Biomarkers in arrhythmogenic right ventricular cardiomyopathy and methods of treatment

Abstract

Provided herein are methods of measuring a response to plakophilin-2 (PKP2) gene therapy in an individual with arrhythmogenic right ventricular cardiomyopathy (ARVC) comprising administering the PKP2 gene therapy to the individual and measuring a metabolic enzyme messenger ribonucleic acid (mRNA) signature or at least one metabolite in a biological sample of the individual.

Description

Attorney Docket No. TYA-078WO BIOMARKERS IN ARRHYTHMOGENIC RIGHT VENTRICULAR CARDIOMYOPATHY AND METHODS OF TREATMENT CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 746,056, filed on January 16, 2025, U. S. Provisional Application No. 63 / 816,362, filed on June 2, 2025, and U. S. Provisional Application No. 63 / 880,194 filed on September 11, 2025, the contents of each of which are incorporated herein by reference in their entireties.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is TYA-078WO_SL.xml. The XML file is 34,935 bytes, was created on January 14, 2026, and is being submitted electronically via USPTO Patent Center.BACKGROUND

[0003] Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Mutations in the desmosome gene Plakophilin-2 (PKP2) lead to reduction in the PKP2 protein and collapse of desmosomes that compromises contractility and electrical stability of cardiomyocytes.SUMMARY

[0004] In an aspect, provided herein are methods of measuring a response to plakophilin-2 (PKP2) gene therapy in an individual with arrhythmogenic right ventricular cardiomyopathy (ARVC). In some embodiments, the method comprises administering the PKP2 gene therapy to the individual. In some embodiments, the method comprises measuring a metabolic enzyme messenger ribonucleic acid (mRNA) signature in a biological sample of the individual. In some embodiments, the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and tri acylglyceride (TAG) dynamics enzyme gene; a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene; and a cardiac contraction gene. In some embodiments, the glycolysis and glucose oxidation enzyme gene comprises one or-1- IPTS / 2.00262535.1more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hk1, Slc16a3, Slc2a1, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slc16a1, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgk1, Mpc1, Mpc2, and PDHB. In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat. In some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH. In some embodiments, the transcriptional regulator of energy metabolism gene comprises one or more of PPAR5, PPARa, PPARy, PPARGCla, Rxra, Ncor2, Gnaq, Nr4a1, and Prdm16. In some embodiments, the mitochondrial dynamics gene comprises one or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, the cardiac contraction gene comprises one or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, the method further comprises measuring at least one metabolite in the biological sample of the individual In some embodiments, the at least one metabolite comprises acylcarnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate. In some embodiments, the at least one metabolite comprises a lipid. In some embodiments, the lipid comprises a triacylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds. In some embodiments, the lipid comprises TAG (54:2). In some embodiments, the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component. In some embodiments, the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2; prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12, 13 -EpOME. In some embodiments, the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin. In some embodiments, the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine. In some embodiments, the PKP2 gene therapy comprises a gene therapy vector comprising a nucleic acid encoding a PKP2 polypeptide or a fragment thereof operatively linked to at least one promoter In some embodiments, the gene-2- IPTS / 2.00262535.1therapy vector comprises a viral vector. 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, and a herpes virus. In some embodiments, the gene therapy vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is an AAV6, an AAV8, an AAV9, an AAVrh7, an AAVrhlO, a derivative, or a pseudotype thereof. In some embodiments, the adeno-associated virus is an AAV9. In some embodiments, the AAV9 comprises a genome comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 7. In some embodiments, the gene therapy vector targets cells in the myocardium, the epicardium, or both. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter directs gene expression in the myocardium, the epicardium, or both. In some embodiments, the cardiac specific promoter is a troponin promoter or an alpha-myosin heavy chain promoter. In some embodiments, the troponin promoter has a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 3. In some embodiments, the promoter is a PKP2 promoter. In some embodiments, the PKP2 promoter has a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 4. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is a beta-actin promoter. In some embodiments, the gene therapy vector further comprises a cardiac specific enhancer. In some embodiments, the cardiac specific enhancer comprises a LMNA enhancer or a MYH7 enhancer. In some embodiments, the gene therapy vector further comprises a 3’ element. In some embodiments, the 3’ element comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 95% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 95% identity to the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 99% identity to the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has the sequence of SEQ ID NO: 2. In some embodiments, the gene therapy vector comprises an expression cassette comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 5. In some embodiments, the individual has at least one mutant copy of a PKP2 gene. In some embodiments, the PKP2 gene therapy is administered intravenously, intracardially, pericardially, or intraarterially-3- IPTS / 2.00262535.1In some embodiments, the mRNA signature or the at least one metabolite is measured about one week, about one month, about two months, about six months, or about one year after administering the PKP2 gene therapy to the individual. In some embodiments, the biological sample comprises cardiac tissue, blood, serum, or plasma.

[0005] In another aspect, provided herein are methods of measuring a response to plakophilin-2 (PKP2) gene therapy in an individual with arrhythmogenic right ventricular cardiomyopathy (ARVC), comprising administering the PKP2 gene therapy to the individual and measuring at least one metabolite in a biological sample of the individual. In some embodiments, the at least one metabolite comprises acylcarnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate. In some embodiments, the at least one metabolite comprises a lipid. In some embodiments, the lipid comprises a triacylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds. In some embodiments, the lipid comprises TAG (54:2). In some embodiments, the metabolite comprises a pro-inflammatory eicosanoid signaling pathway component. In some embodiments, the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2; prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12,13-EpOME. In some embodiments, the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidyl serine, or sphingomyelin. In some embodiments, the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine. In some embodiments, the method further comprises measuring at least one metabolic enzyme messenger ribonucleic acid (mRNA) signature in the biological sample of the subject. In some embodiments, the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and tri acylglyceride (TAG) dynamics enzyme gene; a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene, and a cardiac contraction gene. In some embodiments, the glycolysis and glucose oxidation gene comprises one or more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hkl, Slcl6a3, Slc2al, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slcl6al, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgk1, Mpc1, Mpc2, and PDHB. In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one-4- IPTS / 2.00262535.1or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat. In some embodiments, the transcriptional regulator of energy metaboli sm gene compri ses one or more of PPAR6, PPARa, PPARy, PPARGCla, Rxra, Ncor2, Gnaq, Nr4a1, and Prdm16. In some embodiments, the pro-infl ammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH. In some embodiments, the mitochondrial dynamics gene comprises one or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, the cardiac contraction gene comprises one or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, the PKP2 gene therapy comprises a gene therapy vector comprising a nucleic acid encoding a PKP2 polypeptide or a fragment thereof operatively linked to at least one promoter. In some embodiments, the gene therapy vector comprises a viral vector. 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, and a herpes virus. In some embodiments, the gene therapy vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is an AAV6, an AAV8, an AAV9, an AAVrh7, an AAVrhlO, a derivative, or pseudotype thereof. In some embodiments, the adeno-associated virus is an AAV9. In some embodiments, the AAV9 comprises a genome comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 7. In some embodiments, the gene therapy vector targets cells in the myocardium, the epicardium, or both In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the cardiac specific promoter directs gene expression in the myocardium, the epicardium, or both. In some embodiments, the cardiac specific promoter is a troponin promoter or an alpha-myosin heavy chain promoter. In some embodiments, the troponin promoter has a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 3. In some embodiments, the promoter is a PKP2 promoter. In some embodiments, the PKP2 promoter has a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 4. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is a beta-actin promoter. In some embodiments, the gene therapy vector further comprises a cardiac specific enhancer. In some embodiments, the cardiac specific enhancer comprises a LMNA enhancer or a MYH7 enhancer. In-5- IPTS / 2.00262535.1some embodiments, the gene therapy vector further comprises a 3’ element. In some embodiments, the 3’ element comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 95% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 95% identity to the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 99% identity to the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has the sequence of SEQ ID NO: 2. In some embodiments, the gene therapy vector comprises an expression cassette comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 5. In some embodiments, the individual has at least one mutant copy of a PKP2 gene. In some embodiments, the gene therapy is administered intravenously, intracardially, pericardially, or intraarterially. In some embodiments, the mRNA signature or the at least one metabolite is measured about one week, about one month, about two months, about six months, or about one year after administering the PKP2 gene therapy to the individual. In some embodiments, the biological sample comprises cardiac tissue, blood, serum, or plasma.

[0006] In another aspect, provided herein are methods of identifying an individual with arrhythmogenic right ventricular cardiomyopathy (ARVC), comprising measuring a metabolic enzyme messenger ribonucleic acid (mRNA) signature or at least one metabolite in a biological sample of the individual. In some embodiments, the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and tri acylglyceride (TAG) dynamics enzyme gene; a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene; and a cardiac contraction gene. In some embodiments, the glycolysis and glucose oxidation gene comprises one or more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hk1, Slc16a3, Slc2a1, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slc16a1, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgk1, Mpc1, Mpc2, and PDHB. In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn,-6- IPTS / 2.00262535.1Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat. In some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH. In some embodiments, the transcriptional regulator of energy metabolism gene comprises one or more of PPAR5, PPARa, PPARy, PPARGCla, Rxra, Ncor2, Gnaq, Nr4a1, and Prdm16. In some embodiments, the mitochondrial dynamics gene comprises one or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, the cardiac contraction gene comprises one or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, the at least one metabolite comprises acylcarnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate. In some embodiments, the at least one metabolite comprises a lipid. In some embodiments, the lipid comprises a tri acylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds. In some embodiments, the lipid comprises TAG (54:2). In some embodiments, the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component. In some embodiments, the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2; prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12,13-EpOME. In some embodiments, the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin. In some embodiments, the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine In some embodiments, the biological sample comprises cardiac tissue, blood, serum, or plasma.

[0007] In another aspect, provided herein are kits for use in the methods according to various embodiments described herein. In another aspect, provided herein are kits for measuring a metabolic enzyme messenger ribonucleic acid (mRNA) signature and / or at least one metabolite in a biological sample of an individual with arrhythmogenic right ventricular cardiomyopathy (ARVC) receiving plakophilin-2 (PKP2) gene therapy. In some embodiments, the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and triacylglyceride (TAG) dynamics enzyme gene; a pro-inflammatory-7- IPTS / 2.00262535.1eicosanoid signaling pathway gene, a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene; and a cardiac contraction gene. In some embodiments, the glycolysis and glucose oxidation enzyme gene comprises one or more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hkl, Slcl6a3, Slc2al, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slcl6al, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgkl, Mpcl, Mpc2, and PDHB In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, AcsI4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat. In some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH. In some embodiments, the transcriptional regulator of energy metabolism gene comprises one or more of PPAR6, PPARa, PPARy, PPARGCla, Rxra, Ncor2, Gnaq, Nr4al, and Prdml6. In some embodiments, the mitochondrial dynamics gene comprises one or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, the cardiac contraction gene comprises one or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, the at least one metabolite comprises acylcarnitine, L -lactate, citrate, malate, fumarate, and / or a-ketoglutarate. In some embodiments, the at least one metabolite comprises a lipid. In some embodiments, the lipid comprises a triacylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds. In some embodiments, the lipid comprises TAG (54:2). In some embodiments, the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component. In some embodiments, the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2; prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12,13- EpOME. In some embodiments, the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin. In some embodiments, the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine.-8- IPTS / 2.00262535.1

[0008] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent, applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Various features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0011] FIGS. 1A-1D shows AAV9: PKP2 gene therapy maintained key mRNA signatures of cardiac energy metabolism that was perturbed in Pkp2-cKO ARVC mouse heart. FIG. 1A illustrates the study design to evaluate AAV9: PKP2 efficacy at 51 weeks post tamoxifen induction of Pkp2 deletion in Pkp2-cKO ARVC mouse model. AAV9:mouse PKP2 (AAV9:mPkp2) was dosed at 3E13 and 1E14 vg / kg either 1 week after the induction (the preventive mode of treatment) or at 1E14 vg / kg at 2.5 weeks after induction (the therapeutic mode of treatment). FIG. IB shows two key cardiac energy productions, glucose oxidation and fatty acid b-oxidation was highlighted with enzymes of known points of regulation. Additional mRNA analyses were carried out to examine triacylglycerol (TAG) dynamics, fatty' acid synthesis, and expression of PPAR transcriptional regulators of energy metabolism. FIG. 1C shows relative gene expression of selected genes was measured by RNA-seq Samples were sorted by RV (right ventricle) and LV (left ventricle) chambers and treatment groups. Genes were categorized by gene classes. Left panel shows heatmap of gene-9- IPTS / 2.00262535.1expression of each individual animal and its corresponding chambers presented in scaled log2-transformed. Right panel depicts a scaled average across samples of each treatment group. Number of animals in each treatment group used for RNA sequencing were 9 WT, 4 cKO, 8 cKO + AAV9:mPkp2 at 3E13 vg / kg, 5 cKO + AAV9:mPkp2 at 1E14 vg / kg (the preventive mode), and 6 cKO + AAV9:mPkp2 at 1E14 vg / kg (the therapeutic mode) with both RV and LV collected. FIG. ID shows boxplots of group-wise gene expression of the selected gene classes. Each box shows 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. Sample size is described above.

[0012] FIGS. 2A-2C show that TN-401 gene therapy maintained key cardiac energy metabolism that was perturbed in Pkp2-cKO ARVC mouse model in a dose-dependent manner. FIG. 2A shows the study design to evaluate TN-401 dose-dependent efficacy at week 4 and 9 post tamoxifen induction of Pkp2 gene deletion in Pkp2-cKO mouse model Animals were dosed by TN-401 at 3E13 or 6E13 vg / kg at 1 week before induction and heart tissue collected at week 10 post virus injection. FIG. 2B shows the gene expression of selected genes was measured by RNA-seq.Heatmap of gene expression was sorted by heart chambers (LV vs RV), treatment groups, and gene classes. Left panel shows heatmap of gene expression of each individual animal and its corresponding chambers presented in scaled log2-transformed. Right panel shows a scaled average across samples of each treatment group. Number of samples in each treatment group used for RNA sequencing (for the subsequent integrated omics) were 9 WT, 7 cKO, 6 and 4 cKO+TN-401 at 3E13 and 6E13 vg / kg, respectively with both RV and LV collected. FIG. 2C shows box plots of group-wise gene expression for of the selected gene classes. 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-10- IPTS / 2.00262535.1(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.

