LV chambers, and gene classes.

[0063] FIG. 8G illustrates GSEA showing positively and negatively enriched cardiac gene sets in WT vs Pkp2-cKO.

[0064] FIG. 8H shows RT-qPCR analyses showed expression of a total of mouse Pkp2 mRNA (including mouse transgene mRNA), heart failure marker genes, Nppa. Nppb. and fibrosis genes, Timpl, Coll al , and Col3al, in RV (top row) and LV (bottom row) at 51 weeks post Pkp2 deletion.

[0065] FIG. 9A shows the study design to evaluate AAV9:PKP2 safety in WT CD1 mice.

[0066] FIG. 9B shows body weight progression for 6 weeks.

[0067] FIG. 9C shows heart weight normalized to body weight, % ejection fraction (%EF) and ventricular arrhythmia score at 6 wks.

[0068] FIG. 9D shows neutrophil to lymphocyte ratio at 6 wks.

[0069] FIG. 9E shows liver weight normalized to body weight and live function tests, alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT) at 6 wks. f, Platelet counts and hemoglobin (HGB) amount.

[0070] FIG. 9F shows platelet counts and hemoglobin (HGB) amount.

[0071] FIG. 10A shows percentage ejection fraction at 4 weeks post-induction.

[0072] FIG. 10B shows percentage ejection fraction progression at 4 weeks post-induction.

[0073] FIG. 10C shows right ventricle area normalized to body weight at 4 weeks post-induction.

[0074] FIG. 10D shows right ventricle area normalized to body weight progression at 4 weeks postinduction.

[0075] FIG. 10E shows ventricular arrhythmia score distribution of individual animals at 4 weeks post tamoxifen induction.

[0076] FIG. 10F shows ventricular arrhythmia score progression at 4 weeks post tamoxifen induction. [0077] FIG. 10G shows a Kaplan-Meier survival curve showing that both TN-401 and AAV9:mPkp2 treatment extended median lifespan > 58 weeks vs 4.7 weeks observed in the vehicle treated Pkp2-cKO animals.

[0078] FIG. 10H shows weekly animal weight from start of induction up to 72 weeks post-induction.

[0079] FIG. 11 shows TN-401 showed dose-dependent expression of human PKP2 transgene protein and dose-dependent restoration of desmosome proteins.

[0080] FIG. 12A shows study design describing animal model, virus injections, and timepoints for major functional readouts.

[0081] FIG. 12B shows EF% at 4 weeks post gene deletion.

[0082] FIG. 12C shows RV/BW at 4 weeks post-induction.

[0083] FIG. 12D shows ventricular arrhythmia score distribution of individual animals at 4 weeks post gene deletion.

[0084] FIG. 12E shows a Kaplan-Meier survival curve showing that AAV9:mPkp2 treatment extended life span of Pkp2-cKO mice. DETAILED DESCRIPTION

[0085] Arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by ventricular arrhythmias and progressive cardiac dysfunction. Clinical presentation of ARVC varies from a concealed early phase, a later manifestation of lethal ventricular arrhythmias and ultimately heart failure that requires heart transplant. ARVC has an estimated prevalence in the general population of 1: 1000 to 1:5000 with the mean age of presentation before 40 years old. Arrhythmic sudden cardiac death (SCD) could be the first symptom, often diagnosed postmortem, of mostly young and athletic patients.

[0086] Mutations in the desmosome gene Plakophillin-2. PKP2, are the most frequent cause of genetic ARVC and account for approximately 43% of ARVC cases. Desmosomes are adhesive intercellular connections that play critical roles in heart development and performance. Interactions among desmosome proteins ensures proper structural anchoring and organization of intermediate filaments, cardiac sarcomere, and other organelles. Mutations in PKP2 gene are heterozygous in patients and lead to haploinsufficiency in PKP2 protein levels. Reduction of PKP2 protein at the intercalated discs (ID) disrupts desmosomes and other ID structures such as gap junctions (GJs). Reduction of Connexin 43 (Cx43), a critical component of GJs, results in compromised electrical coupling and heterogeneous conduction between cardiomyocytes. These structural corruptions trigger cell death response, inflammatory infiltration, and metabolic perturbation that underpin clinical manifestations of electrical instability, cardiac structural deterioration, fibrofatty infiltration, and heart failure.

[0087] Clinical management of ARVC patients includes lifestyle modification, pharmacological treatment, catheter ablation, ICDs, and heart transplantation. So far, there is no approved treatment that addresses the underlying genetic cause of this disease. It is technically challenging to apply conventional therapeutic approaches to restore defective large cellular structures such as the desmosome and manage their pleiotropic impact on complex signaling networks. Therefore, a new treatment paradigm that targets the underlying genetic cause of the disease is needed to manage the multiplicity of disease manifestations during disease onset and progression.

[0088] Preclinical results demonstrated that AAV9-based PKP2 gene replacement approach offers significant survival benefit in repairing cellular structures of desmosome, gap junctions (GJs), and Camhandling system, improving cardiac function, reducing ventricular arrhythmia event frequency and severity, and preventing adverse fibrotic remodeling in a dose-dependent fashion in a cardiac-specific Pkp2 knock-out mouse model of ARVC.

[0089] Heterozygous PKP2 mutations in humans are predominantly truncation mutations resulting from nonsense, frameshift, or splice-site mutations. PKP2 mRNAs containing premature stop codons are subjected to surveillance and degradation by nonsense-mediated decay (NMD) machinery. Degradation of the mutated mRNA results in haploinsufficiency as shown by reductions of both mRNA and protein in ARVC patients heart tissues from autopsy, endomyocardial biopsy or explants. Preventive efficacy studies described herein demonstrated that restoration of PKP2 expression correlates with restoration of function in a dose-dependent fashion, suggesting that the cellular level of PKP2 precisely and quantitatively dictates a relationship between the cellular input vs the functional outputs under the condition that is minimally influenced by other undefined genetic and nongenetic factors. This precise dose-function correlation of PKP2 possibly addresses the functional consequence of haploinsufficiency in real human cases and further supports the rationale of an AAV-based gene replacement approach. Furthermore, RNA sequencing analyses provided herein revealed a broad spectrum of functional impact by PKP2 deficiency and destruction of desmosomes. These results strongly support a gene therapy-based intervention that addresses the root cause and its associated pleiotropism. In some cases, mutations in other desmosome genes also lead to destruction of the desmosome, suggesting mechanisms observed herein are likely applied similarly to other desmosome genes in their role of regulating dynamics of desmosome and other multi-united structures such as GJs, sarcomere, and the Ca²⁺-handling system.

Methods for Modulating Gene Expression

[0090] In an aspect, provided herein are methods for restoring gene expression of a plurality of genes in an individual having a heart disease or disorder. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, thereby restoring gene expression of the plurality of genes. In some embodiments, the plurality of genes comprises one or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Pin, Ryr2, Scn5a, Tfn, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the plurality of genes comprises two or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Pin, Ryr2, Scn5a, Tfh, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the plurality of genes comprises three, four, five, six, seven, eight, nine, ten, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Pin, Ryr2, Scn5a, Tfh, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the plurality of genes comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Pin, Ryr2, Scn5a, Tfh, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the plurality of genes comprises 21, 22, 23, 24, 25, 26, 27, 28, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Pin, Ryr2, Scn5a, Tfh, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the plurality of genes comprises Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Pin, Ryr2, Scn5a, Tfh, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, and Ttn.

[0091] In another aspect, provided herein are methods of reducing gene expression of a plurality of genes in an individual having a heart disease or disorder. In some embodiments, the method comprises administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, thereby reducing gene expression of the plurality of genes, wherein the plurality of genes comprises one or more of Collal, Col3al, Mmp2, Nppa, Nppb, P4HA1, Postn, TGF[31, or Timpl. In some embodiments, the plurality of genes comprises two, three, four, five, six, seven, eight, or more of Collal, Col3al, Mmp2, Nppa, Nppb, P4HA1, Postn, TGFpi, or Timpl. In some embodiments, the plurality of genes comprises Collal, Col3al, Mmp2, Nppa, Nppb, P4HA1, Postn, TGFp i . and Timpl.

[0092] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[0093] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, the viral vector comprises a promoter operatively linked to the nucleic acid encoding PKP2. In some case, the promoter is a cardiac specific promoter. In some cases, the promoter or the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some cases, the promoter is a ubiquitous promoter. In some cases, the promoter or the ubiquitous promoter is a beta-actin promoter, a eukaryotic translation elongation factor 1 a promoter, a heat shock protein 70 promoter, or a ubiquitin B promoter. In some cases, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some cases, the viral vector further comprises a cardiac specific enhancer.

[0094] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some cases, the viral vector is an adeno- associated virus. In some cases, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

[0095] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence selected from one or SEQ ID NOs: 13-19. In some cases, the nucleic acid encoding the PKP2 polypeptide has a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%, at least about 95%, at least about 99% or about 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs: 13-19.

[0096] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[0097] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, expression of the plurality of genes is restored or reduced for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months, or longer. [0098] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, one or more symptoms of ARVC or ACM are improved for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months, or longer.

Methods for Monitoring Treatment Efficacy

[0099] In another aspect, provided herein are methods for monitoring efficacy of a treatment for a heart disease or disorder in an individual, the method comprising: (a) administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) operatively linked to a promoter; and (b) measuring expression of one or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tin, TGF i, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring expression of two, three, four, five, six, seven, eight, nine, ten or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfh, TGF i, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring expression of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring expression of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring expression of 31, 32, 33, 34, 35, 36, 37, 38, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfin, TGF i, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring expression of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[00100] In another aspect, provided herein are methods for monitoring efficacy of a treatment for a heart disease or disorder in an individual, the method comprising: (a) administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) operatively linked to a promoter; and (b) measuring expression of one or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acatl, Acly, Acot3, Acsll, Acssl, Actn2, Ank2, Asph, Atp2a2, Bcatl, Bcat2, Cacnalc, Casq2, Cd36, Cldea, Collal, Col3al, Cptlb, Cpt2, Crat, Cs, Cth, Des, Dgatl, Dgat2, Dsc2, Dsg2, Dsp, Echsl, Elovll, Elovl5, Eno3, Fabp3, Fabp3, Fads2, G0s2, Gjal, Gotl, Gpt2, Hadh, Hadha, Hadhb, Hk2, Idh2, Jup, L2hgdh, Lpl, Maprel, Mgll, Mmp2, Mpcl/2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, Oxctl, P4HA1, Pdk4, Pdp2, Pfkm, Plin2, Pin, Polr21, Postn, Ryr2, Scn5a, Sdha, Sdhd, Suclg2, Tfin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, Ttn, or Txn2. In some embodiments, the method comprises measuring expression of two, three, four, five, six, seven, eight, nine, ten or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acatl, Acly, Acot3, Acsll, Acssl, Actn2, Ank2, Asph, Atp2a2, Bcatl, Bcat2, Cacnalc, Casq2, Cd36, Cldea, Collal, Col3al, Cptlb, Cpt2, Crat, Cs, Cth, Des, Dgatl, Dgat2, Dsc2, Dsg2, Dsp, Echsl, Elovll, Elovl5, Eno3, Fabp3, Fabp3, Fads2, G0s2, Gjal, Gotl, Gpt2, Hadh, Hadha, Hadhb, Hk2, Idh2, Jup, L2hgdh, Lpl, Maprel, Mgll, Mmp2, Mpcl/2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, Oxctl, P4HA1, Pdk4, Pdp2, Pfkm, Plin2, Pin, Polr21, Postn, Ryr2, Scn5a, Sdha, Sdhd, Suclg2, Tfin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, Ttn, or Txn2. In some embodiments, the method comprises measuring expression of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acatl, Acly, Acot3, Acsll, Acssl, Actn2, Ank2, Asph, Atp2a2, Bcatl, Bcat2, Cacnalc, Casq2, Cd36, Cldea, Collal, Col3al, Cptlb, Cpt2, Crat, Cs, Cth, Des, Dgatl, Dgat2, Dsc2, Dsg2, Dsp, Echsl, Elovll, Elovl5, Eno3, Fabp3, Fabp3, Fads2, G0s2, Gjal, Gotl, Gpt2, Hadh, Hadha, Hadhb, Hk2, Idh2, Jup, L2hgdh, Lpl, Maprel, Mgll, Mmp2, Mpcl/2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, Oxctl, P4HA1, Pdk4, Pdp2, Pfkm, Plin2, Pin, Polr21, Postn, Ryr2, Scn5a, Sdha, Sdhd, Suclg2, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, Ttn, or Txn2. In some embodiments, the method comprises measuring expression of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acatl, Acly, Acot3, Acsll, Acssl, Actn2, Ank2, Asph, Atp2a2, Bcatl, Bcat2, Cacnalc, Casq2, Cd36, Cldea, Collal, Col3al, Cptlb, Cpt2, Crat, Cs, Cth, Des, Dgatl, Dgat2, Dsc2, Dsg2, Dsp, Echsl, Elovll, Elovl5, Eno3, Fabp3, Fabp3, Fads2, G0s2, Gjal, Gotl, Gpt2, Hadh, Hadha, Hadhb, Hk2, Idh2, Jup, L2hgdh, Lpl, Maprel, Mgll, Mmp2, Mpcl/2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, Oxctl, P4HA1, Pdk4, Pdp2, Pfkm, Plin2, Pin, Polr21, Postn, Ryr2, Scn5a, Sdha, Sdhd, Suclg2, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, Ttn, or Txn2. In some embodiments, the method comprises measuring expression of 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acatl, Acly, Acot3, Acsll, Acssl, Actn2, Ank2, Asph, Atp2a2, Bcatl, Bcat2, Cacnalc, Casq2, Cd36, Cldea, Collal, Col3al, Cptlb, Cpt2, Crat, Cs, Cth, Des, Dgatl, Dgat2, Dsc2, Dsg2, Dsp, Echsl, Elovll, Elovl5, Eno3, Fabp3, Fabp3, Fads2, G0s2, Gjal, Gotl, Gpt2, Hadh, Hadha, Hadhb, Hk2, Idh2, Jup, L2hgdh, Lpl, Maprel, Mgll, Mmp2, Mpcl/2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, Oxctl, P4HA1, Pdk4, Pdp2, Pfkm, Plin2, Pin, Polr21, Postn, Ryr2, Scn5a, Sdha, Sdhd, Suclg2, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, Ttn, or Txn2. In some embodiments, the method comprises measuring expression of 45, 50, 55, 60, 65, 70, 75, 80, 85, or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acatl, Acly, Acot3, Acsll, Acssl, Actn2, Ank2, Asph, Atp2a2, Bcatl, Bcat2, Cacnalc, Casq2, Cd36, Cldea, Collal, Col3al, Cptlb, Cpt2, Crat, Cs, Cth, Des, Dgatl, Dgat2, Dsc2, Dsg2, Dsp, Echsl, Elovll, Elovl5, Eno3, Fabp3, Fabp3, Fads2, G0s2, Gjal, Gotl, Gpt2, Hadh, Hadha, Hadhb, Hk2, Idh2, Jup, L2hgdh, Lpl, Maprel, Mgll, Mmp2, Mpcl/2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, Oxctl, P4HA1, Pdk4, Pdp2, Pfkm, Plin2, Pin, Polr21, Postn, Ryr2, Scn5a, Sdha, Sdhd, Suclg2, Tfn, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, Ttn, or Txn2. In some embodiments, the method comprises measuring expression of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acatl, Acly, Acot3, Acsll, Acssl, Actn2, Ank2, Asph, Atp2a2, Bcatl, Bcat2, Cacnalc, Casq2, Cd36, Cldea, Collal, Col3al, Cptlb, Cpt2, Crat, Cs, Cth, Des, Dgatl, Dgat2, Dsc2, Dsg2, Dsp, Echsl, Elovll, Elovl5, Eno3, Fabp3, Fabp3, Fads2, G0s2, Gjal, Gotl, Gpt2, Hadh, Hadha, Hadhb, Hk2, Idh2, Jup, L2hgdh, Lpl, Maprel, Mgll, Mmp2, Mpcl/2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, Oxctl, P4HA1, Pdk4, Pdp2, Pfkm, Plin2, Pin, Polr21, Postn, Ryr2, Scn5a, Sdha, Sdhd, Suclg2, Tfn, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, Ttn, and Txn2. In some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[00101] In various aspects of methods of monitoring treatment efficacy provided herein, in some cases monitoring treatment efficacy further comprises monitoring cardiac function. In some cases, the method further comprises obtaining an echocardiogram or an electrocardiogram. In some cases, the method further comprises obtaining results of a cardiac stress test. In some cases, the method further comprises measuring one or more of fibrofatty tissue replacement; myocardial atrophy; ventricular dilation; ventricular arrhythmias; sudden cardiac death; exercise-triggered cardiac events; right ventricular cardiomyopathy, dilation, or heart failure; left ventricular cardiomyopathy, dilation, or heart failure; atrial arrhythmias; syncope; palpitations; shortness of breath; or chest pain.

[00102] In various aspects of methods of monitoring treatment efficacy provided herein, in some cases, the viral vector comprises a promoter operatively linked to the nucleic acid encoding PKP2. In some case, the promoter is a cardiac specific promoter. In some cases, the promoter or the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some cases, the promoter is a ubiquitous promoter. In some cases, the promoter or the ubiquitous promoter is a beta-actin promoter, a eukaryotic translation elongation factor 1 a promoter, a heat shock protein 70 promoter, or a ubiquitin B promoter. In some cases, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some cases, the viral vector further comprises a cardiac specific enhancer.

[00103] In various aspects of methods of monitoring treatment efficacy provided herein, in some cases, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some cases, the viral vector is an adeno- associated virus. In some cases, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

[00104] In various aspects of methods of monitoring treatment efficacy provided herein, in some cases, measuring expression comprises one or more of reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), ribonucleic acid sequencing (RNAseq), array hybridization, or probe hybridization. In some embodiments, array hybridization comprises a microarray. In some embodiments, probe hybridization comprises in situ hybridization, Northern blot, or fluorescence activated cell sorting (FACS). In some embodiments, measuring expression comprises one or more of Western blot, immunofluorescence, enzyme-linked immunoassay, or fluorescence activated cell sorting (FACS).

[00105] In various aspects of methods of monitoring treatment efficacy provided herein, in some cases, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some cases, the PKP2 polypeptide has an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 8. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence selected from one or SEQ ID NOs: 13-19. In some cases, the nucleic acid encoding the PKP2 polypeptide has a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%, at least about 95%, at least about 99% or about 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs: 13-19.

[00106] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[00107] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, the method comprises measuring an increase in expression of one or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring an increase in expression of two, three, four, five, six, seven, eight, nine, ten, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring an increase in expression of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring an increase in expression of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring an increase in expression of 31, 32, 33, 34, 35, 36, 37, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring an increase in expression of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, and Ttn. In some cases, the increase in expression is observed for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months or longer.

[00108] In various aspects of methods of restoring or reducing gene expression provided herein, in some cases, the method comprises measuring a decrease in expression of one or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring a decrease in expression of two, three, four, five, six, seven, eight, nine, ten, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring a decrease in expression of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring a decrease in expression of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring a decrease in expression of 31, 32, 33, 34, 35, 36, 37, or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn. In some embodiments, the method comprises measuring a decrease in expression of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tfh, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, and Ttn. In some cases, the decrease in expression is observed for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months or longer. Methods of Restoring Lipid Metabolism and Energy Homeostasis

[00109] In another aspect, provided herein are methods of restoring lipid metabolism and/or regulating energy homeostasis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector restores activity of genes associated with lipid metabolism and/or energy homeostasis in the cardiomyocytes of the individual. In some cases, the genes associated with lipid metabolism and/or energy homeostasis comprise one or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acly, Acot3, Acsll, Acssl, Cd36, Cldea, Cptlb, Cpt2, Crat, Dgatl, Dgat2, Echsl, Elovll, Elovl5, Fabp3, Fabp3, Fads2, G0s2, Hadh, Hadha, Hadhb, L2hgdh, Lpl, Mgll, Mpcl/2, Pdp2, or Plin2. In some cases, the genes associated with lipid metabolism and/or energy homeostasis comprise two, three, four, five, six, seven, eight, nine, ten, or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acly, Acot3, Acsll, Acssl, Cd36, Cldea, Cptlb, Cpt2, Crat, Dgatl, Dgat2, Echsl, Elovll, Elovl5, Fabp3, Fabp3, Fads2, G0s2, Hadh, Hadha, Hadhb, L2hgdh, Lpl, Mgll, Mpcl/2, Pdp2, or Plin2. In some cases, the genes associated with lipid metabolism and/or energy homeostasis comprise 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acly, Acot3, Acsll, Acssl, Cd36, Cldea, Cptlb, Cpt2, Crat, Dgatl, Dgat2, Echsl, Elovll, Elovl5, Fabp3, Fabp3, Fads2, G0s2, Hadh, Hadha, Hadhb, L2hgdh, Lpl, Mgll, Mpcl/2, Pdp2, or Plin2. In some cases, the genes associated with lipid metabolism and/or energy homeostasis comprise 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acly, Acot3, Acsll, Acssl, Cd36, Cldea, Cptlb, Cpt2, Crat, Dgatl, Dgat2, Echsl, Elovll, Elovl5, Fabp3, Fabp3, Fads2, G0s2, Hadh, Hadha, Hadhb, L2hgdh, Lpl, Mgll, Mpcl/2, Pdp2, or Plin2. In some cases, the genes associated with lipid metabolism and/or energy homeostasis comprise Acaa2, Acaca, Acadl, Acadm, Acadsb, Acly, Acot3, Acsll, Acssl, Cd36, Cldea, Cptlb, Cpt2, Crat, Dgatl, Dgat2, Echsl, Elovll, Elovl5, Fabp3, Fabp3, Fads2, G0s2, Hadh, Hadha, Hadhb, L2hgdh, Lpl, Mgll, Mpcl/2, Pdp2, and Plin2.

[00110] In various aspects of methods of restoring lipid metabolism and/or regulating energy homeostasis in cardiomyocytes provided herein, in some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[00111] In various aspects of methods of restoring lipid metabolism and/or regulating energy homeostasis in cardiomyocytes provided herein, in some cases, the viral vector comprises a promoter operatively linked to the nucleic acid encoding PKP2. In some case, the promoter is a cardiac specific promoter. In some cases, the promoter or the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some cases, the promoter is a ubiquitous promoter. In some cases, the promoter or the ubiquitous promoter is a beta-actin promoter, a eukaryotic translation elongation factor 1 a promoter, a heat shock protein 70 promoter, or a ubiquitin B promoter. In some cases, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some cases, the viral vector further comprises a cardiac specific enhancer.

[00112] In various aspects of methods of restoring lipid metabolism and/or regulating energy homeostasis in cardiomyocytes provided herein, in some cases, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some cases, the viral vector is an adeno-associated virus. In some cases, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

[00113] In various aspects of methods of restoring lipid metabolism and/or regulating energy homeostasis in cardiomyocytes provided herein, in some cases, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some cases, the PKP2 polypeptide has an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 8. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence selected from one or SEQ ID NOs: 13-19. In some cases, the nucleic acid encoding the PKP2 polypeptide has a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%, at least about 95%, at least about 99% or about 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs: 13-19.

[00114] In various aspects of methods of restoring lipid metabolism and/or regulating energy homeostasis in cardiomyocytes provided herein, in some cases, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[00115] In various aspects of methods of restoring lipid metabolism and/or regulating energy homeostasis provided herein, in some cases, gene expression is measured using a method comprising one or more of reverse -transcriptase quantitative polymerase chain reaction (RT-qPCR), ribonucleic acid sequencing (RNAseq), array hybridization, or probe hybridization. In some embodiments, array hybridization comprises a microarray. In some embodiments, probe hybridization comprises in situ hybridization, Northern blot, or fluorescence activated cell sorting (FACS). In some embodiments, measuring expression comprises one or more of Western blot, immunofluorescence, enzyme-linked immunoassay, or fluorescence activated cell sorting (FACS).

[00116] In various aspects of methods of restoring lipid metabolism and/or regulating energy homeostasis in cardiomyocytes provided herein, in some cases the method restores lipid metabolism and/or energy homeostasis for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months, or longer.

Methods of Reducing Collagen Synthesis and Fibrosis

[00117] In another aspect, provided herein are methods of reducing collagen synthesis and/or fibrosis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector reduces activity of genes associated with collagen synthesis and/or fibrosis in the cardiomyocytes of the individual. In some cases, the genes associated with collagen synthesis and/or fibrosis comprise one or more of Collal, Col3al, Mmp2, P4HA1, Postn, TGFJ31, or Timpl. In some cases, the genes associated with collagen synthesis and/or fibrosis comprise two, three, four, five, six, or more of Collal, Col3al, Mmp2, P4HA1, Postn, TGFpi, or Timpl. In some cases, the genes associated with collagen synthesis and/or fibrosis comprise Collal, Col3al, Mmp2, P4HA1, Postn, TGFpi, and Timpl.

[00118] In various aspects of methods of reducing collagen synthesis and/or fibrosis in cardiomyocytes provided herein, in some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[00119] In various aspects of methods of reducing collagen synthesis and/or fibrosis in cardiomyocytes provided herein, in some cases, the viral vector comprises a promoter operatively linked to the nucleic acid encoding PKP2. In some case, the promoter is a cardiac specific promoter. In some cases, the promoter or the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some cases, the promoter is a ubiquitous promoter. In some cases, the promoter or the ubiquitous promoter is a beta-actin promoter, a eukaryotic translation elongation factor 1 a promoter, a heat shock protein 70 promoter, or a ubiquitin B promoter. In some cases, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some cases, the viral vector further comprises a cardiac specific enhancer.

[00120] In various aspects of methods of reducing collagen synthesis and/or fibrosis in cardiomyocytes provided herein, in some cases, the viral vector is selected from the group consisting of an adeno- associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some cases, the viral vector is an adeno-associated virus. In some cases, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

[00121] In various aspects of methods of reducing collagen synthesis and/or fibrosis in cardiomyocytes provided herein, in some cases, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some cases, the PKP2 polypeptide has an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 8. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence selected from one or SEQ ID NOs: 13-19. In some cases, the nucleic acid encoding the PKP2 polypeptide has a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%, at least about 95%, at least about 99% or about 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs: 13-19. [00122] In various aspects of methods of reducing collagen synthesis or fibrosis in cardiomyocytes provided herein, in some cases, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[00123] In various aspects of methods of reducing collagen synthesis or fibrosis in cardiomyocytes provided herein, in some cases, gene expression is measured using a method comprising one or more of reverse -transcriptase quantitative polymerase chain reaction (RT-qPCR), ribonucleic acid sequencing (RNAseq), array hybridization, or probe hybridization. In some embodiments, array hybridization comprises a microarray. In some embodiments, probe hybridization comprises in situ hybridization, Northern blot, or fluorescence activated cell sorting (FACS). In some embodiments, measuring expression comprises one or more of Western blot, immunofluorescence, enzyme-linked immunoassay, or fluorescence activated cell sorting (FACS).

[00124] In various aspects of methods of reducing collagen synthesis or fibrosis in cardiomyocytes provided herein, in some cases collagen synthesis and/or fibrosis is reduced for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months, or longer.

Methods of Restoring Calcium Handling in Cardiomyocytes

[00125] In another aspect, provided herein are methods of restoring calcium handling in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector increases expression and/or activity of genes associated with calcium handling in the cardiomyocytes of the individual. In some cases, the genes associated with calcium handling comprise one or more of Ank2, Asph, Atp2a2, Cacnalc, Pin, Ryr2, or Trdn. In some cases, the genes associated with calcium handling comprise two, three, four, five, six, or more of Ank2, Asph, Atp2a2, Cacnalc, Pin, Ryr2, or Trdn. In some cases, the genes associated with calcium handling comprise Ank2, Asph, Atp2a2, Cacnalc, Pin, Ryr2, and Trdn.

[00126] In various aspects of methods of reducing collagen synthesis and/or fibrosis in cardiomyocytes provided herein, in some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[00127] In various aspects of methods of restoring calcium handling in cardiomyocytes provided herein, in some cases, the viral vector comprises a promoter operatively linked to the nucleic acid encoding PKP2. In some case, the promoter is a cardiac specific promoter. In some cases, the promoter or the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some cases, the promoter is a ubiquitous promoter. In some cases, the promoter or the ubiquitous promoter is a beta-actin promoter, a eukaryotic translation elongation factor 1 a promoter, a heat shock protein 70 promoter, or a ubiquitin B promoter. In some cases, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some cases, the viral vector further comprises a cardiac specific enhancer.

[00128] In various aspects of methods of restoring calcium handling in cardiomyocytes provided herein, in some cases, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some cases, the viral vector is an adeno-associated virus. In some cases, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

[00129] In various aspects of methods of restoring calcium handling in cardiomyocytes provided herein, in some cases, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some cases, the PKP2 polypeptide has an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 8. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence selected from one or SEQ ID NOs: 13-19. In some cases, the nucleic acid encoding the PKP2 polypeptide has a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%, at least about 95%, at least about 99% or about 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs: 13-19.

[00130] In various aspects of methods of restoring calcium handling in cardiomyocytes provided herein, in some cases, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[00131] In various aspects of methods of restoring calcium handling in cardiomyocytes provided herein, in some cases, gene expression is measured using a method comprising one or more of reversetranscriptase quantitative polymerase chain reaction (RT-qPCR), ribonucleic acid sequencing (RNAseq), array hybridization, or probe hybridization. In some embodiments, array hybridization comprises a microarray. In some embodiments, probe hybridization comprises in situ hybridization, Northern blot, or fluorescence activated cell sorting (FACS). In some embodiments, measuring expression comprises one or more of Western blot, immunofluorescence, enzyme-linked immunoassay, or fluorescence activated cell sorting (FACS).

[00132] In various aspects of methods of restoring calcium handling in cardiomyocytes provided herein, in some cases calcium handling is restored for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months, or longer.

Methods of Modulating Amino Acid Homeostasis

[00133] In another aspect, provided herein are methods of modulating amino acid homeostasis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector modulates activity of genes associated with amino acid homeostasis in the cardiomyocytes of the individual. In some cases, the genes associated with amino acid homeostasis comprise one or more of Bcatl, Bcat2, Cth, Gotl, or Gpt2. In some cases, the genes associated with amino acid homeostasis comprise two, three, four, or more of Bcatl, Bcat2, Cth, Gotl, or Gpt2. In some cases, the genes associated with amino acid homeostasis comprise Bcatl, Bcat2, Cth, Gotl, and Gpt2.

[00134] In various aspects of methods of modulating amino acid homeostasis in cardiomyocytes provided herein, in some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[00135] In various aspects of methods of modulating amino acid homeostasis in cardiomyocytes provided herein, in some cases, the viral vector comprises a promoter operatively linked to the nucleic acid encoding PKP2. In some case, the promoter is a cardiac specific promoter. In some cases, the promoter or the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some cases, the promoter is a ubiquitous promoter. In some cases, the promoter or the ubiquitous promoter is a beta-actin promoter, a eukaryotic translation elongation factor 1 a promoter, a heat shock protein 70 promoter, or a ubiquitin B promoter. In some cases, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some cases, the viral vector further comprises a cardiac specific enhancer.

[00136] In various aspects of methods of modulating amino acid homeostasis in cardiomyocytes provided herein, in some cases, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some cases, the viral vector is an adeno-associated virus. In some cases, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

[00137] In various aspects of methods of modulating amino acid homeostasis in cardiomyocytes provided herein, in some cases, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some cases, the PKP2 polypeptide has an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 8. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence selected from one or SEQ ID NOs: 13-19. In some cases, the nucleic acid encoding the PKP2 polypeptide has a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%, at least about 95%, at least about 99% or about 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs: 13-19. [00138] In various aspects of methods of modulating amino acid homeostasis in cardiomyocytes provided herein, in some cases, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[00139] In various aspects of methods of modulating amino acid homeostasis in cardiomyocytes provided herein, in some cases, gene expression is measured using a method comprising one or more of reversetranscriptase quantitative polymerase chain reaction (RT-qPCR), ribonucleic acid sequencing (RNAseq), array hybridization, or probe hybridization. In some embodiments, array hybridization comprises a microarray. In some embodiments, probe hybridization comprises in situ hybridization, Northern blot, or fluorescence activated cell sorting (FACS). In some embodiments, measuring expression comprises one or more of Western blot, immunofluorescence, enzyme-linked immunoassay, or fluorescence activated cell sorting (FACS).

[00140] In various aspects of methods of modulating amino acid homeostasis in cardiomyocytes provided herein, in some cases amino acid homeostasis is modulated for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months, or longer.

Methods of Modulating Ketone Homeostasis

[00141] In another aspect, provided herein are methods of modulating ketone homeostasis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector modulates activity of genes associated with ketone homeostasis in the cardiomyocytes of the individual. In some cases, the genes associated with ketone homeostasis comprise one or more of Acatl or Oxctl. In some cases, the genes associated with ketone homeostasis comprise Acatl and Oxctl.

[00142] In various aspects of methods of modulating ketone homeostasis in cardiomyocytes provided herein, in some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[00143] In various aspects of methods of modulating ketone homeostasis in cardiomyocytes provided herein, in some cases, the viral vector comprises a promoter operatively linked to the nucleic acid encoding PKP2. In some case, the promoter is a cardiac specific promoter. In some cases, the promoter or the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some cases, the promoter is a ubiquitous promoter. In some cases, the promoter or the ubiquitous promoter is a beta-actin promoter, a eukaryotic translation elongation factor 1 a promoter, a heat shock protein 70 promoter, or a ubiquitin B promoter. In some cases, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some cases, the viral vector further comprises a cardiac specific enhancer.