[0013] FIGS. 3A-3E show the multiomics identified metabolic and mRNA signatures that were impaired in Pkp2-cKO cardiac tissue and maintained in response to AAV9: PKP2 treatment. FIG.3A shows the multiomic study design for nontargeted metabolomics and RNA sequencing analysis, respectively, to evaluate AAV9: PKP2 efficacy in Pkp2-cKO ARVC mouse model. TN-401 was dosed at 1E14 vg / kg at 2.5 weeks after tamoxifen induction of Pkp2 gene deletion (the therapeutic mode of treatment) and heart tissue was collected 5 weeks after tamoxifen induction and 2.5 weeks after virus injection. FIA-TOF mass spectrometry was used for nontargeted metabolomics. RNA sequencing analysis used the dataset of the therapeutic mode as shown in FIGS. 1A-1D. FIG.3B shows the cluster presentation of integrative metabolomics and RNA sequencing showed significantly changed metabolites in nodes and genes in rods in response to AAV9: PKP2 in the therapeutic mode of treatment when compared to the untreated Pkp2-cKO heart tissue. Metabolites and genes were categorized by functional pathways with highlights in fatty acid homeostasis, TCA cycle, and glycolysis. Boxplots showed expression of significantly changed genes highlighted on the cluster and measured by RNA-seq. 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. FIG. 3C shows the mean values of log2 fold change of metabolites with each column representing cKO vs WT, cKO vs TN-401 treated, and TN-401 treated vs WT, respectively. Metabolites were categorized by functional pathways. FIG. 3D shows shotgun lipidomics quantitatively targeted >1400 lipid species across 19 lipid subclasses using standards and Sciex QTRAP 5500 system with a differential mobility spectrometry (DMS) (SelexION), yielding 981 reported lipid species normalized to wet tissue mass (nmoles / mg).Lipidomics data was subsequently filtered by data occupancy so only lipid species reported in >75% of tissue samples per treatment group were analyzed. After filtering for data occupancy, 691 lipid species from 17 subclasses were analyzed. Lipid species were shown as the average fold change of cKO or TN-401 treated vs WT. Inset box plots show the triacylglyceride (TAG) class average and-11- IPTS / 2.00262535.1TAG 54:2 species average in cardiac tissue of WT, cKO, and TN-401 treated. Black dots represent the class average from each animal, while the midline represents the median of all animals, and the box indicates the interquartile range where the middle 50% of values lie, and the whiskers at the top and bottom of each box represent the range of values outside the interquartile range. Statistical evaluation was performed using ordinary One-Way ANOVA (Tukey’s post-hoc test). FIG. 3E shows that all bulk triacylglycerol species containing any given numbers of carbon or double bonds were averaged and profiled in cardiac tissue of WT, cKO, and TN-401 treated. Number of animals in each treatment group used for RNA sequencing were 9 WT, 4 cKO, 6 cKO treated by AAV9:mPkp2 at 1 E 14 vg / kg (the therapeutic mode). Number of animals in each treatment group used for metabolomics were 9 WT, 8 cKO, 8 cKO treated by TN-401 at 1E14 vg / kg (the therapeutic mode), and 5 WT, 8 cKO, 8 cKO treated by TN-401 at 1E14 vg / kg (the preventive mode).

[0014] FIGS. 4A-4D show that AAV9: PKP2 maintained a broad spectrum of metabolites and their corresponding transcriptional signatures in Pkp2-cKO cardiac tissue. FIG. 4A shows the study designs for nontargeted metabolomics and RNA sequencing, respectively, to evaluate AAV9. PKP2 efficacy in Pkp2-cKO ARVC mouse model. For nontargeted metabolomics, TN-401 was dosed at 1E14 vg / kg at Iweek before induction (the preventive mode of treatment). FIG. 4B shows integrated metabolomics and RNA sequencing showed that AAV9: PKP2 in the preventive mode significantly maintained multiple metabolic pathways with steady-state metabolites and mRNA detected when compared to the untreated Pkp2-cKO cardiac tissue. FIG. 4C shows heatmap mean values of Iog2 fold change of metabolites with each column representing cKO vs WT, cKO vs TN-401 treated, and TN-401 treated vs WT, respectively. Metabolites were categorized by functional pathways, FIG. 4D shows of additional lipid subclasses in cardiac tissue of WT, cKO, and TN-401 treated. Black dots represent the class average from each animal, while the midline represents the median of all animals, and the box indicates the interquartile range where the middle 50% of values lie, and the whiskers at the top and bottom of each box represent the range of values outside the interquartile range. Number of samples in each treatment group used for RNA sequencing and integrated omics were 9 WT, 7 cKO, and 4 cKO+TN-401 at 6E13 vg / kg, respectively, including both RV and LV (the preventive mode). Number of animals in each treatment group used for metabolomics and integrated omics were 5 WT, 8 cKO, 8 cKO+TN-401 at 1E14 vg / kg (the preventive mode).

[0015] FIGS. 5A-5H show that acute PKP2 silencing disrupted lipid homeostasis and led to increased intracellular lipid granules in iPSC cardiomyocytes. FIG. 5A shows the study design to-12- IPTS / 2.00262535.1evaluate lipid homeostasis in response to acute PKP2 silencing and metabolic modulators. FIG. 5B shows the results of FIA-TOF mass spectrometry was used to measure acylcarnitines in response to acute depletion of PKP2 (n = 6 technical replicates, 3 wells of 12-well plate were pooled as 1 technical replicate). The negative control for siRNA-mediated silencing is siNeg. FIG. 5C shows the confocal imaging using two lipid dyes, QBT Faty Acid Uptake (Molecular Devices) for measuring real-time fatty acid uptake (FAU) and LipidSpot 610 for measuring fatty acid storage in fixed iPSC cardiomyocytes after performing FAU. FAU dye in green, storage dye in red, and nuclei in blue. FIG. 5D shows the immunofluorescence post cell fixation showed co-stained of FAU dye and LipidSpot6lO with more cells stained by LipidSpot610 in siPKP2 than siNeg. FIG. 5E shows that the maximum FAU rate, Vmax (Relative Fluorescence Units / minute / cell x 1000), did not change in response to acute silencing of PKP2. Fatty acid storage was increased in response to acute silencing of PKP2 and was not alleviated by 48-hour treatment of isoproterenol, dobutamine, and danicamtiv, respectively. QBT Fatty Acid Uptake: n = 6 technical replicates; LipidSpot 610: n = 18-54 technical replicates FIG. 5F shows that inhibition of [3-oxidation by etomoxir increased intracellular lipid granules in response to PKP2 acute silencing (n = 9-18 technical replicates). FIG. 5G shows that compared to acute silencing of ACAD VL, siPKP2 showed a similar extent of increased lipid granules (n = 9-18 technical replicates). Glucose starvation reduced lipid granules. Inhibition of mitochondrial complex 1 by 40pM phenformin did not increase lipid granules with or without glucose starvation. FIG. 5H shows that inhibition of DGAT1 by AZD7687 reduced amount of lipid granules in response to PKP2 deficiency, suggesting reduction of TAG formation (n = 12 technical replicates). Comparison p values were calculated by Prism unpaired / -test for acylcarnitine levels and Vmax, ordinary One-Way ANOVA (Tukey’s post-hoc test) for all other comparisons: p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data represented as mean ± SD

[0016] FIGS. 6A-6G show that acute PKP2 silencing decreased oxidative and glycolytic metabolism that rendered poorer contractility in human iPSC cardiomyocytes. FIG. 6A shows the study design to evaluate the metabolic status of human iPSC cardiomyocytes in response to acute silencing of PKP2 and metabolic modulators. FIG. 6B shows that acute silencing of PKP2 significantly decreased glycolytic and oxidative metabolism as indicated by decreased glycoATP rate and Max OCR determined by Seahorse MitoStress test (n = 25-53 technical replicates). Left kinetic curves indicated where basal or DMSO / etomoxir-induced ATP rates were calculated Comparison p values were performed by Prism: basal ATP rates by ordinary One-Way ANOVA (Tukey’s post-hoc-13- IPTS / 2.00262535.1test), ***p<0.001, and Max OCR by unpaired t-test, ****p<0.0001. FIG. 6C shows inhibition of p- oxidation by etomoxir or silencing ACADVL decreased fatty acid oxidative metabolism and increased glycolysis determined by Seahorse MitoStress test (n = 24-27 technical replicates).Comparison p values were calculated by ordinary One-Way ANOVA (Tukey’s post-hoc test). FIG.6D shows percent of the initial amount of glucose or lactate in the culturing media as measured by Bioprofile Flex2 in response to acute silencing of PKP2 and a range of phenformin treatment (n = 2- 3 technical replicates, 3 wells of 96-well plate were pooled as 1 technical replicate at both initial time point and final assay time point). FIG. 6E shows Mito ATP and glycoATP rates as determined in response to acute silencing of PKP2 and a range of overnight phenformin treatment in the presence of 1 mM pyruvate (n = 3-6 technical replicates per treatment). Inhibition of mitochondrial complex 1 by phenformin switched from oxidative phosphorylation to glycolysis as represented by decrease mitoATP and increased glycoATP. FIG. 6F shows contractility in response to acute silencing of PKP2 and a range of overnight phenformin treatment was evaluated using SONY live cell imaging and Pulse video analysis (n = 6-16 technical replicates per treatment) (Curi Bio). Switching from oxidative metabolism to glycolysis supported poor contractility as measured by beat rate and contraction velocity. Average nuclear counts from live cells were used to normalize contraction velocity. Phenformin treatment at 120 pM inhibited cell contraction and less data collected (BLOQ = below limit of quantification). Comparison p values shown on d, e, and f were calculated by ordinary Two-Way ANOVA (Sidak's post-hoc test): p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0017] FIGS. 7A-7C show that acute PKP2 vs ACADVL silencing exhibited differential impact on contractility, electrophysiological properties, and calcium transient of iPSC cardiomyocytes. FIG.7A shows cardiac field potential recordings obtained from cardiomyocyte monolayers 7 days post siRNA silencing using the Maestro Pro Microelectrode array (MEA) platform (Axion Biosystems). Raw data were analyzed through AxIS Navigator software and the Cardiac Analysis Tool (Axion Biosystems) (n = 48 technical replicates). FIG. 7B shows the contractility that was evaluated at day 7 post siRNA silencing using SONY live cell imaging and Pulse video analysis (n=54-111 technical replicates) (Curi Bio). Average nuclear counts from live cells were used to normalize contraction velocity. FIG. 7C shows the cardiac calcium transient was evaluated in a multiwell mapping format using CuriBio Nautilai (https: / / www.curibio.com / nautilai). Raw data were analyzed by Curi Bio’s Pulse platform (n = 12- 24 technical replicates). Quantified data are presented as mean± SD.Statistical significance was determined using one-way ANOVA (Tukey’s post-hoc test), performed-14- IPTS / 2.00262535.1in GraphPad Prism. P -values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0018] FIGS. 8A-8G show characterization of isogenic human iPSC-CMs carrying heterozygous and homozygous pathogenic PKP2 mutation. FIG. 8A shows detection of cTnT, PKP2, DSP, and PKG expression by immunofluorescence on mutant isogenic human iPSC cardiomyocytes carrying a pathogenic mutation c.2146G> C: PKP2 heterozygous mutant (PKP2Het, first row), and PKP2 homozygous mutant (PKP2Hom, second row); for the cardiac troponin T (first column), PKP2 (second column), DSP (third column), and PKG (fourth column). Scale bars = 100 mm. FIG. 8B shows quantification of PKP2, DSP, and PKG protein expression for the WT and the two mutant isogenic lines. Results were normalized to the WT protein expression levels; n = 8 technical replicates for PKP2Het; n = 9 technical replicates for WT and PKP2Hom. p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = non-significant. FIG. 8C shows Western Blot images for PKP2 and GAPDH, and FIG. 8D shows the corresponding PKP2 Western Blot protein quantification for WT (n = 3), PKP2Het(n = 2) and PKP2Hom(n = 9) iPSC-CM isogenic lines. Data were normalized to the WT PKP2 protein expression levels. FIG. 8E shows monolayers (n = 24) and engineered heart tissues (EHTs) (n = 10 technical replicates for PKP2Het, n = 16 technical replicates for PKP2Honi) showed reduced contraction amplitude, velocity and prolonged kinetics in the PKP2Homlines using SONY live cell imaging and Pulse video analysis (Curi Bio)55and Mantarray (Curi Bio)57and Pulse video analysis (Curi Bio)55, respectively. FIG. 8F shows monolayers MEA parameter characterized by prolonged field potential duration (FPD) along with depressed spike amplitude, conduction velocity, and spike slope using the Maestro Pro Microelectrode array (MEA) platform (Axion Biosystems)58, n = 15 technical replicates. Quantified data are presented as mean ± SD. FIG. 8G shows FIA-TOF mass spectrometry measurement showed reduced acylcarnitines in PKP2Homcompared to PKP2Het(n = 6 technical replicates, 3 wells of 12-well plate were pooled as 1 technical replicate). Comparison p values were calculated by Prism unpaired t-test and ordinary One-Way ANOVA (Tukey’s post-hoc test) for panel.

[0019] FIGS. 9A-9G show isogenic human iPSC cardiomyocytes carrying a pathogenic PKP2 mutation showed impaired oxidative and glycolytic metabolism and contractile function. FIG. 9A shows PKP2Homwith an increase in lipid granules and no change in lipid uptake compared to WT isogenic cell quantified by confocal imaging using two lipid dyes, QBT Fatty Acid Uptake (Molecular Devices) for measuring real-time fatty acid uptake (FAU) (n = 59-90 technical replicates)-15- IPTS / 2.00262535.1and LipidSpot 610 for measuring fatty acid storage in fixed iPSC cardiomyocytes after performing FAU (n = 12-18 technical replicates). FIG. 9B shows FIA-TOF mass spectrometry measurement with reduced acvlcamitines in PKP2Homcompared to WT isogenic cells (n = 6 technical replicates, 3 wells of 12- well plate were pooled as 1 technical replicate). FIG. 9C shows PKP2Homwith reduced glycolytic and oxidative metabolism evaluated by the Seahorse MitoStress test (n = 14-43 technical replicates). Top kinetic curves indicated where basal or DMSO / etomoxir-induced ATP rates were calculated. FIG. 9D shows monolayers MEA parameters with prolonged field potential duration (FPD) along with depressed spike amplitude, conduction velocity, and spike slope using the Maestro Pro Microelectrode array (MEA) platform (Axion Biosystems)38(n = 15 technical replicates) FIG.9E shows monolayers (n = 24 technical replicates) and engineered heart tissues (EHTs) (n = 16 technical replicates) with reduced contraction amplitude, velocity and prolonged kinetics in the mutant lines, using SONY live cell imaging and Pulse video analysis (Curi Bio) and Mantarray (Curi Bio)57and Pulse video analysis (Curi Bio)35, respectively, p values: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = non-significant. FIG. 9F shows calcium transient recordings from WT and PKP2Hom(n = 12 technical replicates each) assessed with CuriBio Nautilai (https: / / www.curibio.com / nautilai). Parameters displayed are: time to peak from 50% to peak (Tso-Peak), Ca2+transient width at 50% (CaD50), and the rate at which [Ca2+]i reach its peak (Rise Rate). FIG. 9G shows acute PKP2 vs AC DVL silencing showed differential impact on field potential, contractility, and calcium transient of iPSC-CMs (details in Supplementary Fig. 4). Quantified data are presented as mean ± SD. Comparison p values were calculated by Prism unpaired t-test and ordinary One-Way ANOVA (Tukey’s post-hoc test) for panel.

[0020] FIGS. 10A-10F show that enhancing oxidative and glycolytic, metabolism improved contractility but not electrophysiological properties and Ca2+transient of PKP2HomiPSC cardiomyocytes. FIG. 10A shows the study design to evaluate small-molecule metabolic modulators in isogenic Pkp2 homozygous cell model. Monolayers or engineered heart tissues (EHTs) were treated with isoproterenol or seladelpar for 1 and 48 hours before the measurement on contractility, bioenergetics, electrophysiological properties (field potential) and Ca2+’ transient. FIG. 10B shows WT and isogenic PKP2Homcells were treated pharmacologically with DMSO, isoproterenol (ISO), AZD7687 (AZD), etomoxir (ETO), bezafibrate (BEZ), seladelpar (SEL), and phenformin (PHE). Contractility was evaluated at 1 and 48 hours post treatment using SONY live cell imaging and Pulse video analysis (n = 18-48 technical replicates) (Curi Bio)33. Average nuclear counts from live cells-16- IPTS / 2.00262535.1were used to normalize contraction velocity. FIG. 10C shows response to isoproterenol or seladelpar treatment, glycoATP and mitoATP rates and Max OCR of WT and isogenic PKP2Homcells were quantified using the Seahorse MitoStress test (n = 4-18 technical replicates). Statistical significance within the genotypes was determined at 48 hours post post-vehicle control / small molecule administration using Prism one-way ANOVA(Tukey’s post-hoc test). FIG. 10D shows the assessment of normalized twitch force, contraction and relaxation velocity in WT and PKP2Homgenerated 3D-EHTs 1 hour or 48 hours post-vehicle control / small molecule administration (n:::8-11 technical replicates) using Mantarray (Curi Bio)57and Pulse video analysis (Curi Bio). FIG. 10E shows the assessment of field potential parameters spike amplitude and spike slope in WT and PKP2Hommonolayers at 48-day post-vehicle control / small molecule administration using Maestro Pro Microelectrode array (MEA) platform (Axion Biosystems)58. Data were analyzed through AxIS Navigator software and the Cardiac Analysis Tool (Axion Biosystems) (n = 12 technical replicates).FIG. 10F shows the calcium transient recordings from WT and PKP2Hom2-D monolayers (n = 12 technical replicates) or 3D engineered heart tissues (EHTs) (n = 3-4 technical replicates) were evaluated with CuriBio Nautilai (https: / / www. curibio.com / nautilai), multiwell format, and analyzed by Curi Bio’s Pulse platform55. Parameters shown are, time to peak from 50% to peak (Tso-Peak), the rate at which [Ca2+]i reach its peak (Rise Rate), and half-width / Ca2+transient width at 50% (CaD50). Data are presented as mean ± standard deviation (s.d.). Statistical significance was determined using Prism one-way ANOVA(Tukey’s post-hoc test). P -values are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0021] FIGS 11A-11J show that TN -401 treatment restored desmosome protein expression, contractility, electrophysiological properties, Ca2+transient, increased PKP2 expression, and improved bioenergetics and desmosome-associated dysfunctions in PKP2HomiPSC cardiomyocytes.FIG. 11 A shows an exemplary study design to evaluate TN-401 rescue in isogenic PKP2 homozygous cell model. Monolayers or engineered heart tissues (EHTs) were transduced with TN-401 before measurement of bioenergetics, desmosome protein expression, iPSC-CM functions in contractility, electrophysiological properties (field potential), and Ca2+transients. FIG. 11B shows that in response to 15 days of TN-401 treatment of isogenic WT and PKP2Homcells, vehicle / etomoxir (ETO)-induced Max OCR, basal mitoATP and glycoATP rates were quantified using the Seahorse MitoStress test (n = 3-9). Data were normalized to the WT average at each corresponding TN-401 treatment. Statistical significance was determined using Prism one-way-17- IPTS / 2.00262535.1ANOVA (Tukey’s post-hoc test). FIG. 11C shows basal glycolysis, compensatory glycolysis, and %PER from glycolysis were quantified using the Seahorse Glycolytic Rate assay (n = 6-8). Data were normalized to the WT average at each corresponding TN-401 treatment. Statistical significance was determined using Prism one-way ANOVA (Tukey’s post-hoc test). FIG. 11D shows that in response to TN-401 treatment of WT and isogenic PKP2Homcells, Max OCR, glycoATP and mitoATP rates were quantified using the Seahorse MitoStress test (n = 3-9). FIG. 11E shows basal glycolysis, compensatory glycolysis, and %PER from glycolysis using the Seahorse Glycolytic Rate assay (n = 6-8). FIG. 11F shows the representative immunofluorescence images for PKP2 (red) and DSP (green) for the PKP2HomiPSC-CM isogenic line 21 days after transduction with TN-401 at the indicated MOIs. Nuclei were counterstained with Hoechst (blue). Scale bars = 200 mm. FIG. 11G shows quantification of PKP2 and DSP protein expression based on immunofluorescence staining for the WT and PKP2HomiPSC-CM isogenic lines. Protein expression was normalized to WT MOI 0 expression levels for both proteins; n = 7-9. FIG. 11H shows evaluation of contraction displacement and velocity in WT and PKP2IloniiPSC-CMs at day 18 following administration of TN-401 at different MOIs: 0, 10000, 30000, 100000, 300000 and 600000 (n = 15-18) using SONY live cell imaging and Pulse video analysi s (Curi Bio). FIG. 11I shows assessment of twitch force, contraction and relaxation velocity in WT and PKP2Homgenerated 3D-EHTs at day 15 (for WT) and 14 (for PKP2Hom) post-TN-401 administration, MOI = 100k; n = 12 and 3 for WT and WT + TN-401, respectively; n = 9 and 8, and PKP2Homand PKP2Hom+ TN-401, respectively, using Mantarray (Curi Bio)57and Pulse video analysis (Curi Bio). FIG. 11 J shows assessment of field potential parameters using Maestro Pro Microelectrode array (MEA) platform (Axion Biosystems) (top row, n 9-12) and Ca2+transient parameters (bottom row, n = 4-6) in WT and PKP2Hom11 days and 7 days post- TN-401 administration, respectively, using CuriBio Nautilai (www.curibio.com / nautilai), multiwell format, and analyzed by Curi Bio’s Pulse platform. Monolayers were paced at 1.5 Hz during MEA recordings and at 1 Hz during Ca2+transient measurement. Comparison p values were calculated by one-way ANOVA with Post-hoc tests. Quantified data are presented as mean ± SD. p values:*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0022] FIG. 12 shows an exemplary schematic representation of study designs that included mouse model development to recapitulate human ARVC phenotypes. Nontargeted metabolomics and lipidomics identified impaired metabolic pathways that responded to AAV9: PKP2-mediated restoration of PKP2 expression in Pkp2-cKO mouse heart tissue. Integrated metabolomics and-18- IPTS / 2.00262535.1transcriptomics increased confidence in identifying metabolites and enzymes of known points of regulation associated with cardiac energy metabolism. Supported by metabolic dysregulation observed in human ARVC heart, impaired lipid homeostasis, glycolysis, and glucose oxidation were modeled in either monolayer or 3D EHT of iPSC-CMs in response to either acutely silenced PKP2 or isogenic cells carrying a clinically relevant PKP2 mutation. The disclosure herein reports that compromised PKP2 expression led to decreased contractile function and perturbed electrophysiological properties and Ca2+transients in cardiomyocytes. It is further reported that pharmacological stimulation of mitochondrial or glycolytic energetics was found to be associated with partially improved contractility but was decoupled from electrophysiological properties and Ca2+transients. In contrast, TN-401 -mediated PKP2 expression improved not only mitochondrial and glycolytic energetics but also rescued desmosome-associated functions of contractility and electrophysiology.DETAILED DESCRIPTION

[0023] Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease characterized by ventricular arrhythmias and progressive cardiac dysfunction. Mutations in the desmosome gene Plakophilin-2, PKP2, resulted in reduction of PKP2 protein and disruption of desmosomes and gap junctions at the intercalated discs. These structural corruptions trigger cell death response, inflammatory infiltration, and metabolic perturbation that clinically manifested as electrical instability, cardiac structural deterioration, fibrofatty infiltration, and heart failure. The feasibility and the efficacy of adeno-associated virus (AAV)-mediated restoration of PKP2 in ARVC mouse models and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has been reported (Wu, I. et al., AAV9. PKP2 improves heart function and survival in a Pkp2-deficient mouse model of arrhythmogenic right ventricular cardiomyopathy. Commun. Med. 4, 38 (2024); Kyriakopoulou, E. et al., Therapeutic efficacy of AAV-mediated restoration of PKP2 in arrhythmogenic cardiomyopathy. Nat. Cardiovasc. Res. 2, 1261-1276 (2023); van Opbergen C. J. M. et al., AAV-mediated delivery of Plakophilin-2a arrests progression of arrhythmogenic right ventricular cardiomyopathy in murine hearts: preclinical evidence supporting gene therapy in humans. Circ.: Genom. Precis. Med. 17, e004305 (2024); Bradford, W. H. et al., Plakophilin 2 gene therapy prevents and rescues arrhythmogenic right ventricular cardiomyopathy in a mouse model harboring patient genetics. Nat. Cardiovasc. Res. 2, 1246-1261 (2023)). It was also shown that-19- IPTS / 2.00262535.1restoration of PKP2 expression leads to a highly coordinated and durable correction of metabolic mRNA signatures that are responsive to AAV9: PKP2 treatment in a dose-dependent manner.

[0024] The heart consumes fatty acids as the primary energy fuel with the remaining sources being glucose, lactate, ketone bodies and amino acids. In failing heart, long-chain acylcarnitines, mitochondrial fatty acid oxidation (FAO) intermediates, are decreased (Bedi, K. C. et al.. Evidence for intramyocardial disruption of lipid metabolism and increased myocardial ketone utilization in advanced human heart failure. Circ. 133, 706-716 (2016)) accompanied with a metabolic switch from FAO to glucose-based glycolysis and other alternative sources of energy (Schulze, P. C. et al., Lipid use and misuse by the heart. Circ. Res. 11, 1736-1751 (2016); Lopaschuk, G D etal., Cardiac energy metabolism in heart failure. Circ. Res. 128,1487-1513 (2021)). Supported by clinical evidence, deranged cardiac lipid metabolism leads to intramyocardial increases of triacylglycerol (TAG) and toxic lipids that have been linked to cardiac dysfunction in heart failure (Kintscher, U. et al., The role of adipose triglyceride lipase and cytosolic lipolysis in cardiac function and heart failure. Cell Rep. Med. 1, 100001 (2020)). These alterations in cardiac energy metabolism result in less efficient energy production in the heart and ultimately contribute to most forms of myocardial dysfunction (Song, J-P. et al., Elevated plasma P-hydroxybutyrate predicts adverse outcomes and disease progression in patients with arrhythmogenic cardiomyopathy. Sci. Transl. Med. 12, eaay8329 (2020); Previs, M. J. et al., Defects in proteome and metabolome in human hypertrophic cardiomyopathy. Circ. Heart Fail. 15, e009521 (2022)).

[0025] Recent advances in metabolomics and lipidomics expanded our understanding of the mechanistic connection between energy metabolism and cardiomyopathies in human heart. Targeted metabolic characterization revealed significant reductions in long-chain acylcarnitine and FAO and increases in alternative fuel burning in heart tissues from patients with either hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM) (Wang, W. etal.. Metabolic characterization of hypertrophic cardiomyopathy in human heart. Nat. Cardiovasc. Res. 1:445-461 (2022); Flam, E. et al., Integrated landscape of cardiac metabolism in end-stage human nonischemic dilated cardiomyopathy. Nat. Cardiovasc. Res. 1, 817-829 (2022)). However, myocardial TAG content was reported to show no change or decrease in the failing or nonfailing heart tissue of either HCM or DCM (Verdonschot, J. A. J. et al., Metabolic profiling associates with disease severity in nonischemic dilated cardiomyopathy. J. Card. Fail. 26, 212-222 (2020); Basso, C. etal., Ultrastructural evidence of intercalated disc remodeling in arrhythmogenic right ventricular-20- IPTS / 200262535.1cardiomyopathy: an electron microscopy investigation on endomyocardial biopsies. Eur. Heart J.27, 1847-1854 (2006)).

[0026] Unlike DCM and HCM, end-stage ARVC myocardial tissue showed decreased glycolysis and pyruvate oxidation as well as decreased long-chain acylcarnitine and fatty acid oxidation (FAO) (Song, J-P. et al., Elevated plasma β-hydroxybutyrate predicts adverse outcomes and disease progression in patients with arrhythmogenic cardiomyopathy. Sci. Transl. Med. 12, eaay8329 (2020)). A distinct metabolic perturbation reported in ARVC is the intracellular lipid accumulation detected in heart tissue of human or a mouse model and in patient-specific iPSC-CMs (Fujita, S. et al., Markedly increased intracellular lipid droplets and disruption of intercellular junctions in biopsied myocardium from a patient with arrhythmogenic right ventricular cardiomyopathy. Heart Vessels 23, 440-444 (2008); Caspi, O. etal., Modeling of arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent stem cells. Circ.: Cardiovasc. Genet. 6, 557-568 (2013); Lin, Y. et al., Reactivation of PPARa alleviates myocardial lipid accumulation and cardiac dysfunction by improving fatty acid p-oxidation in Dsg2-deficient arrhythmogenic cardiomyopathy. Acta Pharm. Sin. B. 13, 192-203 (2023); Ma, D. et al.. Generation of patient-specific induced pluripotent stem cell-derived cardiomyocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy. Eur. Heart J. 34, 1122-1133 (2013); Kim, C. et al., Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs. Nature 494, 105-110). ARVC patients are subject to restriction of activity and exercise, both found to be associated with accelerating the disease progression and increasing the risk of life-threatening ventricular arrhythmias and sudden cardiac death. Therefore, understanding how ARVC hearts handle the energy metabolism is one of the fundamentals in assessing and managing the arrhythmogenic triggers. Specifically, there is an interest in understanding the detrimental impact on cardiomyocyte energy metabolism due to PKP2 mutations and delineating the intrinsic connection between metabolic remodeling and cardiomyocyte contractility and electrical stability.