[00144] In various aspects of methods of modulating ketone homeostasis in cardiomyocytes provided herein, in some cases, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some cases, the viral vector is an adeno-associated virus. In some cases, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

[00145] In various aspects of methods of modulating ketone homeostasis in cardiomyocytes provided herein, in some cases, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some cases, the PKP2 polypeptide has an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 8. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence selected from one or SEQ ID NOs: 13-19. In some cases, the nucleic acid encoding the PKP2 polypeptide has a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%, at least about 95%, at least about 99% or about 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs: 13-19.

[00146] In various aspects of methods of modulating ketone homeostasis in cardiomyocytes provided herein, in some cases, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[00147] In various aspects of methods of modulating ketone homeostasis in cardiomyocytes provided herein, in some cases, gene expression is measured using a method comprising one or more of reversetranscriptase quantitative polymerase chain reaction (RT-qPCR), ribonucleic acid sequencing (RNAseq), array hybridization, or probe hybridization. In some embodiments, array hybridization comprises a microarray. In some embodiments, probe hybridization comprises in situ hybridization, Northern blot, or fluorescence activated cell sorting (FACS). In some embodiments, measuring expression comprises one or more of Western blot, immunofluorescence, enzyme-linked immunoassay, or fluorescence activated cell sorting (FACS).

[00148] In various aspects of methods of modulating ketone homeostasis in cardiomyocytes provided herein, in some cases ketone homeostasis is modulated for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months, or longer.

Methods of Modulating Glucose Homeostasis and Glycolysis

[00149] In another aspect, provided herein are methods of modulating glucose homeostasis and glycolysis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector modulates activity of genes associated with glucose homeostasis and glycolysis in the cardiomyocytes of the individual. In some cases, the genes associated with glucose homeostasis and glycolysis comprise one or more of Cs, Eno3, Hk2, Idh2, Pdk4, Pfkm, Sdha, Sdhd, or Suclg2. In some cases, the genes associated with glucose homeostasis and glycolysis comprise two, three, four, five, six, seven, eight, or more of Cs, Eno3, Hk2, Idh2, Pdk4, Pfkm, Sdha, Sdhd, or Suclg2. In some cases, the genes associated with glucose homeostasis and glycolysis comprise Cs, Eno3, Hk2, Idh2, Pdk4, Pfkm, Sdha, Sdhd, and Suclg2.

[00150] In various aspects of methods of modulating glucose homeostasis and glycolysis in cardiomyocytes provided herein, in some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[00151] In various aspects of methods of modulating glucose homeostasis and glycolysis in cardiomyocytes provided herein, in some cases, the viral vector comprises a promoter operatively linked to the nucleic acid encoding PKP2. In some case, the promoter is a cardiac specific promoter. In some cases, the promoter or the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some cases, the promoter is a ubiquitous promoter. In some cases, the promoter or the ubiquitous promoter is a beta-actin promoter, a eukaryotic translation elongation factor 1 a promoter, a heat shock protein 70 promoter, or a ubiquitin B promoter. In some cases, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some cases, the viral vector further comprises a cardiac specific enhancer.

[00152] In various aspects of methods of modulating glucose homeostasis and glycolysis in cardiomyocytes provided herein, in some cases, the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some cases, the viral vector is an adeno-associated virus. In some cases, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

[00153] In various aspects of methods of modulating glucose homeostasis and glycolysis in cardiomyocytes provided herein, in some cases, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some cases, the PKP2 polypeptide has an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 8. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence selected from one or SEQ ID Nos: 13-19. In some cases, the nucleic acid encoding the PKP2 polypeptide has a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%, at least about 95%, at least about 99% or about 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID Nos: 13-19. [00154] In various aspects of methods of modulating glucose homeostasis and glycolysis in cardiomyocytes provided herein, in some cases, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[00155] In various aspects of methods of modulating glucose homeostasis and glycolysis in cardiomyocytes provided herein, in some cases, gene expression is measured using a method comprising one or more of reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), ribonucleic acid sequencing (RNAseq), array hybridization, or probe hybridization. In some embodiments, array hybridization comprises a microarray. In some embodiments, probe hybridization comprises in situ hybridization, Northern blot, or fluorescence activated cell sorting (FACS). In some embodiments, measuring expression comprises one or more of Western blot, immunofluorescence, enzyme-linked immunoassay, or fluorescence activated cell sorting (FACS).

[00156] In various aspects of methods of modulating glucose homeostasis and glycolysis in cardiomyocytes provided herein, in some cases glucose homeostasis and glycolysis is modulated for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months, or longer.

Methods of Modulating Nucleic acid Homeostasis

[00157] In another aspect, provided herein are methods of nucleic acid homeostasis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector modulates activity of genes associated with nucleic acid homeostasis in the cardiomyocytes of the individual. In some cases, the genes associated with nucleic acid homeostasis comprise one or more of Polr21 or Txn2. In some cases, the genes associated with nucleic acid homeostasis comprise Polr21 and Txn2.

[00158] In various aspects of methods of modulating nucleic acid homeostasis in cardiomyocytes provided herein, in some cases, the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM). In some cases, the individual has a genome with a mutation in a PKP2 gene. In some cases, the mutation causes haploinsufficiency of PKP2 gene expression. In some cases, the mutation is a nonsense mutation. In some cases, the mutation is a missense mutation.

[00159] In various aspects of methods of modulating nucleic acid homeostasis in cardiomyocytes provided herein, in some cases, the viral vector comprises a promoter operatively linked to the nucleic acid encoding PKP2. In some case, the promoter is a cardiac specific promoter. In some cases, the promoter or the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter. In some cases, the promoter is a ubiquitous promoter. In some cases, the promoter or the ubiquitous promoter is a beta-actin promoter, a eukaryotic translation elongation factor 1 a promoter, a heat shock protein 70 promoter, or a ubiquitin B promoter. In some cases, the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some cases, the viral vector further comprises a cardiac specific enhancer.

[00160] In various aspects of methods of modulating nucleic acid homeostasis in cardiomyocytes provided herein, in some cases, the viral vector is selected from the group consisting of an adeno- associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus. In some cases, the viral vector is an adeno-associated virus. In some cases, the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

[00161] In various aspects of methods of modulating nucleic acid homeostasis in cardiomyocytes provided herein, in some cases, the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8. In some cases, the PKP2 polypeptide has an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 8. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence selected from one or SEQ ID Nos: 13-19. In some cases, the nucleic acid encoding the PKP2 polypeptide has a sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 90%, at least about 95%, at least about 99% or about 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID Nos: 13-19.

[00162] In various aspects of methods of modulating nucleic acid homeostasis in cardiomyocytes provided herein, in some cases, the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

[00163] In various aspects of methods of modulating nucleic acid homeostasis in cardiomyocytes provided herein, in some cases, gene expression is measured using a method comprising one or more of reverse -transcriptase quantitative polymerase chain reaction (RT-qPCR), ribonucleic acid sequencing (RNAseq), array hybridization, or probe hybridization. In some embodiments, array hybridization comprises a microarray. In some embodiments, probe hybridization comprises in situ hybridization, Northern blot, or fluorescence activated cell sorting (FACS). In some embodiments, measuring expression comprises one or more of Western blot, immunofluorescence, enzyme-linked immunoassay, or fluorescence activated cell sorting (FACS).

[00164] In various aspects of methods of modulating nucleic acid homeostasis in cardiomyocytes provided herein, in some cases nucleic acid homeostasis is modulated for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 36 months, at least about 48 months, at least about 60 months, or longer.

Gene Therapy Vectors

[00165] In various aspects of methods provided herein, gene therapy vectors herein comprise nucleic acid sequences or nucleic acid sequences encoding amino acid sequences provided in Table 1 below.

Viral Vectors

[00166] Suitable viral vectors for methods and gene therapy vectors provided herein include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (e.g., Li et al. (1994) Invest Opthalmol Vis Sci 35:2543-2549; Borras et al. (1999) Gene Ther 6:515-524; Li and Davidson, (1995) Proc. Natl. Acad. Sci. 92:7700-7704; Sakamoto et al. (1999) Hum Gene Ther 5: 1088-1097; WO 94/12649; WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (e.g., Ali et al. (1998) Hum Gene Ther 9(1): 81-86, 1998, Flannery et al. (1997) Proc. Natl. Acad. Sci. 94:6916-6921; Bennett et al. (1997) Invest Opthalmol Vis Sci 38:2857-2863; Jomary et al. (1997) Gene Ther 4:683-690; Rolling et al. (1999), Hum Gene Ther 10:641-648; Ali et al. (1996) Hum Mol Genet. 5:591-594; WO 93/09239, Samulski et al. (1989) J. Vir. 63 :3822-3828; Mendelson et al. (1988) Virol. 166: 154-165; and Flotte et al. (1993) Proc. Natl. Acad. Sci. 90: 10613-10617; SV40; herpes simplex virus; human immunodeficiency virus (e.g., Miyoshi et al. (1997) Proc. Natl. Acad. Sci. 94: 10319-10323; Takahashi et al. (1999) J Virol 73 :7812-7816); a retroviral vector (e.g., Murine -Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. Numerous suitable expression vectors are available to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for eukaryotic cells: pXTl, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia), and pAd (Life Technologies). However, any other vector is contemplated for use so long as it is compatible with the methods of the present disclosure.

[00167] The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Viral vectors are contemplated to include control sequences such as promoters for expression of the polypeptide of interest. Although many viral vectors integrate into the host cell genome, if desired, the segments that allow such integration can be removed or altered to prevent such integration. Moreover, in some embodiments, the vectors do not contain a mammalian origin of replication. Non-limiting examples of virus vectors are described below that are contemplated for use in delivering nucleic acids encoding PKP2 into a selected cell. In some embodiments, the viral vector is derived from a replication-deficient virus.

[00168] In general, other useful viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the polypeptide of interest. Non-cytopathic viruses include certain retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. In general, the retroviruses are replication-deficient (e.g., capable of directing synthesis of the desired transcripts, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of polynucleotide in vivo.

[00169] In some embodiments, a polynucleotide encoding PKP2 is housed within an infective virus that has been engineered to express a specific binding ligand. The virus particle will thus bind with specificity to the cognate receptors of the target cell and deliver the contents to the cell. In some embodiments, the virus is modified to impart particular viral tropism, e.g., the virus preferentially infects fibroblasts, heart cells, or more particularly cardiac fibroblasts (CFs). For AAV, in some cases, capsid proteins are mutated to alter the tropism of the viral vector. For example, lentivirus tropism is often modified by using different envelope proteins; this is known as “pseudotyping.”

[00170] In some embodiments, the viral vector is a retroviral vector. Retroviruses often integrate their genes into the host genome, transfer a large amount of foreign genetic material, infect a broad spectrum of species and cell types, and are often packaged in special cell-lines (Miller et al., Am. J. Clin. Oncol., 15(3):216-221, 1992). In some embodiments, a retroviral vector is altered so that it does not integrate into the host cell genome.

[00171] In some embodiments, the recombinant retrovirus comprises a viral polypeptide (e.g., retroviral env) to aid entry into the target cell. Such viral polypeptides are well-established in the art, for example, U.S. Pat. No. 5,449,614. In some embodiments, the viral polypeptide is an amphotropic viral polypeptide, for example, amphotropic env, which aids entry into cells derived from multiple species, including cells outside of the original host species. In some embodiments, the viral polypeptide is a xenotropic viral polypeptide that aids entry into cells outside of the original host species. In some embodiments, the viral polypeptide is an ecotropic viral polypeptide, for example, ecotropic env, which aids entry into cells of the original host species.

[00172] Examples of viral polypeptides capable of aiding entry of retroviruses into cells include, but are not limited to: MMLV amphotropic env, MMLV ecotropic env, MMLV xenotropic env, vesicular stomatitis virus-g protein (VSV-g), HIV-1 env, Gibbon Ape Leukemia Virus (GALV) env, RD114, FeLV-C, FeLV-B, MLV 10A1 env gene, and variants thereof, including chimeras. Yee et al. (1994) Methods Cell Biol, Pt A: 99-1 12 (VSV-G); U.S. Pat. No. 5,449,614. In some cases, the viral polypeptide is genetically modified to promote expression or enhanced binding to a receptor.

[00173] In embodiments, the retroviral construct is derived from a range of retroviruses, e.g., MMLV, HIV-1, SIV, FIV, or other retrovirus described herein. In some embodiments, the retroviral construct encodes all viral polypeptides necessary for more than one cycle of replication of a specific virus. In some cases, the efficiency of viral entry is improved by the addition of other factors or other viral polypeptides. In other cases, the viral polypeptides encoded by the retroviral construct do not support more than one cycle of replication, e.g., U.S. Pat. No. 6,872,528. In such circumstances, the addition of other factors or other viral polypeptides often help facilitate viral entry. In an exemplary embodiment, the recombinant retrovirus is HIV-1 virus comprising a VSV-g polypeptide, but not comprising a HIV 1 env polypeptide.

[00174] In some embodiments, the retroviral construct comprises: a promoter, a multi-cloning site, and/or a resistance gene. Examples of promoters include but are not limited to CMV, SV40, EFla, [3-actin; retroviral LTR promoters, and inducible promoters. In some embodiments, the retroviral construct comprises a packaging signal (e.g., a packaging signal derived from the MFG vector; a psi packaging signal). Examples of some retroviral constructs described in the art include but are not limited to: pMX, pBabeX or derivatives thereof. Onishi et al. (1996) Experimental Hematology, 24:324-329. In some cases, the retroviral construct is a self-inactivating lentiviral vector (SIN) vector. Miyoshi et al. (1998) J. Virol 72( 10): 8150- 8157. In some cases, the retroviral construct is LL-CG, LS-CG, CL-CG, CS-CG, CLG or MFG. Miyoshi et al. (1998) J. Virol 72( 10): 8150-8157; Onishi et al. (1996) Experimental Hematology, 24:324-329; Riviere et al. (1995) Proc. Natl. Acad. Sci., 92:6733-6737.

[00175] In some embodiments, a retroviral vector is constructed by inserting a nucleic acid (e.g., one encoding a polypeptide of interest or an RNA) into the viral genome in the place of some viral sequences to produce a virus that is replication-defective. To produce virions, a packaging cell line containing the gag, pol, and env genes, but without the LTR and packaging components, is constructed (Mann et al., Cell 33: 153-159, 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into a special cell line (e.g., by calcium phosphate precipitation or lipid transfection), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubinstein, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986; Mann et al., Cell, 33: 153-159, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression typically involves the division of host cells (Paskind et al., Virology, 67:242-248, 1975).

[00176] In some embodiments, the viral vector is a lentiviral vector. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Information on lentiviral vectors is available, for example, in Naldini et al., Science 272(5259):263-267, 1996; Zufferey et al., Nat Biotechnol 15(9): 871 -875, 1997; Blomer et al., J Virol. 71(9):6641-6649, 1997; U.S. Patent Nos. 6,013,516 and 5,994,136, each of which is incorporated herein by reference in its entirety. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted to make the vector biologically safe. The lentivirus employed is sometimes replication and/or integration defective.

[00177] Recombinant lentiviral vectors are capable of infecting non-dividing cells and are sometimes used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Patent No. 5,994, 136, which is incorporated herein by reference in its entirety. In some embodiments, the recombinant virus is targeted by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell type. For example, a targetspecific vector is sometimes generated by inserting a nucleic acid segment (including a regulatory region) of interest into the viral vector, along with another gene that encodes a ligand for a receptor on a specific target cell type.

[00178] Some lentiviral vectors are described in the art, see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136 all incorporated herein by reference. In general, these vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. In some cases, a lentiviral vector is introduced into a cell concurrently with one or more lentiviral packaging plasmids, which include, without limitation, pMD2.G, pRSV-rev, pMDLG- pRRE, and pRRL-GOI. Introduction of a lentiviral vector alone or in combination with lentiviral packaging plasmids into a cell, in some embodiments causes the lentiviral vector to be packaged into a lentiviral particle. In some embodiments, the lentiviral vector is a non-integrating lentiviral (NIL) vector. Illustrative methods for generating NIL vectors, such as the D64V substitution in the integrase gene, are provided in US 8,119,119. [00179] 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 (Grunhaus et al., Seminar in Virology 200(2):535-546, 1992)). In some cases, PKP2 is introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, Biotechniques, 17(6): 1110-7, 1994; Cotten et al., Proc Natl Acad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3): 141-64, 1994.).

[00180] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. AAV is an attractive vector system as it has a low frequency of integration and it can infect non-dividing cells, thus making it useful for delivery of polynucleotides into mammalian cells, for example, in tissue culture (Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992) or in vivo. Details concerning the generation and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, each incorporated herein by reference in its entirety.