[0027] The disclosure provided herein started with nontargeted metabolomics of mouse heart tissue from cardiac-specific knock-out, Pkp2-cKO, to identify impaired metabolic pathways that responded to AAV9: PKP2-mediated restoration of PKP2 expression. Functionally, it was investigated how impaired bioenergetics affected contractility, electrophysiological properties, and Ca2+transients using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Both model systems confirmed impaired lipid homeostasis, glycolysis, and glucose oxidation that agreed with-21- IPTS / 2.00262535.1the human data (Song, J-P et al., Elevated plasma P-hydroxybutyrate predicts adverse outcomes and disease progression in patients with arrhythmogenic cardiomyopathy. Sei. Transl. Med. 12, eaay8329 (2020)). Reported herein is PKP2 deficiency led to impaired energy metabolism that contributed to poor contractility of cardiomyocytes. AAV9: PKP2 restored contractility, electrophysiological properties, and Ca2+transient whereas treating PKP 2 -deficient cardiomyocytes with metabolic enhancers improved contractile function but not electrophysiological properties or Ca2+transient. These results suggested structure-mediated functions, such as contractility, electrophysiological properties, and Ca2+’ transient, differentially responded to metabolic perturbance and decoupled energy-responsive contractility from the less responsive Ca2+transient and field potential may be an arrhythmogenic substrate occurring before the overt structural changes. It was concluded that PKP2 integrated the energy metabolism of the cardiomyocyte to contractile function and compromised PKP2 led to metabolic remodeling and cardiac dysfunction at the intracellular level. TN-401, Tenaya Therapeutics’ AAV9:human PKP2 clinical drug candidate, is currently at phase 1 clinical testing to treat PKP2-mutated ARVC patients.

[0028] Early studies demonstrated the efficacy of adeno-associated virus 9 (AAV9)-mediated restoration of PKP2 expression in a cardiac specific knock-out mouse model of Pkp2 and revealed profound changes in metabolic mRNA signatures that were reversed by gene therapy. Accordingly, there is an interest in understanding the functional link between energy metabolism and desmosomes. Shown in the disclosure herein, nontargeted integrated metabolomics and transcriptomics of PKP2-deficient mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were used to identify steady-state metabolites and enzymes associated with impaired lipid homeostasis, glycolysis, and glucose oxidation that aligned with human ARVC metabolic data and also responded to AAV9: PKP2 treatment in Pkp2-cKO mouse heart tissue. Integrated metabolomics and transcriptomics aided the identification of metabolites and enzymes of known points of regulation associated with cardiac energy metabolism. Supported by metabolic dysregulation observed in human ARVC heart, impaired lipid homeostasis, glycolysis, and glucose oxidation were modeled in either monolayer or 3D EHT of iPSC-CMs in response to either acutely silenced PKP2 or isogenic cells carrying a clinically relevant PKP2 mutation

[0029] The disclosure herein reports that compromised PKP2 expression led to decreased contractile function and perturbed electrophysiological properties and Ca2+’ transients in cardiomyocytes. The disclosure further reports that PKP2 deficiency led to an intrinsic impairment in energy metabolismIPTS / 2.00262535.1that contributed to poor cardiomyocyte contractility. However, treating PKP-2 deficient cardiomyocytes with TN-401 (AAV9:human PKP2) improved energy metabolism. In contrast to AAV9: PKP2-mediated restoration of contractility, electrophysiological properties, and Ca2+’ transients, treating PKP2-deficient cardiomyocytes with a metabolic enhancer improved contractility but not electrophysiological properties or Ca2+transients, suggesting differential sensitivity of structure-mediated functions in response to metabolic perturbance. It is also proposed that increased risk of decoupling energy-responsive contractility from less responsive electrical activities is, in some embodiments, a new arrhythmogenic mechanism behind exercise-triggered cardiac adversity in ARVC disease development and progression.Metabolic mRNA Signatures of Treatment Efficacy

[0030] In an aspect, provided herein are methods of measuring a response to plakophilin-2 (PKP2) gene therapy in an individual. In some embodiments, the individual has arrhythmogenic right ventricular cardiomyopathy (ARVC) comprising. In some embodiments, the method comprises administering the PKP2 gene therapy to the individual. In some embodiments, the method comprises measuring a metabolic enzyme messenger ribonucleic acid (mRNA) signature in a biological sample of the individual. In some embodiments, the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and triacylglyceride (TAG) dynamics enzyme gene; a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene, a mitochondrial dynamics gene, and a cardiac contraction gene In some embodiments, maintenance of energy metabolism is an integral part of PKP2 and desmosome functions in cardiomyocytes. In some embodiments, compromised PKP2 and desmosome functions may be implicated in metabolic impairment in cardiomyocyte dysfunctions. In some embodiments, there is an intrinsic functional connection between impaired energy metabolism and desmosome-associated dysfunction.

[0031] In various aspects of m ethods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the glycolysis and glucose oxidation enzyme gene comprises one or more of SLC2Al(solute carrier family 2 member 1 or GLU1 (glucose transporter 1)), SLC2A4 (solute carrier family 2 member 4, or GLU4 (glucose transporter 4)), SLC16A1 (solute carrier family 16 member 1 or MCT1 (monocarboxylate transporter 1)), SLC16A3 (solute carrier family 16 member 3 or MCT4 (monocarboxylate transporter 4)), MPC1 (mitochondrial pyruvate carrier 1), MPC2 (mitochondrial pyruvate carrier 2), PDHA1 (pyruvate dehydrogenase El subunit alpha 1),-23- IPTS / 2.00262535.1Hkl (hexokinase 1), Prkacb (protein kinase A catalytic subunit beta), Pdk4 (pyruvate dehydrogenase kinase 4), Suclg2 (succinate-CoA ligase GDP-forming subunit beta, mitochondrial), Sdhd (succinate dehydrogenase complex subunit D ), Cs (citrate synthase), Pdhb (pyruvate dehydrogenase El subunit beta), Hk2 (hexokinase 2), Sdha (succinate dehydrogenase complex subunit A), Eno3 (enolase 3), Ldhd (lactate dehydrogenase D), Idh2 (isocitrate dehydrogenase 2), Pfkm (phosphofructokinase, muscle), and Pgkl (phosphoglycerate kinase 1) In some embodiments, the glycolysis and glucose oxidation enzyme gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, or more of SLC2A1, SLC2A4, SLC16A1, SLC16A3, MPCL MPC2, PDHA1, Hkl, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Pdhb, Hk2, Sdha, Eno3, Ldhd, Idh2, Pfkm, Mpcl, Mpc2, and Pgkl. In some embodiments, the glycolysis and glucose oxidation enzyme gene comprise SLC2A1, SLC2A4, SLC16A1, SLC16A3, MPC1, MPC2, PDHA1, Hkl, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Pdhb, Hk2, Sdha, Eno3, Ldhd, Idh2, Pfkm, Mpcl, Mpc2, and Pgkl. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of Hkl, SLC16A3, SLC2A1, or Prkacb. In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of SLC2A4, SLC16A1, MPC1, MPC2, PDHA1, Pdk4, Suclg2, Sdhd, Cs, Pdhb, Hk2, Sdha, Eno3, Ldhd, Idh2, Pfkm, Mpcl, Mpc2, or Pgkl

[0032] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36 (cluster of differentiation 36), ACSL1 (acyl-CoA synthetase long-chain family member 1), ACSL6 (acyl-CoA synthetase long-chain family member 6), CPT1 (carnitine palmitoyltransferase 1A), CPT1B (carnitine palmitoyltransferase IB), CPT2 (carnitine palmitoyltransferase 2), DGAT1 (diacylglycerol O-acyltransferase 1), DGAT2 (diacylglycerol O-acyltransferase 2), ACLY (ATP citrate lyase), PLIN2 (perilipin 2), Hmgcl (3 -hydroxymethyl-3 -methylglutaryl-CoA lyase), Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthase 2, mitochondrial), Acsl4 (acyl-CoA synthetase long chain family member 4), Acsl3 (acyl-CoA synthetase long chain family member 3), Acaa2 (acetyl-CoA acyltransferase 2), Acot2 (acyl-CoA thioesterase 2), Slc25a20 (carnitine-acylcamitine translocase or CACT), Hadh (hydroxyacyl-CoA dehydrogenase), Hadhb (trifunctional enzyme subunit beta, mitochondrial), Acadl (acyl-CoA dehydrogenase long chain), Hadha (tri function al enzyme subunit alpha, mitochondrial), Crat (carnitine O-acetyltransferase), Acadm (acyl-CoA dehydrogenase medium chain), Fasn (fatty acid synthase), Bcatl (branched chain-24- IPTS / 2.00262535.1amino acid transaminase 1), Fabp4 (fatty acid binding protein 4), Mgll (monoglyceride lipase), Ffar2 (free fatty acid receptor 2), Abhd5 (alpha / beta-hydrolase domain-containing protein 5), Cidea (cell death inducing DFFA like effector A), Fabp3 (fatty acid binding protein 3), Lpl (lipoprotein lipase), Pnpla2 (patatin-like phospholipase domain-containing protein 2 or ATGL(adipose triglyceride lipase)), G0s2 (G0 / G1 switch gene 2), Bcat2 (branched chain amino acid transaminase 2), Fadsl (fatty acid desaturase 1), Fads2 (fatty acid desaturase 2), Elovll (fatty acid elongase 1), Elovl5 (fatty acid elongase 5), Acaca (acetyl-CoA carboxylase alpha), Plin1 (perilipin 1), and Meat (malonyl-CoA-acyl carrier protein transacylase). In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcatl, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fadsl, Fads2, Elovll, ElovlS, Acaca, Plinl, and Meat. In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of Hmgcl, CPT1A, Acsl4, Fasn, Bcatl, Ffar2, DGAT1, Fadsl, Fads2, PLIN2, Elovll, Elovl5, ACLY, or Acaca. In some embodiments a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of CD36, ACSL1, ACSL6, CPT1B, CPT2, DGAT2, Hmgcs2, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fabp4, Mgll, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Plinl, or Meat.

[0033] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1 (cytochrome p450 family 2 subfamily U member 1), GSTK1 (glutathione S-transferase kappa 1), GSTA3 (glutathione S-transferase alpha 3), COX-1 (mitochondrially encoded cytochrome c oxidase 1), COX-2 (mitochondrially encoded cytochrome c-25- IPTS / 2.00262535.1oxidase 2), DHRS4 (dehydrogenase / reductase 4), PTGES (prostaglandin E synthase), HPGDS (hematopoietic prostaglandin D synthase), TBXAS1 (thromboxane A synthase 1), and sEH (epoxide hydrolase). In some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH. In some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXASI, and sEH. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of FADS2, HPGDS, TBXAS1, GSTK1, GSTA3, PTGES, COX-1, or COX-2. In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of ACSL6, CYP2U1, DHRS4, or sEH.

[0034] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the transcriptional regulator of energy metabolism gene comprises one or more of PPAR5 (peroxisome proliferator activated receptor delta), PPARa (peroxisome proliferator activated receptor alpha), PPARy (peroxisome proliferator activated receptor gamma), PPARGCla (peroxisome proliferator-activated receptor gamma coactivator 1 -alpha), Rxra (retinoid X receptor alpha), Ncor2 (nuclear receptor corepressor 2), Gnaq (guanine nucleotide-binding protein G(q) subunit alpha), Nr4al (nuclear receptor subfamily 4, group A, member 1), and Prdml6 (PR / SET domain 16). In some embodiments, the transcriptional regulator of energy metabolism gene comprises two, three, four, five, six, seven, eight or more of PPAR5, PPARa, PPARy, PPARGCla, Rxra, Ncor2, Gnaq, Nr4al, and Prdm16. In some embodiments, the transcriptional regulator of energy metabolism gene comprises PPAR5, PPARa, PPARy, PPARGCla, Rxra, Ncor2, Gnaq, Nr4al, and Prdml6. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of Rxra, PPAR6, PPARy, Ncor2, Gnaq, or Nr4al. In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of PPARa, PPARGC la, or Prdml6.

[0035] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the mitochondrial dynamics gene comprises one or more of OPA1 (optic atrophy 1), MFN2 (mitofusin 2), MFN1 (mitofusin 1), MFF (mitochondrial fission factor), MED12 (mediator complex subunit 12), NFE2L2 (nuclear factor erythroid 2-like 2), and FIS 1 (mitochondrial fission 1). In some embodiments, the mitochondrial dynamics gene comprises two,-26- IPTS / 2.00262535.1three, four, five, six or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, the mitochondrial dynamics gene comprises OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of MED 12, NFE2L2, or FIS1. In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of 0PA1, MFN2, MFN 1, or MFF.

[0036] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the cardiac contraction gene comprises one or more of ACTA1 (actin alpha 1, skeletal muscle), MYH7 (myosin heavy chain 7), TNNT2 (cardiac troponin T), MYBPC3 (cardiac myosin-binding protein c), TNN13K (troponin 1-interacting kinase), MYH6 (myosin heavy chain 6), MYL2 (myosin light chain 2), TNNI3 (troponin I3, cardiac type), and TNNC1 (troponin C). In some embodiments, the cardiac contraction gene comprises two, three, four, five, six, seven, eight or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, the cardiac contraction gene comprises ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of ACTA1 or MYH7. In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, or TNNC1.

[0037] In various aspects, a gene as provided herein can comprise a human gene. Alternatively, a gene as provided herein can comprise a mouse gene. In some embodiments, a gene as provided herein comprises a mammalian orthologue of a gene as provided herein. For example, a SLC16A1 gene as described herein can describe a human SLC16A1 gene, or it can describe a mammalian orthologue of a human SLC16A1 gene (e.g., a mouse Slcl6al gene). Alternatively, for example, a mouse MpcI gene as described herein can describe a mouse Mpcl gene, or it can describe a mammalian orthologue of a mouse Mpcl gene (e.g., a human MPC1 gene).

[0038] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the method further comprises measuring at least one metabolite in the biological sample of the individual. In some embodiments, the at least one metabolite comprises acyl carnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate. In some embodiments, the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol.27IPTS / 2.00262535.1lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin. In some embodiments, the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine In some embodiments, the at least one metabolite comprises a lipid. In some embodiments, the lipid comprises a triacylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds. In some embodiments, the lipid comprises TAG (54:2) In some embodiments, the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component. In some embodiments, the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2; prostaglandin D2; prostaglandin E2, prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA), 9,10-EpOME (9,10-cis epoxide of linoleic acid); or 12,13-EpOME (12,13-cis epoxide of linoleic acid). In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in one or more of L-lactate, a-ketoglutarate, tri acyl glyceride (TAG), hexosylceramide, prostaglandin A2, prostaglandin D2, prostaglandin E2, prostaglandin F2alpha, prostaglandin H2, or malonic dialdehyde (MDA). In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in one or more of citrate, malate, fumarate, palmitoylcarnitine, oleoylcarnitine, stearoylcarnitine, lysophosphatidylcholine, 9,10-EpOME, or 12,13-EpOME.