[00181] AAV is a replication-deficient parvovirus, the single -stranded DNA genome of which is about 4.7 kb in length including two 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are available in various online databases. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Patent 9,434,928, incorporated herein by reference. Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs. Three AAV promoters (named p5, pl9, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and pi 9), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992). [00182] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and often persists essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). Of particular importance to the present disclosure, AAV, and AAV9 in particular, are capable of infecting cells of the heart, such as myocardium, epicardium, or both (Prasad et al, 2011; Piras et al, 2016; Ambrosi et al., 2019). The AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, in some cases, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, repcap) is replaced with foreign DNA. To generate AAV vectors, in some cases, the rep and cap proteins are provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65 °C for several hours), making cold preservation of AAV less critical. In some cases, AAV is even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection. The AAV vectors of the disclosure include self- complementary, duplexed AAV vectors, synthetic ITRs, and/or AAV vectors with increased packaging compacity. Illustrative methods are provided in US 8,784,799; US 8,999,678; US 9,169,494; US 9,447,433; and US 9,783,824, each of which is incorporated by reference in its entirety.

[00183] AAV DNA in the rAAV genomes is contemplated to be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV-1, AAV -2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV- 10, AAV-11, AAV- 12, AAV-13 and AAV rh74. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Mol. Therapy. 22): 1900-09 (2014). The nucleotide sequences of the genomes of various AAV serotypes are described in the art. AAV vectors of the present disclosure include AAV vectors of serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV39, AAV43, AAV.rh74, and AAV.rh8. Illustrative AAV vectors are provided in US 63/012,703; US 7,105,345; US 15/782,980; US 7,259,151; US 6,962,815; US 7,718,424; US 6,984,517; US 7,718,424; US 6,156,303; US 8,524,446; US 7,790,449; US 7,906,111; US 9,737,618; US App 15/433,322; US 7,198,951, each of which is incorporated by reference in its entirety.

[00184] In some embodiments, the AAV expression vector is pseudotyped to enhance targeting. To promote gene transfer and sustain expression in cardiomyocytes, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV39, AAV43, AAV.rh74, and AAV.rh8, are contemplated for use. In some cases, the AAV2 genome is packaged into the capsid of producing pseudotyped vectors AAV2/5, AAV2/7, and AAV2/8 respectively, as described in Balaji et al. J Surg Res. 184:691-98 (2013). In some embodiments, an AAV9 is used to target expression in myofibroblast-like lineages, as described in Piras et al. Gene Therapy 23:469-478 (2016). 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 Ther. 24:906-13 (2013); Pozsgai et al. Mol Ther. 25:855-69 (2017); Kotterman et al. Nature Reviews Genetics 15:445-51 (2014); and US20160340393A1 to Schaffer et al. In some embodiments, the viral vector is AAV engineered to increase target cell infectivity as described in US20180066285A1. [00185] In some embodiments, the AAV vectors of the disclosure comprise a modified capsid, in particular as capsid engineered to enhance or promote in vivo or ex vivo transduction of cardiac cells, or more particularly cardiomyocytes; or that evade the subject’s immune system; or that have improved biodistribution. Illustrative AAV capsids are provided in US 7,867,484; US 9,233,131; US 10,046,016; WO 2016/133917; WO 2018/222503; and WO 20019/060454, each of which is incorporated by reference in its entirety. In an AAV capsid (or in particular an AAV9 capsid), one or more substitutions are contemplated to increase infectivity towards cells in the myocardium, epicardium, or both. More particularly, in some embodiments, the AAV vectors of the disclosure, optionally AAV9-based vectors, comprise in their capsid proteins one or more substitutions. In some embodiments, the AAV vectors of the disclosure comprise the AAV-A9 capsid and/or serotype. It will be appreciated that these substitutions and insertions are contemplated to be combined together to generate various capsid proteins useful in the present disclosure.

Methods of Producing Viral Vectors

[00186] In general, a viral vector is produced by introducing a viral DNA or RNA construct into a producer cell. In some cases, the producer cell does not express exogenous genes. In other cases, the producer cell is a “packaging cell” comprising one or more exogenous genes, e.g., genes encoding one or more gag, pol, or env polypeptides and/or one or more retroviral gag, pol, or env polypeptides. In some embodiments, the retroviral packaging cell comprises a gene encoding a viral polypeptide, e.g., VSV-g, that aids entry into target cells. In some cases, the packaging cell comprises genes encoding one or more lentiviral proteins, e.g., gag, pol, env, vpr, vpu, vpx, vif, tat, rev, or nef. In some cases, the packaging cell comprises genes encoding adenovirus proteins such as El A or El B or other adenoviral proteins. For example, in some cases, proteins supplied by packaging cells are retrovirus-derived proteins such as gag, pol, and env; lentivirus-derived proteins such as gag, pol, env, vpr, vpu, vpx, vif, tat, rev, and nef; and adenovirus-derived proteins such as El A and El B. In many examples, the packaging cells supply proteins derived from a virus that differs from the virus from which the viral vector is derived. Methods of producing recombinant viruses from packaging cells and their uses are well established; see, e.g., U.S. Pat. Nos. 5,834,256; 6,910,434; 5,591,624; 5,817,491; 7,070,994; and 6,995,009.

[00187] Packaging cell lines include but are not limited to any easily -transfectable cell line. Packaging cell lines are often based on 293T cells, NIH3T3, COS or HeLa cell lines. In some cases, packaging cell lines include SF9 insect cells. Packaging cells are often used to package virus vector plasmids deficient in at least one gene encoding a protein required for virus packaging. Any cells that supply a protein or polypeptide lacking from the proteins encoded by such viral vectors or plasmids are contemplated for use as packaging cells. Examples of packaging cell lines include, but are not limited to: Platinum-E (Plat-E), Platinum-A (Plat- A), BOSC 23 (ATCC CRL 11554) and Bing (ATCC CRL 11270). Morita et al. (2000) Gene Therapy 7(12): 1063-1066; Onishi et al. (1996) Experimental Hematology, 24:324-329; U.S. Pat. No. 6,995,009. Commercial packaging lines are also useful, e.g., Ampho-Pak 293 cell line, Eco-Pak 2-293 cell line, RetroPack PT67 cell line, and Retro-X Universal Packaging System (all available from Clontech).

[00188] Virus vector plasmids (or constructs), include: pMXs, pMxs-IB, pMXs-puro, pMXs-neo (pMXs- IB is a vector carrying the blasticidin-resistant gene instead of the puromycin-resistant gene of pMXs- puro) Kimatura et al. (2003) Experimental Hematology 31 : 1007-1014; MFG Riviere et al. (1995) Proc. Natl. Acad. Sci., 92:6733-6737; pBabePuro; Morgenstern et al. (1990) Nucleic Acids Research 18:3587-3596; LL-CG, CL-CG, CS-CG, CLG Miyoshi et al. (1998) J. Vir. 72:8150-8157 and the like as the retrovirus system, and pAdexl Kanegae et al. (1995) Nucleic Acids Research 23 : 3816-3821 and the like as the adenovirus system. In exemplary embodiments, the retroviral construct comprises blasticidin (e.g., pMXs-IB), puromycin (e.g., pMXs-puro, pBabePuro), or neomycin (e.g., pMXs-neo). Morgenstem et al. (1990) Nucleic Acids Research 18:3587-3596.

Promoters and Enhancers

[00189] In some embodiments, a nucleic acid encoding a PKP2 is operably linked to a promoter and/or enhancer to facilitate expression of PKP2. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive, tissue specific, and inducible promoters, transcription enhancer elements, transcription terminators, etc. are suitable for use in the expression vector (e.g., Bitter et al. (1987) Methods in Enzymology, 153 :516-544).

[00190] Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include CMV, CMV immediate early, HSV thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. In some embodiments, promoters that are capable of conferring cardiac-specific expression will be used, including but not limited to promoters that confer expression in the myocardium, the epicardium, or both (Prasad et al., 2011). Non-limiting examples of suitable cardiac-specific promoters include alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2), cardiac troponin T (cTnT), and cardiac troponin C (cTnC). In some embodiments, a PKP2 or a desmin promoter is used. In some cases, a chimeric promoter with cardiac specific expression is used. In some cases, a cardiac specific enhancer is combined with the promoter. [00191] Examples of suitable promoters for driving expression PKP2 include, but are not limited to, retroviral long terminal repeat (LTR) elements; constitutive promoters such as CMV, HSV1-TK, SV40, EF-la, P-actin, phosphoglycerol kinase (PGK); inducible promoters, such as those containing Tet- operator elements; and cardiac-specific promoters, such as alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2), cardiac troponin T (cTnT), and cardiac troponin C (cTnC). In some embodiments, a PKP2 or a desmin promoter is used. In some embodiments, a chimeric promoter with cardiac specific expression is used. In some cases, a cardiac specific enhancer is combined with the promoter. [00192] In some embodiments, a polynucleotide is operably linked to a cell type-specific transcriptional regulator element (TRE), where TREs include promoters and enhancers. Suitable TREs include, but are not limited to, TREs derived from the following genes: myosin light chain-2, a-myosin heavy chain, AE3, cardiac troponin C, and cardiac actin. Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. N. Y. Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Cell. Biol. 14: 1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.

[00193] Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers often include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences are sometimes produced using recombinant cloning and/or nucleic acid amplification technology, including PCR, in connection with the compositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by reference).

[00194] In some embodiments, the vectors of the disclosure include one or more polyA signals. Illustrative polyA signals useful in the vectors of the disclosure include the short polyA signal and the bGH polyA signal. In some embodiments, the vectors of the disclosure include one or more 3’ elements. Illustrative 3 ’ elements include the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

Gene Therapy Vector Compositions

[00195] To prepare the composition, the vectors and/or the cells are generated, and the vectors or cells are purified as necessary or desired. The vectors, and/or other agents are sometimes suspended in a pharmaceutically acceptable carrier. In some embodiments, the composition is lyophilized. These compounds and cells are often adjusted to an appropriate concentration, and optionally combined with other agents. The absolute weight of a given compound and/or other agent included in a unit dose varies widely. The dose and the number of administrations are contemplated to be optimized by those skilled in the art.

[00196] For example, in some embodiments, about 10²- 10¹⁰ vector genomes (vg) are be administered. In some embodiments, the dose be at least about 10² vg, about 10³ vg, about 10⁴ vg, about 10⁵ vg, about 10⁶ vg, about 10⁷ vg, about 10⁸ vg, about 10⁹ vg, about 10¹⁰ vg, or more vector genomes. In some embodiments, the dose be about 10² vg, about 10³ vg, about 10⁴ vg, about 10⁵ vg, about 10⁶ vg, about 10⁷ vg, about 10⁸ vg, about 10⁹ vg, about 10¹⁰ vg, or more vector genomes. [00197] Daily doses of the compounds vary as well. Such daily doses often range, for example, from at least about 10² vg/day, about 10³ vg/day, about 10⁴ vg/day, about 10⁵ vg/day, about 10⁶ vg/day, about 10⁷ vg/day, about 10⁸ vg/day, about 10⁹ vg/day, about 10¹⁰ vg/day, or more vector genomes per day.

[00198] In some embodiments, the method of the disclosure comprises administering a vector or vector system of the disclosure (e.g. an rAAV vector) by intracardiac injection, intramyocardiac injection, endocardial injection, intracardiac catheterization, or systemic administration. In some embodiments, the subject (e.g., a human) is treated by administering between about IxlO⁸ and about IxlO¹⁵ GC of a vector (e.g., an AAV vector or lentiviral vector) by intracardiac injection, intramyocardiac injection, endocardial injection, intracardiac catheterization, or systemic administration. In some embodiments, the subject is treated by administering between about IxlO⁸ and about IxlO¹⁵ GC, between about IxlO⁸ and about lxlO¹⁵ GC, between about IxlO⁹ and about lxlO¹⁴GC, between about IxlO¹⁰ and about lxlO¹³ GC, between about IxlO¹¹ and about IxlO¹² GC, or between about IxlO¹² and about IxlO¹³ GC of vector. In some embodiments, the subject is treated by administering between about IxlO⁸ and about IxlO¹⁰ GC, between about IxlO⁹ and about IxlO¹¹ GC, between about IxlO¹⁰ and about lxlO¹² GC, between about IxlO¹¹ and about lxlO¹³ GC, between about IxlO¹² and about lxlO¹⁴GC, or between about IxlO¹³ and about IxlO¹⁵ GC of vector. In some embodiments, the subject is treated by administering at least IxlO⁸, at least about IxlO⁹, at least about IxlO¹⁰, at least about IxlO¹¹, at least about IxlO¹², at least about IxlO¹³, or at least about IxlO¹⁵ GC of vector. In some embodiments, the subject is treated by administering at most IxlO⁸, at most about IxlO⁹, at most about IxlO¹⁰, at most about IxlO¹¹, at most about IxlO¹², at most about IxlO¹³, or at most about IxlO¹⁵ GC of vector. In some embodiments, the subject (e.g., a human) is treated by administering between about IxlO⁸ and about IxlO¹⁵ GC/kg of a vector (e.g, an AAV vector or lentiviral vector) by intracardiac injection or systemically. In some embodiments, the subject is treated by administering between about IxlO⁸ and about IxlO¹⁵ GC/kg, between about IxlO⁸ and about IxlO¹⁵ GC/kg, between about IxlO⁹ and about IxlO¹⁴ GC/kg, between about IxlO¹⁰ and about IxlO¹³ GC/kg, between about IxlO¹¹ and about IxlO¹² GC/kg, or between about IxlO¹² and about IxlO¹³ GC/kg of vector. In some embodiments, the subject is treated by administering between about IxlO⁸ and about IxlO¹⁰ GC/kg, between about IxlO⁹ and about IxlO¹¹ GC/kg, between about IxlO¹⁰ and about IxlO¹² GC/kg, between about IxlO¹¹ and about IxlO¹³ GC/kg, between about IxlO¹² and about IxlO¹⁴ GC/kg, or between about IxlO¹³ and about IxlO¹⁵ GC/kg of vector. In some embodiments, the subject is treated by administering at least IxlO⁸, at least about IxlO⁹, at least about IxlO¹⁰, at least about IxlO¹¹, at least about IxlO¹², at least about IxlO¹³, or at least about IxlO¹⁵ GC/kg of vector. In some embodiments, the subject is treated by administering at most IxlO⁸, at most about IxlO⁹, at most about IxlO¹⁰, at most about IxlO¹¹, at most about IxlO¹², at most about IxlO¹³, or at most about IxlO¹⁵ GC/kg of vector. It will be appreciated that the amount of vectors and for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately, in some embodiments, the attendant health care provider will determine proper dosage. A pharmaceutical composition is contemplated to be formulated with the appropriate ratio of each compound in a single unit dosage form for administration.

[00199] The compositions are sometimes formulated for sustained release (for example, using microencapsulation, see WO 94/07529, and/or U.S. Patent No.4, 962, 091). The formulations, where appropriate, are conveniently presented in discrete unit dosage forms and, in some embodiments, are prepared by any of the methods well described in the pharmaceutical arts. Such methods often include the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

[00200] One or more suitable unit dosage forms containing the compounds, in some embodiments, are administered by a variety of routes including parenteral (including subcutaneous, intravenous, intramuscular, and intraperitoneal), intracardially, pericardially, oral, rectal, dermal, transdermal, intrathoracic, intrapulmonary, and intranasal (respiratory) routes.

[00201] The gene therapy vectors provided herein are prepared in many forms that include aqueous solutions, suspensions, tablets, hard or soft gelatin capsules, and liposomes and other slow -release formulations, such as shaped polymeric gels. Administration of gene therapy vectors often involves parenteral or local administration in an aqueous solution. Similarly, compositions containing gene therapy vectors are sometimes administered in a device, scaffold, or as a sustained release formulation. Different types of formulating procedures are described in U.S. Patent No. 6,306,434 and in the references contained therein.

[00202] Vectors, in some embodiments, are formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and are often presented in unit dosage form in ampoules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative. The pharmaceutical compositions often take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and sometimes contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Suitable carriers include saline solution, phosphate buffered saline, and other materials commonly used in the art.

[00203] The compositions sometimes also contain other ingredients such as agents useful for treatment of cardiac diseases, conditions and injuries, such as, for example, an anticoagulant (e.g., dalteparin (fragmin), danaparoid (orgaran), enoxaparin (lovenox), heparin, tinzaparin (innohep), and/or warfarin (coumadin)), an antiplatelet agent (e.g., aspirin, ticlopidine, clopidogrel, or dipyridamole), an angiotensin-converting enzyme inhibitor (e.g., Benazepril (Uotensin), Captopril (Capoten), Enalapril (Vasotec), Fosinopril (Monopril), Eisinopril (Prinivil, Zestril), Moexipril (Univasc), Perindopril (Aceon), Quinapril (Accupril), Ramipril (Altace), and/or Trandolapril (Mavik)), angiotensin II receptor blockers (e.g., Candesartan (Atacand), Eprosartan (Teveten), Irbesartan (Avapro), Losartan (Cozaar), Telmisartan (Micardis), and/or Valsartan (Diovan)), a beta blocker (e.g., Acebutolol (Sectral), Atenolol (Tenormin), Betaxolol (Kerlone), Bisoprolol/hydrochlorothiazide (Ziac), Bisoprolol (Zebeta), Carteolol (Cartrol), Metoprolol (Lopressor, Toprol XL), Nadolol (Corgard), Propranolol (Inderal), Sotalol (Betapace), and/or Timolol (Blocadren)), Calcium Channel Blockers (e.g., Amlodipine (Norvasc, Lotrel), Bepridil (Vascor), Diltiazem (Cardizem, Tiazac), Felodipine (Plendil), Nifedipine (Adalat, Procardia), Nimodipine (Nimotop), Nisoldipine (Sular), Verapamil (Calan, Isoptin, Verelan), diuretics (e.g., Amiloride (Midamor), Bumetanide (Bumex), Chlorothiazide (Diuril), Chlorthalidone (Hygroton), Furosemide (Lasix), Hydro-chlorothiazide (Esidrix, Hydrodiuril), Indapamide (Lozol) and/or Spironolactone (Aldactone)), vasodilators (e.g., Isosorbide dinitrate (Isordil), Nesiritide (Natrecor), Hydralazine (Apresoline), Nitrates and/or Minoxidil), statins, nicotinic acid, gemfibrozil, clofibrate, Digoxin, Digitoxin, Lanoxin, or any combination thereof.

[00204] Additional agents are sometimes included such as antibacterial agents, antimicrobial agents, antiviral agents, biological response modifiers, growth factors; immune modulators, monoclonal antibodies and/or preservatives. The compositions provided herein are contemplated to also be used in conjunction with other forms of therapy.

[00205] The viral vectors described herein are suitable for administration to a subject to treat a disease or disorder. In some embodiments, such a composition is in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient’s physiological condition, whether the purpose of the administration is in response to traumatic injury or for more sustained therapeutic purposes, and other factors described by skilled practitioners. The administration of the compounds and compositions of provided herein, in some embodiments, are administered continuously over a preselected period of time or alternatively are administered in a series of spaced doses. Both local and systemic administration is contemplated. In some embodiments, localized delivery of a viral or non- viral vector is achieved. In some embodiments, localized delivery of cells and/or vectors is used to generate a population of cells within the heart. In some embodiments, such a localized population operates as “pacemaker cells” for the heart.

Definitions

[00206] As used herein, the term “cardiomyopathy” refers to any disease or dysfunction of the myocardium (heart muscle) in which the heart is abnormally enlarged, thickened and/or stiffened. As a result, the heart muscle’s ability to pump blood is usually weakened. The etiology of the disease or disorder is, in some cases, inflammatory, metabolic, toxic, infdtrative, fibroplastic, hematological, genetic, or unknown in origin. There are two general types of cardiomyopathies: ischemic (resulting from a lack of oxygen) and non-ischemic. In some cases, a cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

[00207] ‘ ‘Heart failure (HF) is a complex clinical syndrome that often result from any structural or functional cardiovascular disorder causing systemic perfusion inadequate to meet the body’s metabolic demands without excessively increasing left ventricular filling pressures. It is characterized by specific symptoms, such as dyspnea and fatigue, and signs, such as fluid retention. As used herein, “chronic heart failure” or “congestive heart failure” or “CHF” refer, interchangeably, to an ongoing or persistent forms of heart failure. Common risk factors for CHF include old age, diabetes, high blood pressure and being overweight. CHF is broadly classified according to the systolic function of the left ventricle as HF with reduced or preserved ejection fraction (HFrEF and HFpEF). The term “heart failure” does not mean that the heart has stopped or is failing completely, but that it is weaker than is normal in a healthy person. In some cases, the condition is mild, causing symptoms that are noticeable when exercising, in others, the condition is more severe, causing symptoms that are, in some cases, life-threatening, even while at rest. The most common symptoms of chronic heart failure include shortness of breath, tiredness, swelling of the legs and ankles, chest pain and a cough. In some embodiments, the methods of the disclosure decrease, prevent, or ameliorate one or more symptoms of CHF (e.g., HFrEF) in a subject suffering from or at risk for CHF (e.g., HFrEF). In some embodiments, the disclosure provides methods of treating CHF and conditions that sometimes lead to CHF.

[00208] As used herein “acute heart failure” or “decompensated heart failure” refer, interchangeably, to a syndrome of the worsening of signs and symptoms reflecting an inability of the heart to pump blood at a rate commensurate to the needs of the body at normal fdling pressure. AHF typically develops gradually over the course of days to weeks and then decompensates requiring urgent or emergent therapy due to the severity of these signs or symptoms. In some cases, AHF is the result of a primary disturbance in the systolic or diastolic function of the heart or of abnormal venous or arterial vasoconstriction, but generally represents an interaction of multiple factors, including volume overload. The majority of patients with AHF have decompensation of chronic heart failure (CHF) and consequently much of the discussion of the pathophysiology, presentation, and diagnosis of CHF is directly relevant to an understanding of AHF. In other cases, AHF results from an insult to the heart or an event that impairs heart function, such as an acute myocardial infarction, severe hypertension, damage to a heart valve, abnormal heart rhythms, inflammation or infection of the heart, toxins and medications. In some embodiments, the methods of the disclosure decrease, prevent, or ameliorate one or more symptoms of AHF in a subject suffering from or at risk for AHF. In some embodiments, the disclosure provides methods of treating AHF and conditions that sometimes lead to AHF. In some cases, AHF is the result of ischemia associated with myocardial infarction.

[00209] As used herein, the terms “subject” or “individual” refers to any animal, such as a domesticated animal, a zoo animal, or a human. In some cases, the “subject” or “individual” is a mammal like a dog, cat, horse, livestock, a zoo animal, or a human. Alternatively or in combination, the subject or individual is a domesticated animal such as a bird, a pet, or a farm animal. Specific examples of “subjects” and “individuals” include, but are not limited to, individuals with a cardiac disease or disorder, and individuals with cardiac disorder-related characteristics or symptoms, such as arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

[00210] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5thedition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; IRL Press (1986) Immobilized Cells and Enzymes; Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition (2002) Cold Spring Harbor Laboratory Press; Sohail (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press); Sell (2013) Stem Cells Handbook.

[00211] Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[00212] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of +/- 15 %, or alternatively 10%, or alternatively 5%, or alternatively 2%. 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 described in the art.

[00213] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cardiomyocyte” includes a plurality of cardiomyocytes.

[00214] Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). [00215] ‘ ‘Administration,” “administering” and the like, when used in connection with a gene therapy vector or composition thereof as provided herein refer both to direct administration, which, in some cases includes administration to non-cardiomyocytes in vitro, administration to non-cardiomyocytes in vivo, administration to a subject by a medical professional or by self-administration by the subject and/or to indirect administration, which, in some cases, is the act of prescribing a composition comprising a gene therapy vector provided herein. When used herein in reference to a cell, it refers to introducing a composition to the cell. Typically, an effective amount is administered, which amount is often to be determined by one of skill in the art. Any suitable method of administration is contemplated to be used. In some cases, a gene therapy vector is administered to the cells by, for example, by addition of the gene therapy vector to the cell culture media or injection in vivo to the site of cardiac injury. In some cases, administration to a subject is achieved by, for example, intravascular injection, intramyocardial delivery, and the like.

[00216] 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. In some cases, cardiac cells are derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells.

[00217] The term “cardiomyocyte” or “cardiomyocytes” as used herein refers to sarcomere -containing striated muscle cells, naturally found in the mammalian heart, as opposed to skeletal muscle cells. Cardiomyocytes are characterized by the expression of specialized molecules e.g., proteins like myosin heavy chain, myosin light chain, cardiac a-actinin. The term “cardiomyocyte” as used herein is an umbrella term comprising any cardiomyocyte subpopulation or cardiomyocyte subtype, e.g., atrial, ventricular and pacemaker cardiomyocytes.

[00218] The term “culture” or “cell culture” means the maintenance of cells in an artificial, in vitro environment. A “cell culture system” is used herein to refer to culture conditions in which a population of cells are grown as monolayers or in suspension. “Culture medium” is used herein to refer to a nutrient solution for the culturing, growth, or proliferation of cells. Culture medium is characterized, in some cases, by functional properties such as, but not limited to, the ability to maintain cells in a particular state (e.g., a pluripotent state, a quiescent state, etc.), or to mature cells, such as, in some embodiments, to promote the differentiation of progenitor cells into cells of a particular lineage (e.g., a cardiomyocyte). [00219] As used herein, the term “expression” or “express” refers to the process by which nucleic acids or polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide or nucleic acid is derived from genomic DNA, in some cases, expression includes splicing of the mRNA in a eukaryotic cell. In some cases, the expression level of a gene is determined by measuring the amount of mRNA or protein in a cell or tissue sample.

[00220] As used herein, an “expression cassette” is a DNA polynucleotide comprising one or more polynucleotides or nucleic acids encoding protein(s) or nucleic acid(s) that is configured to express the polynucleotide in a host cell. Typically, expression of the polynucleotide(s) is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such polynucleotides are said to be “operably linked to” or “operatively linked to” the regulatory elements (e.g., a promoter).

[00221] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[00222] ‘ ‘Treatment,” “treating,” and “treat” are defined as acting upon a disease, disorder, or condition with an agent to reduce or ameliorate harmful or any other undesired effects of the disease, disorder, condition and/or their symptoms.

[00223] As used herein, the term “effective amount” and the like refers to an amount that is sufficient to induce a desired physiologic outcome (e.g., treatment of a disease). An effective amount is sometimes administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period which the individual dosage unit is to be used, the bioavailability of the composition, the route of administration, etc. It is understood, however, that specific amounts of the compositions (e.g., gene therapy vectors) for any particular subject depends upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the composition combination, severity of the particular disease being treated and form of administration.

[00224] As used herein, the term “equivalents thereof’ in reference to a polypeptide or nucleic acid sequence refers to a polypeptide or nucleic acid that differs from a reference polypeptide or nucleic acid sequence, but retains essential properties (e.g., biological activity). A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant, in some cases, alters the amino acid sequence of a polypeptide encoded by the reference polynucleotide. In some cases, nucleotide changes result in amino acid substitutions, deletions, additions, fusions and truncations in the polypeptide encoded by the reference sequence. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.

[00225] As used herein, the term “nucleic acid” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides, vectors, probes, and primers. As used herein, the word “polynucleotide” or “nucleic acid” preceded by a gene name (for example, “PKP2 nucleic acid”) refers to a polynucleotide sequence encoding the corresponding protein (for example, a “PKP2 protein”).

[00226] The terms “polypeptide,” “peptide,” and “protein,” are used interchangeably herein and refer to a polymeric form of amino acids of any length, which sometimes include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues, immunologically tagged proteins, and the like. As used herein, the word “protein” preceded by a gene name (for example, “PKP2 protein”) refers to either the native protein or a functional variant thereof. A “native protein” is a protein encoded by a genomic copy of a gene of an organism, preferably the organism for which the vector is intended (e.g., a human, a rodent, a primate, or an animal of veterinary interest), in any of the gene’s functional isoforms or functional allelic variations.

[00227] As used herein, a “functional variant” or “variant” of a protein is a variant with any number of amino acid substitutions, insertions, truncations, or internal deletions that retains the functional attributes of the protein, including, e.g., the protein’s ability to induce, in combination with other factors, organization of desmosomes. In some cases, functional variants are identified computationally, such as variants having only conservative substitutions, or experimentally using in vitro or in vivo assays. [00228] As used herein, a “codon variant” of a polynucleotide sequence is polynucleotide sequence that encodes the same protein as a reference polynucleotide sequence having one or more synonymous codon substitutions. Selection of synonymous codons is within the skill of those in the art, the coding as the genetic code being known. In some cases, codon optimization is performed using a variety of computational tools (such the GENSMART™ Codon Optimization tool available at www.genscript.com). Generally codon optimization is used to increase the expression of protein in a heterologous system, for instance when a human coding sequence is expressed in a bacterial system. The term “codon variant” is intended to encompass both sequences that are optimized in this manner and sequences that are optimized for other purposes, such as removal of CpG islands and/or cryptic start sites. [00229] The term “vector” refers to a macromolecule or complex of molecules comprising a polynucleotide or protein to be delivered to a host cell, either in vitro or in vivo. A vector is sometimes a modified RNA, a lipid nanoparticle (encapsulating either DNA or RNA), a transposon, an adeno- associated virus (AAV) vector, an adenovirus, a retrovirus, an integrating lentiviral vector (LVV), or a non-integrating LW. Thus, as used herein “vectors” include naked polynucleotides used for transformation (e.g. plasmids) as well as any other composition used to deliver a polynucleotide to a cell, included vectors capable of transducing cells and vectors useful for transfection of cells.

[00230] As used herein, the term “viral vector” refers either to a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also cell components in addition to nucleic acid(s).

[00231] The term “genetic modification” refers to a permanent or transient genetic change induced in a cell following introduction of new nucleic acid (i.e., nucleic acid exogenous to the cell). Genetic change is often accomplished by incorporation of the new nucleic acid into the genome of the cardiac cell, or by transient or stable maintenance of the new nucleic acid as an extrachromosomal element. Where the cell is a eukaryotic cell, a permanent genetic change is often achieved by introduction of the nucleic acid into the genome of the cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like.

[00232] FIGs. 1A-1E show siRNA-mediated PKP2 silencing impacted both cellular structure and functions of human iPSC-CMs. FIG. 1A is a heatmap of RNA sequencing analyses (n = 3) from iPSC- CMs harvested 6 days after treatment with siRNAs against PKP2 (siPKP2) or negative control siRNAs (siNeg) highlighting effects on genes encoding components of the desmosome, sarcomere, and ion channels. FIG. IB shows PKP2 silencing led to reduction in protein expression of DSP, JUP, DES, and MyBPC3 in response to reduced PKP2 protein (Western blot on the left panel) and reduction in SCN5A mRNA in response to reduced PKP2 mRNA (RT-qPCR on the right panel). FIG. 1C shows PKP2 silencing resulted in disappearance of PKP2 and DSP protein from the cellular membrane (top two rows, day 10, n=5 technical replicates; IXM confocal microscope) and cell disarray in patterned iPSC-CMs (bottom two rows, day 10, n=3 technical replicates; Leica DMi8 microscope). Immunofluorescent staining: PKP2 in green, DSP in red, and nuclei in blue. FIG. ID shows PKP2 silencing led to defective contraction as quantified by contraction velocity analyzed by DANA Solution Pulse analysis software. FIG. IE shows that upon PKP2 silencing, reductions in gene expression of ion channels led to depressed beat period, amplitude, and propagation of electrical signal detected as an extracellular field potential from the cardiac monolayers using Axion Biosystems Microelectrode array (MEA) plates.

[00233] FIGs. 2A-2E show AAV:hPKP2 transgene restored the expression level of DSP protein and rescued contraction velocity post PKP2 silencing in Human iPSC-CMs. FIG. 2A shows a schematic representation of the 1 st generation and the 2nd generation AAV expression cassette of PKP2a. Key 3 ’ elements in AAV expression cassette include Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and bovine growth hormone polyadenylation signal (bGH). FIG. 2B shows Western blot analysis demonstrating that the second generation of AAV:hPKP2 is expressed in iPSC-CMs in a dose-dependent fashion by applying viruses at different multiplicity of infection (MOI). FIG. 2C shows day 10 of PKP2 silencing and day 8 of AAV transduction, GFP expression of the first generation of PKP2 expression cassette was used to label AAV transduced iPSC-CMs. Codon optimization allows the transgene PKP2 resistant to siRNA-mediated silencing. The immunofluorescent mini panels show cells were stained for GFP, PKP2, DSP and nuclei, respectively, with the bottom large panel showing merged channels. Yellow inset was magnified to highlight two GFP cells expressing transgene PKP2 (grey color) at the junction of each other and at the junction of other non-GFP neighbors. FIG. 2D shows day 10 of PKP2 silencing and day 8 of AAV transduction, the left bar graph summarized the percentage of cells without GFP (n=12 technical replicates) and with GFP (n=36 technical replicates). The right graph showed restored DSP protein expression quantified by total intensity of immunofluorescence signal post PKP2 silencing in the absence (n=6 technical replicates) or the presence (n=12-18 technical replicates) of AAV:hPKP2 transgene. Quantified data were presented as mean ± s.d. Statistical significance was evaluated by ordinary One-Way ANOVA (Tukey’s post-hoc test). FIG. 2E shows AAV:hPKP2 showed rescue of contraction velocity post PKP2 silencing in iPSC-CMs (n=18-27 technical replicates). Cell contractility was recorded from day 3 to 8 post AAV transduction and analyzed by Pulse video analysis (Curi Bio). Average nuclear counts from live cells were used to normalize contraction velocity. Quantified data were presented as mean ± s.d. Statistical significance was evaluated by ordinary Two- Way ANOVA (Tukey’s post-hoc test). P value: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[00234] FIGs. 3A-3E show Pkp2-cKO ARVC mouse model recapitulated the majority of human ARVC clinical manifestations. FIG. 3A shows Pkp2-cKO ARVC mice (aMyHC-Cre-ER(T2), Pkp2 ^/fl) at ~3 months of age were injected with tamoxifen to induce cardiac knock-out of the Pkp2 gene. Representative immunoblots showed reduction of desmosome proteins PKP2, DSP, PKG, and GJ protein, Cx43. FIG. 3B shows Pkp2-cKO mice developed spontaneous PVCs as observed during 30 minutes of continuous recording of EKG. FIG. 3C shows Pkp2-cKO mice started to develop biventricular dilatation at 2 weeks post tamoxifen induction. RV area (left panel) and LV internal diameter end diastole (LVIDd, right panel) were normalized to body weight. FIG. 3D shows LV performance measured by % ejection fraction sharply declined at 2 weeks post tamoxifen induction. FIG. 3E shows a Kaplan-Meier survival curve showing a sharp decline of survival of Pkp2 cKO mice beginning 3 weeks post tamoxifen induction. Animals showed severe symptoms including sudden death, edema, reduced activity, and reduced tolerance to isoflurane beginning 3 weeks post induction. P value: Student’s t-test. Error bar: s.e.m. *p<0.05, **p<0.01, ***p<0.001 vs. WT.