[0039] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the PKP2 gene therapy comprises a gene therapy vector comprising a nucleic acid encoding a PKP2 polypeptide or a fragment thereof operatively linked to at least one promoter. In some embodiments, the gene therapy vector comprises a viral vector. 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, and a herpes virus. In some embodiments, the gene therapy vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is an AAV6, an AAV8, an AAV9, an AAVrh7, an AAVrhlO, a derivative, or a pseudotype thereof. In some embodiments, the adeno-associated virus is any adeno-associated virus described herein. In some embodiments, the adeno-associated virus is an AAV9. In some embodiments, the AAV9 comprises a genome comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 7. In some embodiments, the AAV9 comprises a genome comprising or consisting of the nucleotide sequence of SEQ ID NO:-28- IPTS / 2.00262535.17. In some embodiments, the gene therapy vector targets cells in the myocardium, the epicardium, or both.

[0040] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the promoter is selected as suitable for driving expression of the nucleic acid encoding the PKP2 polypeptide in the heart or cardiac tissue. In some embodiments, the promoter is a cardiac specific promoter. In some embodiments, the promoter or the cardiac specific promoter directs gene expression in the myocardium, the epicardium, or both. In some embodiments, the cardiac specific promoter is a troponin promoter or an alpha-myosin heavy chain promoter. In some embodiments, the troponin promoter comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 3. In some embodiments, the troponin promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the promoter is a PKP2 promoter. In some embodiments, the PKP2 promoter comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 4. In some embodiments, the PKP2 promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is a betaactin promoter.

[0041] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the gene therapy vector further comprises a cardiac specific enhancer. In some embodiments, the cardiac specific enhancer comprises a LMNA enhancer or a MYH7 enhancer. In some embodiments, the gene therapy vector further comprises a 3’ element. In some embodiments, the 3’ element comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

[0042] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the nucleic acid encoding the PKP2 polypeptide has any suitable sequence encoding a wild-type PKP2 polypeptide. In some embodiments, the PKP2 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide comprises or-29- IPTS / 2.00262535.1consists of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a codon optimized sequence. In some embodiments, the nucleic acid encoding the PKP2 polypeptide comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide comprises or consists of the sequence ofSEQ ID NO: 2.

[0043] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the gene therapy vector comprises any nucleic acid sequence provided herein. In some embodiments, the gene therapy vector comprises an expression cassette comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 5. In some embodiments, the gene therapy vector comprises an expression cassette comprising or consisting of the nucleotide sequence SEQ ID NO: 5. In some embodiments, the gene therapy vector comprises an expression cassette comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 6. In some embodiments, the gene therapy vector comprises an expression cassette comprising or consisting of the nucleotide sequence SEQ ID NO: 6.

[0044] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the individual has at least one mutant copy of a PKP2 gene In some embodiments, the individual is haploinsufficient for PKP2 In some embodiments, the individual has at least one mutant copy of a PKP2 gene but has no symptoms of ARVC or cardiomyopathy.

[0045] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the PKP2 gene therapy is administered via any suitable route, such as any route disclosed elsewhere herein In some embodiments, the PKP2 gene therapy is administered intravenously, intracardially, pericardially, or intraarterially.

[0046] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the mRNA signature or the at least one metabolite is measured about one week, about one month, about two months, about six months, or about one year after administering the PKP2 gene therapy to the individual

[0047] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the biological sample comprises any suitable tissue for measuring a-30- IPTS / 2.00262535.1response to PKP2 gene therapy. In some embodiments, the biological sample comprises cardiac tissue, blood, serum, or plasma.Metabolite Signatures of Treatment Efficacy

[0048] In additional aspects, provided herein are methods of measuring a response to plakophilin-2 (PKP2) gene therapy in an individual. In some embodiments, the individual has arrhythmogenic right ventricular cardiomyopathy (ARVC). In some embodiments, the method comprises administering the PKP2 gene therapy to the individual. In some embodiments, the method comprises measuring at least one metabolite in a biological sample of the individual.

[0049] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the at least one metabolite comprises acylcarnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate. In some embodiments, the at least one metabolite comprises one, two, three, four, five, or more of acylcarnitine, L-lactate, citrate, malate, fumarate, or a-ketoglutarate. In some embodiments, the at least one metabolite comprises acylcarnitine, L-lactate, citrate, malate, fumarate, and a-ketoglutarate.

[0050] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the at least one metabolite comprises a lipid. In some embodiments, the lipid comprises a triacylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds. In some embodiments, the lipid comprises TAG (54:2).

[0051] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component. In some embodiments, the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2; prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12,13-EpOME.

[0052] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin. In some embodiments, the at least one metabolite comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more of acylcamitine, a cholesterol-31- IPTS / 2.00262535.1ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and sphingomyelin. In some embodiments, the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin,

[0053] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine. In some embodiments, the at least one metabolite comprises one, two, or more of palmitoylcarnitine, oleoylcarnitine, and stearoylcarnitine. In some embodiments, the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, and stearoylcarnitine.

[0054] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the method further comprises measuring at least one metabolic enzyme messenger ribonucleic acid (mRNA) signature in the biological sample of the subject. In some embodiments, the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and triacylglyceride (TAG) dynamics enzyme gene; a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene; and a cardiac contraction gene.

[0055] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the glycolysis and glucose oxidation enzyme gene comprises one or more of SLC2A1, SLC2A4, SLC16A1, SLC16A3, MPC1, MPC2, PDHA1, Hkl, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Pdhb, Hk2, Sdha, Eno3, Ldhd, Idh2, Pfkm, Mpc1, Mpc2, and Pgk1. In some embodiments, the glycolysis and glucose oxidation enzyme gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one twenty-two, or more of SLC2A1, SLC2A4, SLC16A1, SLC16A3, MPC1, MPC2, PDHA1, Hk1, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Pdhb, Hk2, Sdha, Eno3, Ldhd, Idh2, Pfkm, Mpc1, Mpc2, and Pgk1. In some embodiments, the glycolysis and glucose oxidation enzymeIPTS / 2.00262535.1gene comprises SLC2A1, SLC2A4, SLC16A1, SLC16A3, MPC1, MPC2, PDHA1, Hk1, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Pdhb, Hk2, Sdha, Eno3, Ldhd, Idh2, Pfkm, Mpc1, Mpc2, and Pgk1.

[0056] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PL1N2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat. In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty -one twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty -nine, forty, forty-one, or more of CD36, ACSL1, ACSL6, CPT1 A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat. In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises CD36, ACSLL ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat.

[0057] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH. In some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH. In some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH.-33- IPTS / 2.00262535.1

[0058] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the transcriptional regulator of energy metabolism gene comprises one or more of PPARδ, PPARα, PPARγ, PPARGCl1α, Rxra, Ncor2, Gnaq, Nr4a1, and Prdm16. In some embodiments, the transcriptional regulator of energy metabolism gene comprises two, three, four, five, six, seven, eight, or more of PPARδ, PPARα, PPARγ, PPARGCl1α, Rxra, Ncor2, Gnaq, Nr4a1, and Prdm16. In some embodiments, the transcriptional regulator of energy metabolism gene comprises PPARδ, PPARα, PPARγ, PPARGCl1α, Rxra, Ncor2, Gnaq, Nr4a1, and Prdm16,

[0059] In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of Hkl, SLC16A3, SLC2A1, or Prkacb. In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of SLC2A4, SLC16A1, MPC1, MPC2, PDHA1, Pdk4, Suclg2, Sdhd, Cs, Pdhb, Hk2, Sdha, Eno3, Ldhd, Idh2, Pfkm, Mpcl, Mpc2, or Pgkl. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of Hmgcl, CPT1A, Acsl4, Fasn, Bcatl, Ffar2, DGAT1, Fadsl, Fads2, PLIN2, Elovll, Elovl5, ACLY, or Acaca. In some embodiments a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of CD36, ACSL1, ACSL6, CPT1B, CPT2, DGAT2, Hmgcs2, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fabp4, Mgll, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Plinl, or Meat. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of FADS2, HPGDS, TBXAS1, GSTK1, GSTA3, PTGES, COX-1, or COX-2. In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of ACSL6, CYP2U1, DHRS4, or sEH. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in expression of one or more of Rxra, PPAR5, PPARy, Ncor2, Gnaq, or Nr4al. In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in expression of one or more of PPARa, PPARGCla, or Prdm 16. In some embodiments, a positive response to PKP2 gene therapy is indicated by an increase in one or more of L-lactate, a-ketoglutarate, triacylglyceride (TAG), hexosylceramide, prostaglandin A2, prostaglandin D2, prostaglandin E2, prostaglandin F2alpha, prostaglandin H2, or malonic dialdehyde (MDA). In some embodiments, a positive response to PKP2 gene therapy is indicated by a decrease in one or more of citrate, malate, fumarate, palmitoylcarnitine, oleoylcarnitine, stearoylcarnitine, lysophosphatidylcholine, 9,10-EpOME, or 12, 13 -EpOME.-34- IPTS / 2.00262535.1

[0060] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the mitochondrial dynamics gene comprises one or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, the mitochondrial dynamics gene comprises two, three, four, five, six or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, the mitochondrial dynamics gene comprises OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1.

[0061] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the cardiac contraction gene comprises one or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, the cardiac contraction gene comprises two, three, four, five, six, seven, eight, or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, the cardiac contraction gene comprises ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1.

[0062] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the PKP2 gene therapy comprises a gene therapy vector comprising a nucleic acid encoding a PKP2 polypeptide or a fragment thereof operatively linked to at least one promoter. In some embodiments, the gene therapy vector comprises a viral vector. 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, and a herpes virus. In some embodiments, the gene therapy vector is an adeno-associated virus. In some embodiments, the adeno-associated virus is an AAV6, an AAV8, an AAV9, an AAVrh7, an AAVrh10, a derivative, or a pseudotype thereof In some embodiments, the adeno-associated virus is any adeno-associated virus described herein. In some embodiments, the adeno-associated virus is an AAV9, In some embodiments, the AAV9 comprises a genome comprising a nucleotide sequence with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 7. In some embodiments, the AAV9 comprises a genome comprising or consisting of the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the gene therapy vector targets cells in the myocardium, the epicardium, or both.

[0063] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the promoter is selected as suitable for driving expression of the nucleic acid encoding the PKP2 polypeptide in the heart or cardiac tissue. In some embodiments, the-35- IPTS / 2.00262535.1promoter is a cardiac specific promoter. In some embodiments, the promoter or the cardiac specific promoter directs gene expression in the myocardium, the epicardium, or both. In some embodiments, the cardiac specific promoter is a troponin promoter or an alpha-myosin heavy chain promoter. In some embodiments, the troponin promoter comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 3. In some embodiments, the troponin promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the promoter is a PKP2 promoter. In some embodiments, the PKP2 promoter comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 4 In some embodiments, the PKP2 promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is a beta¬ actin promoter.

[0064] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the gene therapy vector further comprises a cardiac specific enhancer. In some embodiments, the cardiac specific enhancer comprises a LMNA enhancer or a MYH7 enhancer. In some embodiments, the gene therapy vector further comprises a 3’ element. In some embodiments, the 3’ element comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

[0065] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the nucleic acid encoding the PKP2 polypeptide has any suitable sequence encoding a wild-type PKP2 polypeptide. In some embodiments, the PKP2 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid encoding the PKP2 polypeptide comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide comprises or consists of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide has a codon optimized sequence In some embodiments, the nucleic acid encoding the PKP2 polypeptide comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID-36- IPTS / 2.00262535.1NO: 2. In some embodiments, the nucleic acid encoding the PKP2 polypeptide comprises or consists of the nucleotide sequence of SEQ ID NO: 2.

[0066] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the gene therapy vector comprises any nucleic acid sequence provided herein. In some embodiments, the gene therapy vector comprises an expression cassette comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 5. In some embodiments, the gene therapy vector comprises an expression cassette comprising or consisting of the nucleotide sequence of SEQ ID NO: 5. In some embodiments, the gene therapy vector comprises an expression cassette comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 6. In some embodiments, the gene therapy vector comprises an expression cassette comprising or consisting of the nucleotide sequence of SEQ ID NO: 6.

[0067] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the individual has at least one mutant copy of a PKP2 gene. In some embodiments, the individual is haploinsufficient for PKP2. In some embodiments, the individual has at least one mutant copy of a PKP2 gene but has no symptoms of A RV C or cardiomyopathy,

[0068] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the PKP2 gene therapy is administered via any suitable route, such as any route disclosed elsewhere herein. In some embodiments, the PKP2 gene therapy is administered intravenously, intracardially, pericardially, or intraarterially.

[0069] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the mRNA signature or the at least one metabolite is measured about one week, about one month, about two months, about six months, or about one year after administering the PKP2 gene therapy to the individual.

[0070] In various aspects of methods of measuring a response to PKP2 gene therapy provided herein, in some embodiments, the biological sample comprises any suitable tissue for measuring a response to PKP2 gene therapy. In some embodiments, the biological sample comprises cardiac tissue, blood, serum, or plasma.Diagnostic Methods

[0071] In another aspect, provided herein are methods of identifying an individual with arrhythmogenic right ventricular cardiomyopathy. In some embodiments, the method comprises-37- IPTS / 2.00262535.1measuring a metabolic enzyme messenger ribonucleic acid (mRNA) signature. In some embodiments, the method comprises measuring at least one metabolite in a biological sample of the individual.

[0072] In various aspects of method of identifying an individual with ARVC provided herein, in some embodiments, the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and tri acyl glyceride (TAG) dynamics enzyme gene; a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene; and a cardiac contraction gene.

[0073] In various aspects of method of identifying an individual with ARVC provided herein, in some embodiments, the glycolysis and glucose oxidation enzyme gene comprises one or more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hkl, Slcl6a3, Slc2al, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slcl6al, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgkl, Mpcl, Mpc2, and PDHB, In some embodiments, the glycolysis and glucose oxidation enzyme gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one twenty-two, or more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hkl, Slcl6a3, Slc2al, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slc16a1, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgk1, Mpc1, Mpc2, and PDHB. In some embodiments, the glycolysis and glucose oxidation enzyme gene comprises SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hk1, Slc16a3, Slc2a1, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slc16a1, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgk1, Mpc1, Mpc2, and PDHB.

[0074] In various aspects of method of identifying an individual with ARVC provided herein, in some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat. In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one twenty -two, twenty -three, twenty -four, twenty-five, twenty-six, twenty-seven,-38- IPTS / 2.00262535.1twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty- six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat. In some embodiments, the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fads1, Fads2, Elovl1, Elovl5, Acaca, Plin1, and Mcat.

[0075] In various aspects of method of identifying an individual with ARVC provided herein, in some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH. In some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises two, three, four, five, six, seven, eight, or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH. In some embodiments, the pro-inflammatory eicosanoid signaling pathway gene comprises ACSL6, FADS2, CYP2U1, GSTKI, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH.