[00235] FIGs. 4A-4E show an early and single dose of AAV9:PKP2 significantly reduced arrhythmias, improved cardiac function, and prolonged life span to 1.5-year post AAV administration. FIG. 4A shows the study design to evaluate AAV:hPKP2 or AAV9:mPkp2 efficacy using Pkp2-cKO ARVC mouse model. AAV9 was injected three weeks before gene deletion, AAV:hPKP2 at 3E13 vector genomes per kilogram bodyweight (vg/kg) and AAV9:mPkp2 at 5E13 vg/kg. Echocardiograph and EKG data were collected at week 3 and week 4 post gene deletion. FIG. 4B shows raw EKG traces demonstrating a significant contrast in spontaneous arrhythmias in Pkp2-cKO mice in the absence and the presence of AAV9:mPKP2 treatment. PVCs, premature ventricular contractions; NSVT, non-sustained ventricular tachycardia. The right graph summarizes averaged arrhythmia scores representing frequency, duration, and severity of ventricular arrhythmias. AAV9:PKP2 treatment resulted in significantly improved arrhythmia scores. FIG. 4C shows AAV9:PKP2 treatment of Pkp2-cKO mice demonstrated efficacy in reducing RV dilation as estimated by RV area normalized to body weight and FIG. 4D shows maintaining left ventricular ejection fraction at 4 weeks post gene deletion. FIG. 4E shows a Kaplan- Meier survival curve demonstrating that AAV:hPKP2 extended life span of Pkp2-cKO mice after 72 weeks post gene deletion. Numbers in paratheses showed dead vs live animals by the time of takedown. Animals treated by AAV9:mPkp2 (in green line) were taken down early for exploratory studies. P value: Student’s t-test. Error bar: s.e.m.; ****, pO.OOOl.

[00236] FIGs. 5A-5E show AAV:hPKP2 dose-dependently reduced arrhythmias, improved heart structure and cardiac function, restored expression of desmosome proteins and Cx43 and prevented development of fibrosis in Pkp2-cKO mouse. FIG. 5A shows the study design to evaluate dosedependent efficacy of AAV:hPKP2 using Pkp2-cKO ARVC mouse model. Mice were injected with AAV9:PKP2 at 1E13, 3E13, or 1E14 vg/kg at one week after tamoxifen induction of cardiac Pkp2 gene deletion. At 4 weeks post tamoxifen induction (3 weeks post AAV9 injection), animals were sacrificed for expression and histological evaluation. FIG. 5B shows AAV9:PKP2 demonstrated a dose-dependent response at 3 weeks in preventing RV dilation as estimated by RV area normalized to body weight, preventing decline of % LV ejection fraction, and trending improvement in arrhythmia scores. FIG. 5C shows semi-quantitative Western blot analyses showed restoration of PKP2, JUP, and DSP protein at 3 weeks post AAV treatment. Statistical significance was estimated by ordinary One-Way ANOVA. FIG. 5D shows immune-histochemistry for the gap junction protein, Connexin-43 (Cx43), in heart tissue sections showing restoration of Cx43 expression at intercalated discs (ID) at 3 weeks post AAV treatment (top panels). Red arrows indicate ID. Trichrome staining showed a significant reduction of fibrosis, muscle (red) and fibrosis (blue), in heart sections at 3 weeks post AAV treatment (bottom panel). Yellow arrows highlight areas with fibrosis in Pkp2-cKO mouse heart. The percentage of collagen-positive tissue was quantified and shown in the right graph. FIG. 5E shows RT-qPCR analyses of RV tissue at 3 weeks post AAV treatment showed expression of hPKP2 transgene and suppression of heart failure markers (Nppa) (Nppb did not show statistical significance) and fibrosis genes (Collet! , Col3al, Timpl). Gapdh was used as internal control. Statistical significance was estimated by ordinary One-Way ANOVA (Tukey’s post-hoc test). Quantified data were presented as mean ± s.e.m. P value: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[00237] FIGs. 6A-6H show a single dose of AAV9:PKP2 after overt cardiomyopathy halted disease progression via reversed adverse right ventricular remodeling, improved LV function, prevented arrhythmia worthening, and reduced mortality. FIG. 6A shows a study design to evaluate AAV9:mPkp2 efficacy using Pkp2-cKO ARVC mouse model with delivery at 2.5 weeks after Pkp2 cardiac gene deletion by tamoxifen induction. FIG. 6B shows that HBSS-treated Pkp2-cKO ARVC mice died within 6 weeks of cardiac Pkp2 gene deletion, in contrast, AAV9:mPkp2 treatment significantly reduced mortality and extended life span of Pkp2-cKO mice to 51 weeks. Numbers in paratheses show dead vs live animals by the time of takedown. FIG. 6C and FIG. 6F show that AAV9:mPkp2 at 9 weeks post gene deletion and 6.5 weeks post treatment improved left ventricle ejection fraction by 36%; FIG. 6D and FIG. 6G show reversed right ventricle enlargement by 31% and restored RV size similar to that of WT animals (RV size was normalized to body weight, mm2/g); and FIG. 6E and FIG. 6F show prevention of further worsening of arrhythmias by 33.3%, all three readouts relative to HBSS treated Pkp2-cKO ARVC mice. In FIG. 6F, FIG. 6G, and FIG. 6H the top bar graphs show EF%, RV size, and arrhythmia score at 4 weeks post gene deletion and 1.5 weeks post treatment. The bottom bar graphs show multiple comparisons between treatment groups at different time points. P value for all bar graphs in FIG. 6F, FIG. 6G, and FIG. 6H: statistical significance of EF% or RV area was evaluated with ordinary One-Way ANOVA (Tukey’s post-hoc test) and arrhythmia scores with nonparametric Kruskal- Wallis test with Dunn’s correction. All quantified data were presented as mean ± s.e.m.

[00238] FIGs. 7A-7E show AAV:hPKP2 restored expression of genes encoding desmosome, sarcomere, and Ca²⁺ handling system and attenuated expression of genes encoding adverse remodeling factors in a highly coordinated and quantitative fashion. FIG. 7A shows the study design to evaluate AAV:hPKP2 dose-dependent efficacy at week 4 and 9 post tamoxifen induction of Pkp2 cardiac gene deletion in Pkp2- cKO ARVC mouse model. AAV:hPKP2 was dosed 1 week before induction after initial baseline readings of body weight, echocardiography, and EKG. FIG. 7B shows human PKP2 transgene mRNA levels at two doses (Low (L), 3E13 vg/kg; High (H), 6E13 vg/kg) were quantified in copy number per ng of total LV RNA (top panel). Human PKP2 transgene protein levels at two doses were compared to the levels of endogenous mouse Pkp2 in WT and in Pkp2-cKO mouse post cardiac gene deletion by semi- quantitative Western blot (WB) (bottom panel). FIG. 7C shows TN-401 treatment at 3E13 or 6E13 vg/kg at week 9 post gene deletion showed comparable efficacy in EF%, RV area (mm²/g, normalized to body weight), LV mass (mg/g, normalized to body weight), and arrhythmia scores. Statistical significance of EF% or RV area was evaluated with ordinary One-Way ANOVA (Tukey’s post-hoc test) and 4 to 9-week arrhythmia scores with ordinary Two-Way ANOVA (Tukey’s post-hoc test). Quantified data were presented as mean ± s.e.m. FIG. 7D shows a heatmap of gene expression analyses sorted by heart chambers (LV vs RV) clustered gene classes in response to treatment groups. FIG. 7E shows volcano plots from differential gene expression analysis showed changes in gene expression between treatment groups ofWT vs HBSS (top graph) and TN-401 high dose vs HBSS treated animals (bottom graph). Genes highlighted in red were selected from each gene class. FIG. 7F shows boxplots showing group-wise gene expression for each representative gene of the selected gene classes. In the figure, Pkp2 indicates the endogenous mouse gene while PKP2 indicates the human transgene expressed from the gene therapy construct. Each box showed the distribution of expression values in the following manner: the midline represented the median expression value, the box indicated the interquartile range where the middle 50% of values lie, and the whiskers at the top and bottom of each box represented the range of values outside the interquartile range. The black dots represent values that fall outside the 2nd and 3rd quartiles. Values were log 2 of TPM (Transcripts Per Million) and were aggregated from LV and RV. Comparison p values were calculated by Student’s t-test: p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[00239] FIGs. 8A-8H show long-term durable expression of AAV9:mPkp2 significantly reduced mortality after disease onset and sustained a broad spectrum of pathways that were perturbed in Pkp2- cKO ARVC mouse and restored by the gene therapy. FIG. 8A shows a study design to evaluate AAV9:mPkp2 efficacy in reducing mortality at 51 weeks post tamoxifen induction of Pkp2 deletion in Pkp2-cKO ARVC mouse model. AAV9:mPkp2 was dosed at 1E13, 3E13, and 1E14 vg/kg either 1 week before the induction (the preventive mode) or at 1E14 vg/kg at 2.5 weeks after induction (the therapeutic mode). FIG. 8B shows a Kaplan-Meier curve illustrating percent survival for each mode of treatment for 51 weeks post Pkp2 deletion. Numbers in paratheses show dead vs live animals by the time of takedown. FIG. 8C shows a Principal Component Analysis showing clusters of gene transcripts from WT (animals taken down at 51 weeks post induction), untreated Pkp2-cKO animals (animals taken down at 4 weeks post induction), and AAV9:mPkp2 treated animals (animals taken down at 51 weeks post induction). FIG. 8D shows volcano plots highlighted numbers of down-regulated genes in blue and numbers of up- regulated genes in red between pair-wise comparisons of untreated vs WT, preventive vs WT, and therapeutic vs WT. FIG. 8E shows Gene Set Enrichment Analysis (GSEA) demonstrating top 10 positively and negatively enriched cardiac gene sets in WT vs Pkp2-cKO. These enriched gene sets were used to compare preventive vs Pkp2-cK(). therapeutic vs Pkp2-cK(). preventive vs WT, therapeutic vs WT, and preventive vs therapeutic. Pathways were filtered for BH-corrected Q value < 0.25. FIG. 8F shows heatmap of RNA sequencing results showing relative expression of selected genes categorized in treatment groups, cardiac RV and LV chambers, and gene classes. Each column depicted a scaled mean value taken across samples with the same treatment group. Animal numbers from each treatment group used for RNA sequencing were 9 WT, 4 cKO, 2 at 1E13 vg/kg (not included on the Heatmap), 8 at 3E13 vg/kg, 5 at 1E14 vg/kg, and 6 at 1E14 vg/kg (the therapeutic mode) with both RV and LV collected. FIG. 8G shows GSEA demonstrating positively and negatively enriched cardiac gene sets in WT vs Pkp2-cKO. These enriched gene sets were used to compare preventive vs Pkp2-cKO, therapeutic vs Pkp2-cKO, preventive vs WT, therapeutic vs WT, and preventive vs therapeutic. Pathways in the middle and bottom heatmap were filtered for BH-corrected Q value < 0.25. The top heatmap showed known gene sets reported in ARVC literature; middle and bottom heatmaps showed annotated gene sets of biological pathways and Gene Ontology groups (C2 and C5 clusters, respectively) from Human MSigDB database (v2023. 1.Hs) . FIG. 8H shows RT-qPCR analyses showed expression of a total of mouse Pkp2 mRNA (including mouse transgene mRNA), heart failure marker genes, Nppa. Nppb. and fibrosis genes, Timpl, Coll al , and Col3al, in RV (top row) and LV (bottom row) at 51 weeks post Pkp2 deletion.

Statistical evaluation was performed using ordinary One-Way ANOVA (Tukey’s post-hoc test); P values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[00240] FIGs. 9A-9F show AAV9:PKP2 6wks safety study in WT mouse showed no adverse effects at <10X of efficacious dose. FIG. 9A shows the study design to evaluate AAV9:PKP2 safety in WT CD1 mice. Mice were injected with AAV9:PKP2 at 1E14 and 3E14 vg/kg, respectively, after baseline readings of body weight, echocardiography, and EKG. Readings post virus injection were recorded at 3 and 6 weeks, respectively, including echocardiography of B-mode, M-Mode (RV, LV), structure (LV internal diameters) and 30 -min ECG for quantifying arrythmias and evaluating electrophysiological parameters. Mice were sacrificed in week 6 and tissues and blood samples were collected. FIG. 9B shows body weight progression for 6 weeks. FIG. 9C shows heart weight normalized to body weight, % ejection fraction (%EF) and ventricular arrhythmia score at 6 wks. FIG. 9D shows neutrophil to lymphocyte ratio at 6 wks. FIG. 9E shows liver weight normalized to body weight and live function tests, alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT) at 6 wks. FIG. 9F shows platelet counts and hemoglobin (HGB) amount.

[00241] FIGs. 10A-AH show results after AAV9 was injected at three weeks before, right after, or 1 weeks after induction of Pkp2 gene deletion. TN-401 was dosed at 3E13 vector genomes per kilogram body weight (vg/kg) and AAV9:mPkp2 at 5E13 vg/kg. Echocardiograph (Echo) and electrocardiogram (EKG) data were collected at week 3 and week 4 post gene deletion. FIG. 10A shows EF% and FIG. 10B shows EF% progression at 4 weeks post-induction. RV chamber dilation was measured by RV area normalized to body weight. FIG. 10C shows RV/BW and FIG. 10D shows RV/BW progression at 4 weeks post-induction. EF% and RV dilation in response to AAV9:PKP2 treatment was statistically evaluated with ordinary One-Way ANOVA (Tukey’s post-hoc test). Animals that were euthanized or died prior to the last echo were excluded from the EF progression and LV Mass graphs. Incidence of ventricular arrhythmia was quantified during 30 minutes of recording of anesthetized animals. The frequency and severity of the spontaneous arrhythmia were recorded and categorized based on the grading chart in Table 2. FIG. 10 E shows ventricular arrhythmia score distribution of individual animals at 4 weeks post tamoxifen induction and FIG. 10F shows ventricular arrhythmia score progression. Statistical significance in response to AAV9:PKP2 treatment was evaluated using Kruskal- Wallis test with Dunn’s correction. FIG. 10G shows Kaplan-Meier survival curve showed that both TN- 401 and AAV9:mPkp2 treatment extended median lifespan > 58 weeks vs 4.7 weeks observed in the vehicle treated Pkp2-cKO animals. Pkp2-cKO animals treated 3 weeks before induction with AAV9:mPkp2 were euthanized at 32 weeks for exploratory ex-vivo studies. Numbers in paratheses showed dead vs live animals by the time of takedown. FIG. 10H shows results from animals that were weighed weekly from start of induction up to 72 weeks post-induction, at which time the animals were taken down for terminal cardiac assessment. The vehicle-treated Pkp2-cKO animals reached the humane endpoint by 6 weeks post-induction. There was no concerning decline in body weight for the treatment groups (all animals, including the ones that were found dead or euthanized were included). HBSS served as vehicle control. Error bar: s.e.m.

[00242] FIG. 11 shows dose- dependent efficacy of TN-401 which was evaluated in Pkp2-cKO mouse model. Mice were injected with TN-401 at 1E13, 3E13, or 1E14 vg/kg at one week after tamoxifen induction of cardiac Pkp2 gene deletion. At 4 weeks post tamoxifen induction (3 weeks post AAV9 injection), animals were sacrificed for expression and histological evaluation. Immunoblot analysis showed a dose-dependent expression of human PKP2 protein and restoration of endogenous JUP and DSP protein in LV. Below the graphs, raw immunoblot images were shown and * indicates animal found dead before protein analysis.