[0076] In various aspects of method of identifying an individual with ARVC provided herein, in some embodiments, the transcriptional regulator of energy metabolism gene comprises one or more of PPAR5, PPARa, PPARy, PPARGCla, Rxra, Ncor2, Gnaq, Nr4al, and PrdmI6. In some embodiments, the transcriptional regulator of energy metabolism gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of PPARδ, PPARα, PPARγ, PPARGCl1α, Rxra, Ncor2, Gnaq, Nr4a1, and Prdm16. In some embodiments, the transcriptional regulator of energy metabolism gene comprises PPARδ, PPARα, PPARγ, PPARGCl1α, Rxra, Ncor2, Gnaq, Nr4a1, and Prdm16.

[0077] In various aspects of method of identifying an individual with ARVC provided herein, in some embodiments, the mitochondrial dynamics gene comprises one or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, the mitochondrial dynamics gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of OPA1, MFN2,IPTS / 2.00262535.1MFN1, MFF, MED12, NFE2L2, and FIS1. In some embodiments, the mitochondrial dynamics gene comprises OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1.

[0078] In various aspects of method of identifying an individual with ARVC provided herein, in some embodiments, the cardiac contraction gene comprises one or more of ACTA 1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, the cardiac contraction gene comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of ACTA1, M YH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1. In some embodiments, the cardiac contraction gene comprises ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1.

[0079] In various aspects of method of identifying an individual with ARVC provided herein, in some embodiments, the method further comprises measuring at least one metabolite in the biological sample of the individual. In some embodiments, the at least one metabolite comprises acylcarnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate. In some embodiments, the at least one metabolite comprises acyl carnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanol amine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin. In some embodiments, the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine. In some embodiments, the at least one metabolite comprises a lipid. In some embodiments, the lipid comprises a triacylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds. In some embodiments, the lipid comprises TAG (54:2) In some embodiments, the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component. In some embodiments, the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2; prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12, 13 -EpOME.

[0080] In some embodiments, a diagnosis of ARVC is indicated by a decrease in expression of one or more of Hkl, SLC16A3, SLC2A1, orPrkacb. In some embodiments, a diagnosis of ARVC is indicated by an increase in expression of one or more of SLC2A4, SLC16A1, MPC1, MPC2, PDHA1, Pdk4, Suclg2, Sdhd, Cs, Pdhb, Hk2, Sdha, Eno3, Ldhd, Idh2, Pfkm, Mpcl, Mpc2, orPgkl. In some embodiments, a diagnosis of ARVC is indicated by a decrease in expression of one or more of Hmgcl, CPT1A, Acsl4, Fasn, Bcatl, Ffar2, DGAT1, Fadsl, Fads2, PLIN2, Elovll, Elovl5,-40- IPTS / 2.00262535.1ACLY, or Acaca. In some embodiments a diagnosis of ARVC is indicated by an increase in expression in expression of one or more of CD36, ACSL1, ACSL6, CPT1B, CPT2, DGAT2, Hmgcs2, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acad], Hadha, Crat, Acadm, Fabp4, Mgll, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Plinl, or Meat. In some embodiments, a diagnosis of ARVC is indicated by a decrease in expression of one or more of FADS2, HPGDS, TBXAS1, GSTK1, GSTA3, PTGES, COX-1, or COX-2, in some embodiments, a diagnosis of ARVC is indicated by an increase in expression of one or more of ACSL6, CYP2U1, DHRS4, or sEH, In some embodiments, a diagnosis of ARVC is indicated by a decrease in expression of one or more of Rxra, PPAR8, PPARy, Ncor2, Gnaq, orNr4al. In some embodiments, a diagnosis of ARVC is indicated by an increase in expression of one or more of PPARa, PPARGCla, or Prdml6. In some embodiments, a diagnosis of ARVC is indicated by a decrease in one or more of L-lactate, a- ketoglutarate, triacylglyceride (TAG), hexosyl ceramide, prostaglandin A2, prostaglandin D2, prostaglandin E2, prostaglandin F2alpha, prostaglandin H2, or malonic dialdehyde (MDA). In some embodiments, a diagnosi s of ARVC is indicated by an increase in one or more of citrate, mal te, fumarate, palmitoylcarnitine, oleoylcarnitine, stearoylcamitine, lysophosphatidylcholine, 9,10-EpOME, or 12, 13 -EpOME.

[0081] In various aspects of method of identifying an individual with ARVC provided herein, in some embodiments, the biological sample comprises any suitable tissue for measuring a response to PKP2 gene therapy. In some embodiments, the biological sample comprises cardiac tissue, blood, serum, or plasma.RNA Signature Assays

[0082] Ribonucleic acid (RNA) gene expression is determined, in part, by detecting and / or quantifying one or more gene expression products present in cellular material obtained from a subject. Cellular material may be obtained from a tissue sample isolated from an organ. The tissue sample may be isolated by any suitable means. In some instances, cellular material is obtained from blood, urine, tear, sweat, hair, plasma, and / or serum sample from the subject. In some embodiments, the one or more gene expression product is fully or partially isolated and / or purified from other cellular materi al prior to or during the detection and / or quantification of the gene expression product. In some embodiments, the gene expression product is an RNA molecule. In some embodiments, the gene expression product is a polypeptide.-41- IPTS / 2.00262535.1

[0083] In some embodiments, a microarray is employed for detection and / or quantification of a gene expression product in a gene expression profile. The manufacture and use of biochips such as those involving microarrays, also known as bioarrays, are known in the art (For reviews of Biochips and microarrays see, e.g., Kallioniemi O. P., " Biochip technologies in cancer research," Ann Med, Mar; 33(2): 142 7 (2001); and Rudert F., " Genomics and proteomics tools for the clinic," Curr Opin. Mol. Ther., Dec; 2(6):63342 (2000)). Furthermore, a number of biochips for expression analysis are commercially available (See e.g., microarrays available from Sigma-Genosys (The Woodl nds, Tex.); Affymetrix (Santa Clara, Calif), and Full Moon Biosystems (Sunnyvale, Calif.)). In some embodiments, such microarrays are analyzed using blotting techniques similar to those discussed below for conventional techniques of detecting polynucleotides and polypeptides. In some embodiments, detailed protocols for hybridization conditions are available through manufacturers of microarrays. In some embodiments, a microarray provides for the detection and analysis of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 100, 200, 250, 500, 750, 1000, 2500, 5000, 7500, 10,000, 12,500, 25,000, 50,0000, or 100,000 gene expression products.Information regarding the detection and / or quantification of a gene expression profile is a product and / or arti cle of the microarray methods provided herein.

[0084] In some embodiments, for microarray expression analysis, isolated and / or purified RNA is amplified. Isolated and / or purified RNA is an article and / or product of the microarray expression analysis. Amplified RNA is an article and / or product of the microarray expression analysis. In some embodiments, the amplified RNA is then used for hybridization to sequence specific nucleic acid probes on a biochip. Hybridized RNA to sequence specific nucleic probes on a biochip is an article and / or product of the microarray expression analysis. In some embodiments, amplification is performed using a commercially available kit, such as MessageAMp™ RNA kit (Ambion Inc ). In some embodiments, isolated and / or purified RNA is labeled before contacting the biochip such that binding to the target array can be detected using streptavidin. In some embodiments, isolated and / or purified RNA is labeled with a detectable moiety, including, but not limited to, a fluorescent moiety, a dye, or a ligand, such as biotin. Labeled RNA is an article and / or product of the microarray expression analysis. In some embodiments, the nucleic acid probes of the microarray bind specifically to genes provided herein.

[0085] In some embodiments, hybridization of amplified nucleic acids to probes on a microarray is typically performed under stringent hybridization conditions. Conditions for hybridization reactions-42- IPTS / 2.00262535.1are well known in the art and are available from microarray suppliers For example, in some embodiments, hybridization of a nucleic acid molecule with probes found on a microarray is performed under moderately stringent or highly stringent physiological conditions, as are known in the art. For example, in some embodiments, hybridization on a microarray is performed according to manufacturer's (Affynietrix) instructions. For example, in some embodiments, hybridization is performed for 16 hours at 45 °C in a hybridization buffer, such as 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween 20, In some embodiments, washes are performed in a low stringency buffer ((6XSSPE, 0.01% Tween 20) at 25 °C followed by a high stringency buffer (100 mM MES, 0. IM [Nar], 0.01% Tween 20) at 5 °C. Tn some embodiments, washes are performed using progressively higher stringency conditions: 2>< SSC / 0.1% SDS at about room temperature (hybridization conditions); 0.2>< SSC / 0.1% SDS at about room temperature (low stringency conditions);0.2*SSC / 0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 xSSC at about 68°C (high stringency conditions). In some embodiments, washing is carried out using only one of these conditions, for example, high stringency conditions. In some embodiments, washing is carried out using each of the conditions. In some embodiments, washing is earned out using each of the conditions, for 10 to 15 minutes each, in the order listed above, optionally repeating any or all of the steps listed.

[0086] In some embodiments, other microfluidic devices and methods for analyzing gene expression products, including those in which more than one gene expression product can be analyzed simultaneously and those involving high-throughput technologies, are used for the methods provided herein. Information obtained during and after the analysis of gene expression products is an article and / or product of the methods provided herein.

[0087] Quantitative measurement of gene expression levels using bioarrays is also known in the art, and typically involves a modified version of a traditional method for measuring expression as described herein. For example, such quantitation can be performed by measuring a phosphor image of a radioactive-labeled probe binding to a spot of a microarray, using a phospohor imager and imaging software. Information regarding the measured gene expression levels is an article and / or product of the methods provided herein.

[0088] In some embodiments, the determined gene expression profile of a subject is analyzed by comparing it to a set of reference gene expression profiles. The reference set of gene expression profiles comprise gene expression products recovered from reference subjects with a known-43- IPTS / 2.00262535.1characteristic (e g., ARVC treated with PKP2 gene therapy). A deviation or correlation between the reference profiles and the subject gene expression profile is used to establish a characterization of PKP2 gene therapy treatment. Many statistical techniques are known in the art, which can be used to determine whether a statistically significant difference or correlation in gene expression between a subject’s gene expression profile and a reference gene expression profile is observed at a 90% or preferably a 95% confidence level. In some embodiments, a statistical software program or module performed on a computer processor is used to determine whether a statistically significant difference or correlation in gene expression is observed at a given confidence level. Information regarding the statistical significance of a difference or correlation in gene expression level is an article and / or product of the statistical techniques provided herein.

[0089] In some embodiments, a RNAse protection assay is used where RNA is the gene expression product to be detected in the method. In this procedure, a labeled antisense RNA probe is hybridized to the complementary polynucleotide in the sample. The remaining unhybridized single-stranded probe is degraded by ribonuclease treatment. The hybridized, double stranded probe is protected from RNAse digestion. After an appropriate time, the products of the digestion reaction are collected and analyzed on a gel. In some embodiments, a software program or module performed on a computer processor is involved in the analysis of the digestion reaction products. Information regarding the analysis of digested reaction products is an article and / or product of the RNAse protection assay. As used herein, " RNA probe" refers to a ribonucleotide capable of hybridizing to RNA in a sample of interest. Those skilled in the art will be able to identify and modify the RNAse protection assay specific to the polynucleotide to be measured, for example, probe specificity, hybridization temperatures, and quantity of nucleic acid can be altered individually or collectively in part or in full. Additionally, a number of commercial kits are available, for example, RiboQuant™ Multi -Probe RNAse Protection Assay System (Pharmingen, Inc., San Diego, Calif.).

[0090] In another embodiment, an RNA molecule in a sample is analyzed by a blotting procedure, typically a Northern blot procedure. For blotting procedures RNA molecules are separated on a gel and then probed with a complementary' polynucleotide to the sequence of interest. For example, RNA is separated on a gel, transferred to nitrocellulose and probed with complementary DNA to one of the genes disclosed herein. In some embodiments, the complementary probe is labeled radioactively or chemically. In some embodiments, the complementary labeled probe is detected and the Northern blot analyzed using a software program or module performed on a computer processor.-44- IPTS / 2.00262535.1Information regarding the analysis of an RNA molecule is an article and / or product of the blotting procedure described herein.

[0091] In some embodiments, detection of an RNA molecule includes size fractionation. Methods of size fractionating RNA are well known to those of skill in the art, such as by gel electrophoresis, including polyacrylamide gel electrophoresis (PAGE), For example, in some embodiments, the gel is a denaturing 7 M or 8 M urea-polyacrylamide-formamide gel. In some embodiments, size fractionating the RNA molecule is accomplished by chromatographic methods known to those of skill in the art. In some embodiments, the chromatograph is produced and / or analyzed by a software program or module performed on a computer processor. Information regarding the detection of a RNA molecule, including a chromatograph, is an article and / or product of the size fractionating methods described herein.

[0092] In some embodiments, the detection of RNA is performed by using radioactively labeled probes. In some embodiments, any radioactive label is employed which provides an adequate signal. Other labels include ligands, colored dyes, and fluorescent molecules, which, in some embodiments, serve as a specific binding pair member for a labeled ligand, and the like. The labeled preparations are used to probe for a RNA molecule by the Southern or Northern hybridization techniques, for example. RNA obtained from samples are transferred to filters that bind polynucleotides. After exposure to the labeled polynucleotide probe, which will hybridize to RNA nucleotide fragments, the binding of the radioactive probe to RNA fragments is identified by autoradiography. In some embodiments, the autoradiograph image is analyzed using a software program or module performed on a computer processor. The analyzed image is an article and / or product of the RNA detection methods provided herein. The particular hybridization technique is not essential to the performance of the method provided. As improvements are made in hybridization techniques, they can readily be applied in the method of the invention.

[0093] In some embodiments, probes for use in the methods provided selectively hybridize to a target gene or gene expression product. In some embodiments, the probes are spotted on a bioarray using methods known in the art. As used herein, the term "selective hybridization" or "selectively hybridize," refers to hybridization under moderately stringent or highly stringent conditions such that a nucleotide sequence preferentially associates with a selected nucleotide sequence over unrelated nucleotide sequences to a large enough extent to be useful in detecting expression of a gene. It will be recognized that some amount of non-specific hybridization is unavoidable, but is acceptable-45- IPTS / 2.00262535.1provide that hybridization to a target nucleotide sequence is sufficiently selective such that it can be distinguished over the non-specific cross-hybridization, for example, at least about 2-fold more selective, generally at least about 3-fold more selective, usually at least about 5-fold more selective, and particularly at least about 10-fold more selective, as determined, for example, by an amount of labeled oligonucleotide that binds to target nucleic acid molecule as compared to a nucleic acid molecule other than the target molecule, particularly a substantially similar (i.e., homologous) nucleic acid molecule other than the target nucleic acid molecule.

[0094] In some embodiments, conditions that allow for selective hybridization are determined empirically, or estimated based, for example, on the relative GC: AT content of the hybridizing oligonucleotide and the sequence to which it is to hybridize, the length of the hybridizing oligonucleotide, and the number, if any, of mismatches between the oligonucleotide and sequence to which it is to hybridize (see, for example, Sambrook et al., " Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989)). An example of progressively higher stringency conditions is as follows: 2xSSC / 0.1% SDS at about room temperature (hybridization conditions); 0.2>< SSC / 0.1% SDS at about room temperature (low stringency conditions); 0.2>< SSC / 0.1% SDS at about 42EC (moderate stringency conditions), and 0.1*SSC at about 68EC (high stringency conditions). In some embodiments, washing is carried out using only one of these conditions, eg., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.