[00243] FIGs. 12A-12E shows AAV9:mPkp2 dose-dependent efficacy in preserving ejection fraction and right ventricle size, reducing arrhythmia scores, and improved lifespan relative to vehicle -treated Pkp2-cKO animals. FIG. 12A shows the study design describing animal model, virus injections, and timepoints for major functional readouts. AAV9:mPkp2 was injected one week after gene deletion at dose range of 1E13, 3E13, and 1E14 vg/kg. Echocardiograph (Echo) and electrocardiogram (EKG) data were collected at 4 weeks post gene deletion. FIG. 12B shows EF% at 4 weeks post gene deletion. RV chamber dilation was measured by RV area normalized to body weight. FIG. 12C shows RV/BW at 4 weeks post-induction. EF% and RV dilation in response to AAV9:mPkp2 treatment were statistically evaluated with ordinary One-Way ANOVA (Tukey’s post-hoc test). Incidence of ventricular arrhythmia was quantified during 30 minutes of recording of anesthetized animals. The frequency and severity of the spontaneous arrhythmia were recorded and categorized based on the grading chart in Table 2. FIG. 12D shows ventricular arrhythmia score distribution of individual animals at 4 weeks post gene deletion. Statistical significance in response to AAV9:mPkp2 treatment was evaluated using Kruskal-Wallis test with Dunn’s correction. FIG. 12E shows Kaplan-Meier survival curve showed that AAV9:mPkp2 treatment extended life span of Pkp2-cKO mice with median life span to 35 weeks at IE 13 vg/kg and > 50 weeks for both 3E13 and 1E14 vg/kg. Error bar: s.e.m.

EXAMPLES

[00244] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1: AAV:PKP2 corrected disease phenotypes in a human iPSC-derived cardiomyocytes (iPSC- CMs) model.

[00245] To model ARVC disease and identify the molecular, structural, and functional signatures that are fundamental to the disease mechanisms, RNA sequencing analyses of iPSC-CMs were carried out after acute silencing of PKP2 expression. These studies revealed that the desmosome functions as a signaling hub connecting key structures in cardiomyocytes such that reduction in PKP2 expression led to downregulation of structural and functional gene expression encoding components of desmosomes, sarcomeres, intermediate filaments, and ion channels (FIG. 1A). Down-regulation of protein or mRNA was observed for desmoplakin (DSP), plakoglobin (JUP), myosin-binding protein C3 (MyBPC3), desmin (DES), and sodium voltage-gated channel a subunit 5 (SCN5A) (FIG. IB). PKP2 deficiency resulted in structural disappearance of PKP2 and DSP from the cellular membrane and caused cell-to-cell disarray of patterned iPSC-CMs (FIG. 1C). In addition, PKP2 deficiency perturbed both contractile (FIG. ID) and electrophysiological properties of iPSC-CMs (FIG. IE).

[00246] The 1st generation AAV expression cassette was used for iPSC-CM-based studies and the 2nd generation for in vivo mouse efficacy studies (FIG. 2A). Dose-dependent protein expression was evident in iPSC-CMs driven by a cardiac-specific troponin T promoter (FIG. 2B). AAV:human PKP2 (AAV:hPKP2) restored DSP expression post PKP2 silencing when compared to the reduced DSP protein intensity without AAV rescue (FIG. 2C and FIG. 2D). AAV:hPKP2 restored contractility as quantified by contraction velocity when compared to the reduced contraction velocity without AAV rescue (FIG. 2E). Using human iPSC-CMs as a cell model for ARVC, AAV:hPKP2 restored desmosomes and rescued contractility in PKP2 -deficient iPSC-CMs, suggesting PKP2 governs intrinsic cellular properties of cardiomyocytes.

Example 2: Pkp2-cKO ARVC mouse model recapitulated the majority of human ARVC clinical manifestations

[00247] A mouse conditional knockout model was used to assess the feasibility and the efficacy of AAV- mediated PKP2 gene replacement. Consistent with the early observations of this model, tamoxifen- induced cardiac deletion of both alleles of Pkp2 in adult mice at 1 week did not show overt structural and functional changes. Weekly monitoring and tissue collection at the end of the study showed disruption of desmosomes and GJs (FIG. 3A), severe spontaneous premature ventricular contractions (PVCs) (FIG. 3B), biventricular dilatation (FIG. 3C), and a sharp decline in cardiac function (FIG. 3D) and survival (FIG. 3E) after 3-4 weeks of induced cardiac knock-out of Pkp2. These phenotypes recapitulated human ARVC clinical manifestations. However, unlike in humans, heterozygous disruption of Pkp2 in mouse hearts did not result in cardiac phenotypes that closely recapitulated human ARVC symptoms. Thus, homozygous Pkp2-cKO mice were used as a model of human ARVC.

Example 3: AAV9:PKP2 treatment largely attenuated disease development and disease progression to mortality in Pkp2-cKO ARVC mouse

[00248] To determine whether an AAV9 expression cassette (FIG. 2A, the second generation) encoding either the human or mouse orthologs of PKP2 could counteract the effects of cardiac Pkp2 gene deletion, Pkp2-cKO mice were given a single systemic dose via retro-orbital injection of TN-401 (AAV9:hPKP2 (AAV9: human PKP2) at 3E13 vg/kg) or AAV9:mPkp2 (AAV9:mouse Pkp2 at 5E13 vg/kg) 3 weeks prior to tamoxifen induction of cardiac Pkp2 gene deletion (FIG. 4A). Hank's Balanced Salt Solution (HBSS) was used as the carrier buffer for TN-401 or AAV9:mPkp2 to prevent aggregation of capsids. It was administered as the vehicle control to WT and to Pkp2-cKO animals. There were 4 experimental groups: ‘WT’ and ‘cKO’ were treated with the vehicle and Pkp2-cKO animals treated with either ‘TN- 401’ or ‘AAV9:mPkp2’ as shown in FIG. 4A.

[00249] At 4 weeks post gene deletion and 7 weeks post AAV delivery, both human and mouse orthologs prevented ventricular arrhythmia event frequency and severity as summarized by a ventricular arrhythmia score (FIG. 4B, Table 2, an overall composite score estimating arrhythmia burden), prevented right ventricular remodeling (FIG. 4C), and prevented decline in left ventricular function (FIG. 4D), which were prominent features of Pkp2-cKO mice at this timepoint. TN-401 (AAV9:hPKP2) demonstrated significant efficacy in preventing ARVC development and in extending lifespan by >58 weeks, far beyond the 4.7 weeks observed in the HBSS-treated Pkp2-cKO animals, with survival comparable to the natural lifespan of the WT control animals as monitored up to 72 weeks (FIG. 4E). In this same study, efficacy of AAV9:mPkp2 in Pkp2-cKO mice was evaluated at 3 intervention timepoints and concluded that there were no significant differences in efficacy readouts of EF, RV dilation, arrhythmias, and prolonged lifespan of more than 50% of the treated animals by 50 weeks (FIGs. 10A-10H). Overall, these results showed that either the mouse or human ortholog of PKP2 was sufficient to prevent the detrimental cardiac and survival phenotypes of Pkp2-cK0 mice when delivered in the AAV9 vector.

[00250] To assess the dose response to AAV9:hPKP2 (TN-401, FIGS. 5A-5E) or AAV9:mPkp2 (FIGS. 12A-12E), Pkp2-cKO mice were given single systemic treatments one week after tamoxifen induction of cardiac Pkp2 gene deletion (FIG. 5A) and sacrificed at 4 weeks post induction (3 weeks post AAV treatment) for histological and expression analyses. AAV9:hPKP2 treatment of Pkp2-cKO mice showed dose-dependent efficacy in reducing RV dilation as estimated by RV area normalized to body weight, in preventing decline of LV ejection fraction, and a trending dose-dependent reduction in arrhythmias (FIG. 5B). This dose-dependent efficacy was confirmed with larger cohorts of animals in significantly improving LV ejection fraction, reducing RV area and arrhythmia burden, and improving survival (FIGS. 12A-12E).

[00251] At molecular level, left ventricle heart tissue showed dose-dependent protein expression of human PKP2 (FIG. 5C, top panel; Western blot images in FIG. 11) as well as dose-dependent restoration of DSP and JUP, two additional desmosome proteins that were decreased in Pkp2-cKO mice. Connexin 43 (Cx43), a gap junction protein present at intercalated discs, was reduced in Pkp2-cKO mice, as shown by immunohistochemistry of heart tissue, and was restored in Pkp2-cKO mice treated with AAV9:hPKP2 (FIG. 5D, top row). AAV9:hPKP2 treatment also significantly reduced fibrosis development and collagen deposition in both right ventricle and left ventricle (FIG. 5D, bottom row and quantification shown in the graph). In addition, quantitative analyses of molecular signatures supported that AAV9:hPKP2 treatment reduced mRNA expression of heart failure markers, fibrosis, and tissue remodeling genes in both left and right ventricles (FIG. 5E).

[00252] Overall, TN-401 (AAV9:hPKP2) or AAV9:mPkp2 treatment supported a dose-dependent improvement in ARVC phenotypes and efficacy in the Pkp2-cKO mouse model of ARVC.

AAV9:hPKP2 in this dose-escalation study demonstrated efficacy at doses > 3E13 vg/kg in preventing adverse right ventricular remodeling, and improving ventricular function, fibrosis, and electrophysiological properties.

[00253] The preventive mode of treatment, AAV9:PKP2 dosing before overt structural changes, had demonstrated significant benefit of early intervention in largely preventing disease development and extending lifespan. To further examine whether ARVC disease progression could be slowed down or attenuated by restoration of PKP2 expression after overt structural changes, the therapeutic mode of treatment, animals were dosed at 2.5 weeks after inducing cardiac knock-out of Pkp2 (FIG. 6A). At 2.5 weeks, overt structural changes were observed that coincided with a rapid development of RV dilatation, LVEF decline, and significant ventricular arrhythmias (FIGS. 3A-3E). Note that the rapid mortality presented by this mouse model (within 3-6 weeks of tamoxifen induction) combined with the relatively slow time course of AVV9 transduction and transgene expression make it challenging to perform the therapeutic mode of treatment. However, at 9 week post induction, AAV9:mPkp2 treatment prevented further decline and even improved the left ventricle function (FIG. 6C and FIG. 6F), reduced and reversed right ventricle enlargement when compared to the WT level (p=0.6856, ns) (FIG. 6D and FIG. 6G), and attenuated the progression of arrhythmias when compared to the untreated and the treated at 4 weeks (FIG. 6E and FIG. 6H). Significantly, PKP2 gene therapy blunted early development of heart failure and reduced mortality of this mouse model of ARVC throughout one year follow-up with a median lifespan by >50 weeks (FIG. 6B), which is comparable to the survival benefit observed in the preventive mode of treatment (FIG. 12E).

Example 4: Restoration of PKP2 expression led to a highly coordinated and durable correction of PKP2- associated transcriptional networks beyond desmosome

[00254] It was rather surprising to observe that restoration of a single desmosome component, PKP2, led to significant survival benefits, improved cardiac function, reversed adverse RV remodeling, reduced ventricular arrhythmias, and prevented fibrosis. It was asked whether “on-target” PKP2 effects possibly extend beyond its effects on the desmosome by evaluating PKP2 dose-dependent response, specifically at the transcriptional level. There is no reported study that reveals whether 1) PKP2 dynamically coordinates its gene expression with other desmosome members, and 2) to what extent PKP2 quantitively dictates the state of disease progression. To obtain a deeper understanding, two large-scale RNA sequencing analyses were conducted.

[00255] Pkp2-cKO mice were given a single systemic dose of TN-401 (AAV9:hPKP2) one week before tamoxifen induction of cardiac Pkp2 gene deletion (FIG. 7A) and cardiac function and arrhythmias were evaluated at 4 and 9 weeks post induction. Mice were sacrificed at 9 weeks post induction and heart tissues were collected for RNA sequencing and quantification of PKP2 RNA and protein expression. At a 2-fold expression difference between low, 3E13 vg/kg and high, 6E13 vg/kg doses at 9 weeks (FIG. 7B), no significant dose-dependent difference was observed in key readouts of EF%, RV dilatation, and arrhythmia score, although the less critical readouts of LV mass to body weight and QT intervals did show dose dependence and one out of six animals at the high dose vs 5 out of nine animals at the low dose had arrhythmia scores >1 at 9 weeks post induction (FIG. 7C). Specific gene classes were evaluated including desmosome, gap junctions (GJs), sarcomere, ion channels and Ca²⁺ handling systems, heart failure markers, and fibrosis, that have been previously demonstrated to be significant contributors to disease mechanisms (FIG. 7D). Comparison between WT vs HBSS treated Pkp2-cKO animals showed significant changes in gene expression in these classes and an extensive reversal of these changes in response to TN-401 (AAV9:hPKP2) (FIG. 7E, genes of interest marked in red). Intriguingly, RNA sequencing analysis at the transcriptional level showed a positive dose correlation to TN-401 (AAV9:hPKP2) among structural genes encoding desmosomes, Cx43, sarcomeres, ion channels and Ca²⁺ handling proteins (FIG. 7F). When examining expression of heart failure markers and fibrosis genes, a negative dose correlation to TN-401 (AAV9:hPKP2) was observed (FIG. 7F). Therefore, while key functional readouts of efficacy could not be distinguished between dose levels of 3E13 and 6E13 vg/kg, the 2-fold difference in PKP2 transcript levels achieved by these two doses did result in quantitative and dose-dependent changes in transcriptional signatures described above. Based on this observation, it is believed that identification of key genes can be informative in associating a transcriptional signature with a particular phase of ARVC disease progression and therefore, may facilitate patient stratification in a more quantitative and precise manner, particularly in early ‘concealed’ phase when structural changes are not evident.

[00256] Transcriptome analysis showed that TN-401 (AAV9:hPKP2) restored expression of structural genes and attenuated expression of genes encoding adverse remodeling factors in a highly coordinated and quantitative fashion. It was asked whether such transcriptional response can be sustained to attenuate disease progression and therefore, extend survival over a longer duration.

[00257] As shown earlier in FIGs 6A-6E, a single dose of AAV9:mPkp2 treatment after overt cardiomyopathy haled disease progression via reversed adverse right ventricular remodeling, improved LV function, prevented arrhythmia worthening, and extended life span to 51 weeks post induction of Pkp2 deletion. Heart tissues collected at 51 weeks post induction of Pkp2 deletion were analyzed by RNA sequencing (FIG. 8A). Compared to intervention before overt structural changes (the preventive mode), AAV9:PKP2 intervention after overt structural change (the therapeutic mode) showed comparable efficacy in extending life span at the same dose, 1E14 vg/kg (FIG. 8B). Exploratory transcriptional analysis by Principle Component Analysis (PCA) showed that transcriptional profiles of AAV9:mPkp2-treated Pkp2-cKO animals were clustered close to WT and distant from non-treated animals, suggesting a normalization of transcriptional landscape close to WT in response to the treatment (FIG. 8C). While the transcriptional profile of low-dose treated animals showed a partial recovery pattern, the transcriptional profiles of the high-dose treated animals effectively overlapped with that of WT samples. In addition, when comparing the total number of differentially up- or down-regulated genes relative to the WT animals, the preventive mode and to a lesser degree, the therapeutic mode of intervention showed a significant normalization compared to that between Pkp2-cKO and WT animals (FIG. 8D). When comparing HBSS-treated Pkp2-cKO animals vs WT, the significant negatively enriched gene sets identified by Gene Set Enrichment Analysis (GSEA) were enriched with mitochondrial dysfunction, cardiac muscle contraction, and cardiac muscle conduction. The top significant positively enriched gene sets were predominantly fibrosis related. Both modes of intervention showed significant reversal of these enriched gene sets with the preventive mode supporting the most complete reversal (FIG. 8E). Surprisingly, the long-term survival benefit offered by either mode of intervention was supported by a broad spectrum of sustained correction of gene expression encoding components of the desmosomes, sarcomeres, ion channels and calcium handling systems, and also multiple pathways that regulate metabolism, fibrosis, inflammation, and apoptosis as shown (FIG. 8F and FIG. 8G). Once again, both modes of intervention showed significant reversal of these enriched gene sets with the preventive mode effect being most complete (FIG. 8G). Quantitative RT-PCR validated that at the same dose, IE 14 vg/kg, each mode of intervention maintained a similar level of Pkp2 transgene expression at 51 weeks, suggesting the mode of intervention does not change the durability of the transgene expression (FIG. 8H). Expression of heart failure genes (Nppa and Nppb) and fibrosis genes Timpl, Coll al . and ColCal) were significantly lowered by both modes of treatments at 1E14 vg/kg. In agreement with the observation shown by RNA-seq analyses, these genes were expressed higher in therapeutic mode than in the preventive mode among age-matched animals.