[0095] In another embodiment, provided are methods for obtaining gene expression data from amplified nucleic acids that compensates for variability in amplification reactions. In this method, relative expression of a target gene and a control gene is compared to obtain relevant expression data In some embodiments, the expression comparison is accomplished by utilizing a software program or module performed on a computer processor. Accordingly, in certain embodiments, a ACt value is determined in order to identify gene expression changes. In some embodiments, this value and method is used to identify differential gene expression in any sample containing cellular material, including tissue obtained from the tape stripped methods provided herein. Such method is especially useful, where it is relatively difficult to obtain sufficient RNAfrom a control sample. In some embodiments, the ACt value is determined using a software program or module performed on-46- IPTS / 2.00262535.1a computer processor. The ACt value and related gene expression data are articles and / or products of the method provided herein.

[0096] The Ct value is the experimentally determined number of amplification (e.g. PCR) cycles required to achieve a threshold signal level (statistically significant increase in signal level (e.g fluorescence) over background) for mRNAxand a reference or control mRNA (Gibson, Heid et al.1996; Heid, Stevens et al. 1996). The Ct values are typically determined using a target nucleic acid (e.g mRNAx) primer and probe set, and a reference or control mRNA primer and probe set A A Ct value is calculated by calculating a difference in the number of amplification cycles required to reach a threshold signal level between the target nucleic acid molecule and the reference or control nucleic acid molecule. A difference in the A Ct value at a target area versus another area of a subject's skin, such as a normal area, or an unaffected area, is indicative of differential gene expression of the target nucleic acid molecule at the target area. A difference in the A Ct value at a target area versus another area from a reference sample with a melanoma characteristic, is indicative of differential gene expression of the target nucleic acid molecule at the target area. Using this value, differential expression is detected by comparing expression of the target nucleic acid molecule with expression of a control nucleic acid molecule. Using this value, correlated expression is detected by comparing expression of the target nucleic acid molecule with expression of a control nucleic acid molecule. In some embodiments, the comparison is performed by a software program or module performed on a computer. The A Ct value is useful for characterizing the physiologic state of the skin without reference to a calibration site. Such methods provide the advantage that it is not necessary to obtain a nucleic acid sample from a control site, where it may be difficult to obtain sufficient nucleic acid molecules. In some embodiments, the ACt value is determined using a software program or module performed on a computer processor. The ACt value and information regarding the characteristic of a target area of skin are articles and / or products of the method provided herein.

[0097] Accordingly, provided herein is a method for detecting a difference or correlation between gene expression profiles in a subject and a reference or control sample. In certain aspects, the method is used to detect an expression level for one or more genes described herein to assist in a characterization of a treatment for?\RVC. In some embodiments the gene expression product is a nucleic acid, such as RNA, which is then amplified. In some embodiments, RNA from a reference or control sample is isolated and amplified. In some instances, the reference or control RNA is isolated and / or amplified at a different point in time than the RNA obtained from the subject. In some-47- IPTS / 2.00262535.1embodiments, information regarding the amplified reference or control RNA is maintained in a reference or control profile for comparison to information obtained from the subject. In some embodiments, a A Ctvalue is obtained by calculating a difference in the number of amplification cycles required to reach a threshold signal level between the RNA obtained from the subject and the reference or control RNA, wherein a difference in the A Ct value is indicative of a characteristic of the pigmented skin lesion in a subject. In some embodiments, a A Ct value is obtained by calculating a difference in the number of amplification cycles required to reach a threshold signal level between the RNA obtained from the subject and the reference or control RNA, wherein a correlation in the A Ct value is indicative of a characteristic of the pigmented skin lesion in a subject. In some embodiments, the target and control nucleic acids are identified using a software program or module performed on a computer processor. In some embodiments, A Ct value is obtained using a software program or module performed on a computer processor. In some embodiments, the A Ct values are determined in the same amplification experiment (e.g. using separate reaction wells on the same multi-well reaction plate) using similar reaction conditions to other reactions In some embodiments, the ACt value is determined using a software program or module performed on a computer processor. The z\Ct value and information regarding the characteristic of a pigmented skin lesion are articles and / or products of the method provided herein.

[0098] In some embodiments, the method for detecting an RNA signature is used along with the other embodiments provided herein to characterize an expected response to PKP2 gene therapy or to determine a response to a PKP2 gene therapy.

[0099] In some embodiments, the gene expression profile is determined by identifying and / or quantifying one or more polypeptide products present in cellular material obtained from a subject. Cellular material may be obtained from a tissue sample isolated from an individual. In some instances, cellular material is obtained from a blood, urine, tear, sweat, hair, plasma, and / or serum sample from the subject. In some embodiments, a polypeptide is fully or partially isolated and / or purified from other cellular material prior to or during the detection of the polypeptide. In some instances, a polypeptide is isolated and / or purified from other cellular materials by standard protein purification techniques including, but limited to, ammonium sulfate precipitation, ion exchange chromatography, size exclusion chromatography, affinity chromatography, immunoprecipitation, ultracentrifugation, hydrophobicity chromatography, and any combination thereof. The isolated and / or partially purified polypeptides are articles and / or products of the methods described herein.-48- IPTS / 2.00262535.1Information regarding the identification and / or quantification of a polypeptide are articles and / or products of the methods described herein.[00100 j In some embodiments, polypeptide products expressed from the genes elsewhere herein are detected and / or quantified in a sample to determine a gene expression profile, some embodiments, the levels of such polypeptide gene expression products are indicative of successful of treatment with a PKP2 gene therapy or a response to treatment with a PKP2 gene therapy when compared to reference or control polypeptide products in a similar sample. In some instances, the sample is a tissue sampled from the heart of a subject. In this regard, the sample, as described herein, is used as a source to isolate polypeptides For example, in some embodiments, following tissue collection, cells isolated from tissue are lysed by any number of means, and polypeptides are obtained from the cells. In some embodiments, these polypeptides are identified and / or quantified using detection methods known to those of skill in the art, for example by protein microarrays, ELISA, immunohistochemistry, immunophenotyping, fluorescent in situ hybridization (FISH), mass spectrometry, absorbance measurement, and / or any combination thereof. In some embodiments, polypeptide gene expression products are identified and / or quantified using either polyclonal or monoclonal antibodies specific for the protein expression product. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). In some embodiments, the gene expression profile is obtained and / or analyzed using a software program or module performed on a computer processor. The gene expression profile and information regarding the gene expression profile are articles and / or products of the methods described herein.Metabolite Analysis

[0101] Metabolomic features involve the analysis of small molecules or metabolites present in a biological sample from an individual before and / or after treatment with PKP2 gene therapy. Metabolites are sensitive to changes in cellular processes and can reflect alterations in metabolic pathways associated with ARVC. Metabolomic biomarkers can be identified through techniques like mass spectrometry or nuclear magnetic resonance spectroscopy, enabling the detection of ARVC- specific metabolic signatures. In some embodiments, biomarkers derived from metabolomic features comprise metabolite profiling, ARVC specific metabolic signatures, metabolic pathway biomarkers, drug response biomarkers, environmental exposure markers, or other related biomarkers.-49- IPTS / 2.00262535.1Compositions and Kits for RNA Signature and / or Metabolite Analysis

[0102] In various aspects, provided is a composition, kit, or system to be used for the methods according to various embodiments described herein, i.e., methods of measuring a response to PKP2 gene therapy in an individual and / or methods of identifying an individual with arrhythmogenic right ventricular cardiomyopathy In some embodiments, provided is a composition, kit, or system to measure a mRNA signature and / or at least one metabolite in a biological sample of an individual. In some embodiments, the composition, kit, or system comprises a microarray, or any suitable means known to one skilled in the art, for detection and / or quantification of a gene expression product in a gene expression profile In some embodiments, the composition, kit, or system comprises a microarray, for measuring small molecules or metabolites present in a biological sample from an individual. In some embodiments, the composition, kit, or system comprises reagents (e.g., probes, buffers) for detection and / or quantification of the gene expression products and / or metabolites in a biological sample from an individual.Samples

[0103] In some embodiments of the various methods described herein, the sample is from a subject. A subject may be any animal, including but not limited to, a cow, a pig, a mouse, a rat, a chicken, a cat, a dog, etc., and is usually a mammal, such as a human Sample polynucleotides are often isolated from a cell-free sample from a subject, such as a tissue sample, bodily fluid sample, or organ sample, including, for example, blood sample, or fluid sample containing nucleic acids (e.g., saliva). In some cases, the sample is treated to remove cells, or polynucleotides are isolated without a cellular extractions step (e.g., to isolate cell-free polynucleotides, such as cell-free DNA), Other examples of sample sources include those from blood, urine, feces, nares, the lungs, the gut, other bodily fluids or excretions, materials derived therefrom, or combinations thereof In some embodiments, the sample is a blood sample or a portion thereof (e.g., blood plasma or serum).Serum and plasma may be of particular interest, due to the relative enrichment for cardiovascul r DNA associated with the higher rate of malignant cell death among such tissues. In some embodiments, a sample from a single individual is divided into multiple separate samples (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more separate samples) that are subjected to methods of the disclosure independently, such as analysis in duplicate, triplicate, quadruplicate, or more. Where a sample is from a subject, the reference sequence may also be derived from the subject, such as a consensus sequence from the sample under analysis or the sequence of polynucleotides from another sample or-50- IPTS / 2.00262535.1tissue of the same subject. For example, a blood sample may be analyzed for cfDNA mutations, while cellular DNA from another sample (e.g., buccal or skin sample) is analyzed to determine the reference sequence.

[0104] Polynucleotides may be extracted from a sample according to any suitable method. A variety of kits are available for extraction of polynucleotides, selection of which may depend on the type of sample, or the type of nucleic acid to be isolated. Examples of extraction methods are provided herein, such as those described with respect to any of the various aspects disclosed herein. In one example, the sample may be a blood sample, such as a sample collected in an EDTA tube (e.g., BD Vacutainer). Plasma can be separated from the peripheral blood cells by centrifugation (e.g., 10 minutes at 1900×g at 4°C). Plasma separation performed in this way on a 6mL blood sample will typically yield 2.5 to 3 mL of plasma. Circulating cell-free DNA can be extracted from a plasma sample, such as by using a QIAmp Circulating Nucleic Acid Kit (Qiagene), according to the manufacturer’s protocol. DNA may then be quantified (e.g., on an Agilent 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent)). As an example, yield of circulating DNA from such a plasma sample from a healthy person may range from 1ng to 10ng per mL of plasma, with significantly more in disease (e.g., ARVC) patient samples.

[0105] In some embodiments, the plurality of polynucleotides comprises cell-free polynucleotides, such as cell-free DNA (cfDNA) or cell-free RNA (cfRNA). Cell-free DNA circulates in both healthy and diseased individuals. Cell-free RNA circulates in both healthy and diseased individuals. Cell-free DNA may be obtained from a variety of sources. One common source is blood samples of a subject. However, cfDNA or other fragmented DNA may be derived from a variety of other sources For example, urine and stool samples can be a source of cfDNA. Cell-free RNA may be obtained from a variety of sources.

[0106] In some embodiments, polynucleotides are subjected to subsequent steps (e.g., circularization and amplification) without an extraction step, and / or without a purification step. For example, a fluid sample may be treated to remove cells without an extraction step to produce a purified liquid sample and a cell sample, followed by isolation of DNA from the purified fluid sample. A variety of procedures for isolation of polynucleotides are available, such as by precipitation or non-specific binding to a substrate followed by washing the substrate to release bound polynucleotides. Where polynucleotides are isolated from a sample without a cellular-51- IPTS / 2.00262535.1extraction step, polynucleotides will largely be extracellular or “cell-free” polynucleotides. For example, cell-free polynucleotides may include cell-free DNA (also called “circulating” DNA). Gene Therapy Vectors

[0107] In another aspect, there are provided gene therapy vectors comprising a plakophilin 2 gene operatively linked to at least one promoter In some cases, the gene therapy vector comprises a viral vector. In some cases, the viral vector is any suitable viral vector for treating a heart disease or condition. In some cases, the viral vector is suitable for delivering a gene to cells in the myocardium, the epicardium, or both. 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, and a herpes virus. In some cases, the gene therapy vector is an adeno-associated virus. In some cases, the adeno- associated virus is selected from the group consisting of an AAV6, an AAV8, and an AAV9, or a derivative thereof. In some cases, the adeno-associated virus is an AAV9 or a derivative thereof. In some cases, the AAV9 comprises a genome comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 7. In some cases, the adeno-associated virus is a derivative of AAV6, AAV8, or AAV9, optimized for transducing cells according to methods of treatment herein. In some cases, the derivative is any AAV described in U S. Patent Application No.63 / 012,703, which is hereby incorporated by reference in its entirety.

[0108] In some embodiments of gene therapy vectors provided herein, PKP2 is expressed by any promoter suitable for expression in the affected cells and tissues, for example cardiomyocytes. In some cases, PKP2 is expressed by a promoter that is active in cells of the myocardium, the epicardium, or both. For example, in some cases, the promoter is a cardiac specific promoter. In some cases, the cardiac specific promoter is a troponin promoter or an alpha-myosin heavy chain promoter. In some cases, the promoter is a PKP2 promoter. In some cases, a cardiac specific enhancer is combined with the promoter. In some cases, the troponin promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 3. In some cases, the PKP2 promoter comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 4. In some cases, the promoter is a constitutive promoter. In some cases, the constitutive promoter is a beta-actin promoter.-52- IPTS / 2.00262535.1

[0109] In some embodiments of gene therapy vectors provided herein the nucleic acid encoding the PKP2 gene has any suitable sequence encoding a PKP2 polypeptide for example, any nucleic acid encoding a polypeptide having a sequence of SEQ ID NO: 8 For example, in some cases, the PKP2 gene has a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 1. In some cases, the PKP2 gene has a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 2. In some cases, the nucleic acid sequence encoding the PKP2 gene is codon optimized.

[0110] In some embodiments of gene therapy vectors provided herein, the gene therapy vector comprises a 3’ element. In some embodiments, the 3’ element stabilizes the transcriptional product of the gene therapy vector (e g., the PKP2 transcript). In some embodiments, the 3’ element comprises a bovine growth hormone (BGH) polyadenylation sequence. In some embodiments, the 3’ element comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

[0111] In various embodiments of gene therapy vectors provided herein, the gene therapy vector comprising a PKP2 gene is formulated in a composition comprising a pharmaceutically acceptable carrier or excipient. For example, in some cases, the pharmaceutically acceptable carrier or excipient comprises a buffer, a polymer, a salt, or a combination thereof.