[00258] It was concluded that long-term restoration of PKP2 expression by gene replacement approach was correlated with sustained restoration of a broad spectrum of structural genes and pathways, supporting a notion that early intervention is the key to restore PKP2-associated intrinsic transcriptional networks and their functions and increase overall cardiomyocyte fitness to effectively mitigate adverse maladaptive remodeling such as fibrosis as early as possible. These results strongly support that PKP2- associated transcriptional networks can be used to quantitively evaluate the extent of disease progression and gene therapy efficacy at the molecular level.

Example 5: More than IQx of efficacious dose of AAV9:PKP2 is safe in WT CD1 mice

[00259] Safety evaluation of AAV9:PKP2 in WT mice for 6 weeks (FIG. 9A) showed no adverse effects at > lOx efficacy dose on body weight (FIG. 9B), heart weight and ventricular functions (FIG. 9C), neutrophil to lymphocyte ratio (FIG. 9D), liver weight and enzyme levels (FIG. 9E), and platelet count and hemoglobin levels (FIG. 9F). Histological analyses showed no AAV9:PKP2-related changes in heart, lung, liver, pancreas, brain, kidneys, and skeletal muscle examined.

[00260] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for restoring gene expression of a plurality of genes in an individual having a heart disease or disorder, the method comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, thereby restoring gene expression of the plurality of genes, wherein the plurality of genes comprise one or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Pin, Ryr2, Scn5a, Tfin, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn.

2. A method of reducing gene expression of a plurality of genes in an individual having a heart disease or disorder, the method comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, thereby reducing gene expression of the plurality of genes, wherein the plurality of genes comprises one or more of Col lai, Col3al, Mmp2, Nppa, Nppb, P4HA1, Postn, TGFpi, or Timpl.

3. The method of claim 1 or claim 2, wherein the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

4. The method of any one of claims 1 to 3, wherein the individual has a genome with a mutation in a PKP2 gene.

5. The method of any one of claims 1 to 4, wherein the promoter is a cardiac specific promoter.

6. The method of claim 5, wherein the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter.

7. The method of any one of claims 1 to 4, wherein the promoter is a ubiquitous promoter.

8. The method of any one of claims 1 to 7, wherein the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

9. The method of any one of claims 1 to 8, wherein the viral vector further comprises a cardiac specific enhancer.

10. The method of any one of claims 1 to 9, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus.

11. The method of any one of claims 1 to 10, wherein the viral vector is an adeno-associated virus.

12. The method of claim 10 or claim 11, wherein the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

13. The method of any one of claims 1 to 12, wherein the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8.

14. The method of any one of claims 1 to 13, wherein the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

15. The method of any one of claims 1 to 14, wherein the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

16. The method of any one of claims 1 to 15, wherein expression of the plurality of genes is restored or reduced for at least 12 months.

17. The method of any one of claims 1 to 16, wherein one or more symptoms of ARVC or ACM are improved for at least 12 months.

18. A method for monitoring efficacy of a treatment for a heart disease or disorder in an individual, the method comprising: (a) administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) operatively linked to a promoter; and (b) measuring expression of one or more of Actn2, Ank2, Asph, Atp2a2, Cacnalc, Casq2, Collal, Col3al, Des, Dsc2, Dsg2, Dsp, Gjal, Jup, Maprel, Mmp2, Mybpc3, Myh6, Myh7, Myl2, Myl3, Myoz2, Nppa, Nppb, P4HA1, Pin, Postn, Ryr2, Scn5a, Tin, TGFpi, Timpl, Tnncl, Tnni3, Tnnt2, Tpml, Trdn, or Ttn.

19. The method of claim 18, wherein the heart disease or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

20. The method of claim 18 or claim 19, wherein the individual has a genome with a mutation in a PKP2 gene.

21. The method of any one of claims 18 to 20, wherein the promoter is a cardiac specific promoter.

22. The method of claim 21, wherein the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter.

23. The method of any one of claims 18 to 20, wherein the promoter is a ubiquitous promoter.

24. The method of any one of claims 18 to 23, wherein the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

25. The method of any one of claims 18 to 24, wherein the viral vector further comprises a cardiac specific enhancer.

26. The method of any one of claims 18 to 25, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus.

27. The method of any one of claims 18 to 26, wherein the viral vector is an adeno- associated virus.

28. The method of claim 26 or claim 27, wherein the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

29. The method of any one of claims 18 to 28, wherein the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8.

30. The method of any one of claims 18 to 29, wherein the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

31. The method of any one of claims 18 to 30, wherein the method further comprises measuring one or more of fibrofatty tissue replacement; myocardial atrophy; ventricular dilation; ventricular arrhythmias; sudden cardiac death; exercise-triggered cardiac events; right ventricular cardiomyopathy, dilation, or heart failure; left ventricular cardiomyopathy, dilation, or heart failure; atrial arrhythmias; syncope; palpitations; shortness of breath; or chest pain.

32. The method of any one of claims 18 to 31, wherein the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

33. The method of any one of claims 18 to 32, wherein (b) comprises RT-qPCR, RNAseq, array hybridization, or probe hybridization.

34. The method of any one of claims 18 to 33, wherein the method comprises measuring an increase in expression of one or more of Cacnalc, Ryr2, Asph, Atp2a2, Ank2, Casq2, Scn5a, Des, Jup, Dsp, Dsg2, Dsc2, Maprel, Myh7, Mybpc3, Myoz2, Actn2, Tpml, Myl3, Tnncl, Myh6, Pin, Tnni3, Myl2, Tfh, Tnnt2, Trdn, Ttn, Gjal, Nppa, Nppb, TGFpi, P4HA1, Collal, Col3al, Mmp2, Postn, or Timpl.

35. The method of any one of claims 18 to 33, wherein the method comprises measuring a decrease in expression of one or more of Cacnalc, Ryr2, Asph, Atp2a2, Ank2, Casq2, Scn5a, Des, Jup, Dsp, Dsg2, Dsc2, Maprel, Myh7, Mybpc3, Myoz2, Actn2, Tpml, Myl3, Tnncl, Myh6, Pin, Tnni3, Myl2, Tfh, Tnnt2, Trdn, Ttn, Gjal, Nppa, Nppb, TGFpi, P4HA1, Collal, Col3al, Mmp2, Postn, or Timpl.

36. A method of restoring lipid metabolism and/or regulating energy homeostasis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector restores activity of genes associated with lipid metabolism and/or energy homeostasis in the cardiomyocytes of the individual.

37. The method of claim 36, wherein the genes associated with lipid metabolism and/or energy homeostasis comprise one or more of Acaa2, Acaca, Acadl, Acadm, Acadsb, Acly, Acot3, Acsll, Acssl, Cd36, Cldea, Cptlb, Cpt2, Crat, Dgatl, Dgat2, Echsl, Elovll, Elovl5, Fabp3, Fabp3, Fads2, G0s2, Hadh, Hadha, Hadhb, L2hgdh, Lpl, Mgll, Mpcl/2, Pdp2, or Plin2.

38. The method of claim 36 or claim 37, wherein the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

39. The method of any one of claims 36 to 38, wherein the individual has a genome with a mutation in a PKP2 gene.

40. The method of any one of claims 36 to 39, wherein the promoter is a cardiac specific promoter.

41. The method of claim 40, wherein the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter.

42. The method of any one of claims 36 to 39, wherein the promoter is a ubiquitous promoter.

43. The method of any one of claims 36 to 42, wherein the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

44. The method of any one of claims 36 to 43, wherein the viral vector further comprises a cardiac specific enhancer.

45. The method of any one of claims 36 to 44, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus.

46. The method of any one of claims 36 to 45, wherein the viral vector is an adeno- associated virus.

47. The method of claim 45 or claim 46, wherein the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

48. The method of any one of claims 36 to 47, wherein the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8.

49. The method of any one of claims 36 to 48, wherein the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

50. The method of any one of claims 36 to 49, wherein the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

51. The method of any one of claims 36 to 50, wherein the method restores lipid metabolism and/or energy homeostasis for at least 12 months.

52. A method of reducing collagen synthesis and/or fibrosis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector reduces activity of genes associated with collagen synthesis and/or fibrosis in the cardiomyocytes of the individual.

53. The method of claim 52, wherein the genes associated with collagen synthesis and/or fibrosis in cardiomyocytes comprise one or more of Collal, Col3al, Mmp2, P4HA1, Postn, TGFpi, or Timpl.

54. The method of claim 52 or claim 53, wherein the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

55. The method of any one of claims 52 to 54, wherein the individual has a genome with a mutation in a PKP2 gene.

56. The method of any one of claims 52 to 55, wherein the promoter is a cardiac specific promoter.

57. The method of claim 56, wherein the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter.

58. The method of any one of claims 52 to 55, wherein the promoter is a ubiquitous promoter.

59. The method of any one of claims 52 to 58, wherein the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

60. The method of any one of claims 52 to 59, wherein the viral vector further comprises a cardiac specific enhancer.

61. The method of any one of claims 52 to 60, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus.

62. The method of any one of claims 52 to 61, wherein the viral vector is an adeno- associated virus.

63. The method of claim 61 or claim 62, wherein the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

64. The method of any one of claims 52 to 63, wherein the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8.

65. The method of any one of claims 52 to 64, wherein the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

66. The method of any one of claims 52 to 65, wherein the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

67. The method of any one of claims 52 to 66, wherein collagen synthesis and/or fibrosis is reduced for at least 12 months.

68. A method of modulating amino acid homeostasis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector modulates activity of genes associated with amino acid homeostasis in the cardiomyocytes of the individual.

69. The method of claim 68, wherein the genes associated with modulating amino acid homeostasis in cardiomyocytes comprise one or more of Bcatl, Bcat2, Cth, Gotl, or Gpt2.

70. The method of claim 68 or claim 69, wherein the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

71. The method of any one of claims 68 to 70, wherein the individual has a genome with a mutation in a PKP2 gene.

72. The method of any one of claims 68 to 71, wherein the promoter is a cardiac specific promoter.

73. The method of claim 72, wherein the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter.

74. The method of any one of claims 68 to 71, wherein the promoter is a ubiquitous promoter.

75. The method of any one of claims 68 to 74, wherein the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

76. The method of any one of claims 68 to 75, wherein the viral vector further comprises a cardiac specific enhancer.

77. The method of any one of claims 68 to 76, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus.

78. The method of any one of claims 68 to 77, wherein the viral vector is an adeno- associated virus.

79. The method of claim 77 or claim 78, wherein the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

80. The method of any one of claims 68 to 79, wherein the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8.

81. The method of any one of claims 68 to 80, wherein the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

82. The method of any one of claims 68 to 81, wherein the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

83. The method of any one of claims 68 to 82, wherein amino acid homeostasis is modulated for at least 12 months.

84. A method of modulating ketone homeostasis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector modulates activity of genes associated with ketone homeostasis in the cardiomyocytes of the individual.

85. The method of claim 84, wherein the genes associated with modulating ketone homeostasis in cardiomyocytes comprise one or more of Acatl or Oxctl.

86. The method of claim 84 or claim 85, wherein the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

87. The method of any one of claims 84 to 86, wherein the individual has a genome with a mutation in a PKP2 gene.

88. The method of any one of claims 84 to 87, wherein the promoter is a cardiac specific promoter.

89. The method of claim 88, wherein the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter.

90. The method of any one of claims 84 to 87, wherein the promoter is a ubiquitous promoter.

91. The method of any one of claims 84 to 90, wherein the viral vector comprises a 3 ’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

92. The method of any one of claims 84 to 91, wherein the viral vector further comprises a cardiac specific enhancer.

93. The method of any one of claims 84 to 92, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus.

94. The method of any one of claims 84 to 93, wherein the viral vector is an adeno- associated virus.

95. The method of claim 93 or claim 94, wherein the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

96. The method of any one of claims 84 to 95, wherein the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8.

97. The method of any one of claims 84 to 96, wherein the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

98. The method of any one of claims 84 to 97, wherein the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

99. The method of any one of claims 84 to 98, wherein ketone homeostasis is modulated for at least 12 months.

100. A method of modulating glucose homeostasis and glycolysis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector modulates activity of genes associated with glucose homeostasis and glycolysis in the cardiomyocytes of the individual.

101. The method of claim 100, wherein the genes associated with modulating glucose homeostasis and glycolysis in cardiomyocytes comprise one or more of Cs, Eno3, Hk2, Idh2, Pdk4, Pfkm, Sdha, Sdhd, or Suclg2.

102. The method of claim 100 or claim 101, wherein the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

103. The method of any one of claims 100 to 102, wherein the individual has a genome with a mutation in a PKP2 gene.

104. The method of any one of claims 100 to 103, wherein the promoter is a cardiac specific promoter.

105. The method of claim 104, wherein the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter.

106. The method of any one of claims 100 to 103, wherein the promoter is a ubiquitous promoter.

107. The method of any one of claims 100 to 106, wherein the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

108. The method of any one of claims 100 to 107, wherein the viral vector further comprises a cardiac specific enhancer.

109. The method of any one of claims 100 to 108, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus.

110. The method of any one of claims 100 to 109, wherein the viral vector is an adeno- associated virus.

111. The method of claim 109 or claim 110, wherein the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

112. The method of any one of claims 100 to 111, wherein the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8.

113. The method of any one of claims 100 to 112, wherein the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

114. The method of any one of claims 100 to 113, wherein the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

115. The method of any one of claims 100 to 114, wherein glucose homeostasis and glycolysis is modulated for at least 12 months.

116. A method of modulating nucleic acid homeostasis in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector modulates activity of genes associated with nucleic acid homeostasis in the cardiomyocytes of the individual.

117. The method of claim 116, wherein the genes associated with modulating nucleic acid homeostasis in cardiomyocytes comprise one or more of Polr21 or Txn2.

118. The method of claim 116 or claim 117, wherein the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

119. The method of any one of claims 116 to 118, wherein the individual has a genome with a mutation in a PKP2 gene.

120. The method of any one of claims 116 to 119, wherein the promoter is a cardiac specific promoter.

121. The method of claim 120, wherein the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter.

122. The method of any one of claims 116 to 119, wherein the promoter is a ubiquitous promoter.

123. The method of any one of claims 116 to 122, wherein the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

124. The method of any one of claims 116 to 123, wherein the viral vector further comprises a cardiac specific enhancer.

125. The method of any one of claims 116 to 124, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus.

126. The method of any one of claims 116 to 125, wherein the viral vector is an adeno- associated virus.

127. The method of claim 125 or claim 126, wherein the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

128. The method of any one of claims 116 to 127, wherein the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8.

129. The method of any one of claims 116 to 128, wherein the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

130. The method of any one of claims 116 to 129, wherein the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

131. The method of any one of claims 116 to 130, wherein modulating nucleic acid homeostasis is modulated for at least 12 months.

132. A method of restoring calcium handling in cardiomyocytes of an individual in need thereof comprising administering to the individual a viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, wherein administration of the viral vector increases activity of genes associated with calcium handling in the cardiomyocytes of the individual.

133. The method of claim 132, wherein the genes associated with calcium handling in cardiomyocytes comprise one or more of Ank2, Asph, Atp2a2, Cacnalc, Pin, Ryr2, or Trdn.

134. The method of claim 132 or claim 133, wherein the individual has heart disease or disorder comprising arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic cardiomyopathy (ACM).

135. The method of any one of claims 132 to 134, wherein the individual has a genome with a mutation in a PKP2 gene.

136. The method of any one of claims 132 to 135, wherein the promoter is a cardiac specific promoter.

137. The method of claim 136, wherein the cardiac specific promoter is a PKP2 promoter, a troponin promoter, or an alpha-myosin heavy chain promoter.

138. The method of any one of claims 132 to 135, wherein the promoter is a ubiquitous promoter.

139. The method of any one of claims 132 to 138, wherein the viral vector comprises a 3’ element comprises a Woodchuck Hepatitis Virus Posttransciptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

140. The method of any one of claims 132 to 139, wherein the viral vector further comprises a cardiac specific enhancer.

141. The method of any one of claims 132 to 140, wherein the viral vector is selected from the group consisting of an adeno-associated virus, an adenovirus, a lentivirus, a pox virus, a vaccinia virus, or a herpes virus.

142. The method of any one of claims 132 to 141, wherein the viral vector is an adeno- associated virus.

143. The method of claim 141 or claim 142, wherein the adeno-associated virus is selected from the group consisting of an AAV6, an AAV.rh74, an AAV8, and an AAV9.

144. The method of any one of claims 132 to 143, wherein the PKP2 polypeptide has an amino acid sequence of SEQ ID NO: 8.

145. The method of any one of claims 132 to 144, wherein the nucleic acid encoding the PKP2 polypeptide has a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

146. The method of any one of claims 132 to 145, wherein the viral vector is administered in a pharmaceutically acceptable carrier or excipient comprising a buffer, a polymer, a salt, or a combination thereof.

147. The method of any one of claims 132 to 146, wherein calcium handling is restored for at least 12 months.

148. A viral vector comprising a nucleic acid encoding a plakophilin 2 (PKP2) polypeptide operatively linked to a promoter, for use in a method according to any one of claims 1 to 147.