[0112] In some embodiments, gene therapy vectors herein comprise nucleic acid sequences provided in Table 1 below.Table 1: Seo uencesName Sequence SEQ ID NO: Human atggcagcccccggcgccccagctgagtacggctacatccggaccgtcctgggccagcagatcct 1 PKP2 gggacaactggacagctccagcctggcgctgccctccgaggccaagctgaagctggcggggagc agcggccgcggcggccagacagtcaagagcctgcggatccaggagcaggtgcagcagaccctc gcccggaagggccgcagctccgtgggcaacggaaatcttcaccgaaccagcagtgttcctgagtat gtctacaacctacacttggttgaaaatgattttgttggaggccgttcccctgttcctaaaacctatgacat gctaaaggctggcacaactgccacttatgaaggtcgctggggaagaggaacagcacagtacagct cccagaagtccgtggaagaaaggtccttgaggcatcctctgaggagactggagatttctcctgacag cagcccggagagggctcactacacgcacagcgattaccagtacagccagagaagccaggctggg cacaccctgcaccaccaagaaagcaggcgggccgccctcctagtgccaccgagatatgctcgttcc gagatcgtgggggtcagccgtgctggcaccacaagcaggcagcgccactttgacacataccacag acagtaccagcatggctctgttagcgacaccgtttttgacagcatccctgccaacccggccctgctca cgtaccccaggccagggaccagccgcagcatgggcaacctcttggagaaggagaactacctgac ggcagggctcactgtcgggcaggtcaggccgctggtgcccctgcagcccgtcactcagaacagggcttccaggtcctcctggcatcagagctccttccacagcacccgcacgctgagggaagctgggccca-53- IPTS / 2.00262535.1gtgtcgccgtggattccagcgggaggagagcgcacttgactgtcggccaggcggccgcaggggg aagtgggaatctgctcactgagagaagcactttcactgactcccagctggggaatgcagacatggag atgactctggagcgagcagtgagtatgctcgaggcagaccacatgctgccatccaggatttctgctg cagctactttcatacagcacgagtgcttccagaaatctgaagctcggaagagggttaaccagcttcgt ggcatcctcaagcttctgcagctcctaaaagttcagaatgaagacgttcagcgagctgtgtgtggggc cttgagaaacttagtattgaagacaatgacaacaaattggaggtggctgaactaaatggggtacctc ggctgctccaggtgctgaagcaaaccagagacttggagactaaaaaacaaataacaggtttgctgtg gaatttgtcatctaatgacaaactcaagaatctcatgataacagaagcattgcttacgctgacggagaa tatcatcatccccttttctgggtggcctgaaggagactacccaaaagcaaatggtttgctcgattttgac atattctacaacgtcactggatgcctaagaaacatgagttctgctggcgctgatgggagaaaagcgat gagaagatgtgacggactcattgactcactggtccattatgtcagaggaaccattgcagattaccagc cagatgacaaggccacggagaattgtgtgtgcattcttcataacctctcctaccagctggaggcagag ctcccagagaaatattcccagaatatctatattcaaaaccggaatatccagactgacaacaacaaaag tatggatgttttggcagtcgaagcaggaaagtaaaagagcaataccaggacgtgccgatgccgga ggaaaagagcaaccccaagggcgtggagtggctgtggcattccattgttataaggatgtatctgtcct tgatcgccaaaagtgtccgcaactacacacaagaagcatccttaggagctctgcagaacctcacggc cggaagtggaccaatgccgacatcagtggctcagacagttgtccagaaggaaagtggcctgcagc acacccgaaagatgctgcatgttggtgacccaagtgtgaaaaagacagccatctcgctgctgagga atctgtcccggaatctttctctgcagaatgaaattgccaaagaaactctccctgatttggtttccatcattc ctgacacagtcccgagtactgaccttctcattgaaactacagcctctgcctgttacacattgaacaacat aatccaaaacagttaccagaatgcacgcgaccttctaaacaccgggggcatccagaaaattatggcc attagtgcaggcgatgcctatgcctccaacaaagcaagtaaagctgcttccgtccttctgtattctctgt gggcacacacggaactgcatcatgcctacaagaaggctcagtttaagaagacagattttgtcaacag ccggactgccaaagcctaccactcccttaaagactgaHuman atggctgctcctggtgctcctgccgagtacggctacatcagaacagtgctgggccagcagatcctgg 2 PKP2 gacagctggattctagctctctggccctgccttctgaggccaagctgaaactggccggcagttctgga (codon agaggcggccagacagtgaagtccctgcggatccaagaacaggtgcagcagaccctggccagaa optimized) agggcagatcttctgtcggcaacggcaacctgcacagaaccagctctgtgcccgagtacgtgtaca atctgcacctggtggaaaacgacttcgtcggcggcagatcccctgtgcctaagacctacgatatgctg aaggccggcaccaccgccacctatgaaggcagatggggaagaggcacagcccagtacagcagc cagaaaagcgtggaagagagaagcctgcggcaccctctgcggagactggaaatcagccctgatag cagcccagagagagcccactacacccacagcgactaccagtactcccagagatctcaggccggcc acacactgcaccaccaagagtctagaagggccgctctgctggtgcctcctagatacgccagatctga gatcgtgggcgtgtccagagccggcacaacaagcagacagagacacttcgacacctaccaccggc agtatcagcacggcagcgtgtccgataccgtgttcgatagcatccccgccaatcctgctctgctgaca taccctagacctggcacctccagatccatgggcaatctgctggaaaaagagaactacctgaccgcc ggactgaccgtgggacaagttcgacctctggttcctctgcagcccgtgacacagaacagagccagc agaagcagctggcaccagtccagcttccacagcaccagaacactgagagaagctggccctagcgt ggccgtggattcttctggtagaagggctcacctgacagttggccaagcagctgcaggcggaagcgg aaatctgctgaccgagagaagcaccttcaccgacagccagctgggcaacgccgacatggaaatga cactggaacgggccgtgtccatgctggaagccgatcacatgctgcccagcagaattagcgccgctg ccacctttatccagcacgagtgcttccagaagtctgaggcccggaagagagtgaaccagctgagag gcatcctgaagctgctgcagctcctgaaggtgcagaacgaggatgtgcagagggctgtgtgtgggg ccctgagaaatctggtgttcgaggacaacgacaacaagctggaagtggccgagctgaacggcgtg ccaagactgctgcaggttctgaaacagacccgcgacctggaaacaaagaagcagatcaccggcctgctctggaacctgagcagcaacgacaagctgaagaacctgatgatcacagaggccctgctgaccctIPTS / 2.00262535.1gacagagaacatcatcatccctttcagcggctggcccgagggcgattaccctaaagctaatggcctg ctggacttcgacatcttctacaacgtgaccggctgcctgagaaacatgtctagcgctggcgccgatg gcagaaaggccatgagaagatgtgacggcctgatcgacagcctggtgcactatgtgcggggcaca atcgccgattaccagcctgatgataaggccaccgagaactgcgtgtgcatcctgcacaacctgagct accagctggaagcagagctgcccgagaagtacagccagaacatctacatccagaaccggaacatc cagaccgacaacaacaagagcatcggctgcttcggcagccgcagccggaaagtgaaagaacagt accaggacgtgcccatgcctgaggaaaagtctaaccccaaaggcgtggaatggctgtggcacagc atcgtgatccggatgtacctgagcctgatcgccaagagcgtgcggaattacacccaagaggcatctc tgggcgccctgcagaatctgacagcaggatctggccctatgcctacctctgtggctcagaccgtggt gcagaaagagtctggcctgcagcacacccggaagatgctgcatgtgggagatcccagcgtgaaga aaaccgccatcagcctgctgagaaacctgagccggaatctgtctctgcagaatgagatcgccaaag agacactgcccgacctggtgtctatcatccctgacaccgtgcctagcaccgacctgctgattgagaca acagccagcgcctgctacaccctgaacaacatcattcagaactcctaccagaacgcccgcgatctgc tgaacacaggcggcatccagaaaatcatggccatctctgccggcgacgcctacgcctctaacaagg cctctaaagccgccagcgtgctgctgtattctctgtgggcccataccgagctgcaccatgcctataag aaggcccagttcaaaaagaccgacttcgtgaacagccggaccgccaaggcctaccactctctgaaa gatpcTNT gtcatggagaagacccaccttgcagatgtcctcactggggctggcagagccggcaacctgcctaag 3 Promoter gctgctcagtccattaggagccagtagcctggaagatgtctttacccccagcatcagttcaagtggag cagcacataactcttgccctctgccttccaagattctggtgctgagacttatggagtgtcttggaggttg ccttctgccccccaaccctgctcccagctggccctcccaggcctgggttgctggcctctgctttatcag gattctcaagagggacagctggtttatgttgcatgactgttccctgcatatctgctctggttttaaatagct tatctgagcagctggaggaccacatgggcttatatggcgtggggtacatgttcctgtagcctgtccct ggcacctgccaaaatagcagccaacaccccccacccccaccgccatccccctgccccacccgtcc cctgtcgcacattcctccctccgcagggctggctcaccaggccccagcccacatgcctgcttaaagc cctctccatcctctgcctcacccagtccccgctgagactgagcagacgcctccaPKP2 catctcagcatcatggttggatgtttccacctggctacataagcaagctttacacaaggtgtaatttgcct 4 promoter aaatagtggtccattctattggggtgggagcaattgcttccaggactcacatccatatggctcccacta gccatgtggcctgctgacaaagggtggcggaactgtcactactctgttgtccacgctttcagtcctttg gtttcctcttcactccctggacgctcatgtaaaaagggaggccatatacctgtgcattgtgtgt.ctaagc attcagtgtgtgtctaaaggcagaagggtgtgggtaggaaaacaaagacgagggaagctgcgttct ccaaacacttcagacttgagtaagtggggttttgcagcaattgagtgatttgagggaaagtgaacatac aaacccaagcaatcaaagggaatattatcttaataccagggatacatgtttttctttctgcctcttaagtcc aaagaggcaaatcaggacaagtggctttggttgtaaactttaaggtcaaggatcctttctgttgagctta gctctcaagttctcagtagtcaactgcggtgaaacataattaatagcacgataaatacaagttgtggaa gattcgattgaaagttggaggccctctccgtggatctctctacaaagagcctgtaataaagaggactta atcaacgtagcagggctatttaaaaagcatcgtctattaaaattcatttcttctctagagcctcttgttgg agttctctgtgtgggtgtgttcgtaagagaggaatgggttagcaagagtactgggtacaatttgtgtat ccaagagaaaacagaagctctcaatgaggaagaacatatgtttctgggactgcatctgtgcaaaaag tacatagtcctgacgttgtactaagaaaaaaaacactctctttagaaagtcttttatttcacacgttatcttc ttggcacatttccctcatattgccctttccgcctgaccaaatagccctttctcaccctcaggtccaggaa aaccaggaaacgtttccaacagtgcgacaaagcctgactaaccagacatactactcgctcggggatc ccggaggcaagcctcagtccaagaacaggagtgactctcgagggctcacctgcctgcagggcagc ccctccctgcatcgagcggaaatccatcctgtccagcgcggggcgtgggcagagcggggcgcgg ccccggcaggcggtatccgctgggactccgacaacgtgcgcgaccccaggcgaaccgcgcccctctccccacctccccgcgggcgggtacaagtctccaggtgtccgcgcgctcagcgggtccggcccgIPTS / 2.00262535.1cccccgcccccgcccccgggcccgactgcgcgtgcccggccggagccgcgccccctcctcagg gaaggccgggcgtccggcccacgaggccgagctcccccccggcccgggcctctcaccggcgcg gggggcgggccaggggcggggccggactcgagcggggcggggctcgcgccagcgcccccag ctccgtggcggcttcgcccgcgagtccagaggcaggcgagcagctcggtcgcccccaccggccccAAV ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcg 5 Human cccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct PKP2a tgtagttaatgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgccc Expression ttaagtcatggagaagacccaccttgcagatgtcctcactggggctggcagagccggcaacctgcc Cassette caaggctgctcagtccattaggagccagtagcctggaagatgtctttacccccagcatcagttcaagt (pcTnT ggagcagcacataactcttgccctctgccttccaagattctggtgctgagacttatggagtgtcttgga promoter, ggttgccttctgccccccaaccctgctcccagctggccctcccaggcctgggttgctggcctctgcttt codon atcaggattctcaagagggacagctggtttatgttgcatgactgttccctgcatatctgctctggttttaa optimized) atagcttatctgagcagctggaggaccacatgggcttatatggcgtggggtacatgttcctgtagcctt gtccctggcacctgccaaaatagcagccaacaccccccacccccaccgccatccccctgccccac ccgtcccctgtcgcacattcctccctccgcagggctggctcaccaggccccagcccacatgcctgct taaagccctctccatcctctgcctcacccagtccccgctgagactgagcagacgcctccagccacca tggctgctcctggtgctcctgccgagtacggctacatcagaacagtgctgggccagcagatcctggg acagctggattctagctctctggccctgccttctgaggccaagctgaaactggccggcagttctggaa gaggcggccagacagtgaagtccctgcggatccaagaacaggtgcagcagaccctggccagaaa gggcagatcttctgtcggcaacggcaacctgcacagaaccagctctgtgcccgagtacgtgtacaat ctgcacctggtggaaaacgacttcgtcggcggcagatcccctgtgcctaagacctacgatatgctga aggccggcaccaccgccacctatgaaggcagatggggaagaggcacagcccagtacagcagcc agaaaagcgtggaagagagaagcctgcggcaccctctgcggagactggaaatcagccctgatagc agcccagagagagcccactacacccacagcgactaccagtactcccagagatctcaggccggcca cacactgcaccaccaagagtctagaagggccgctctgctggtgcctcctagatacgccagatctgag atcgtgggcgtgtccagagccggcacaacaagcagacagagacacttcgacacctaccaccggca gtatcagcacggcagcgtgtccgataccgtgttcgatagcatccccgccaatcctgctctgctgacat accctagacctggcacctccagatccatgggcaatctgctggaaaaagagaactacctgaccgccg gactgaccgtgggacaagttcgacctctggttcctctgcagcccgtgacacagaacagagccagca gaagcagctggcaccagtccagcttccacagcaccagaacactgagagaagctggccctagcgtg gccgtggattcttctggtagaagggctcacctgacagttggccaagcagctgcaggcggaagcgga aatctgctgaccgagagaagcaccttcaccgacagccagctgggcaacgccgacatggaaatgac actggaacgggccgtgtccatgctggaagccgatcacatgctgcccagcagaattagcgccgctgc cacctttatccagcacgagtgcttccagaagtctgaggcccggaagagagtgaaccagctgagagg catcctgaagctgctgcagctcctgaaggtgcagaacgaggatgtgcagagggctgtgtgtggggc cctgagaaatctggtgttcgaggacaacgacaacaagctggaagtggccgagctgaacggcgtgc caagactgctgcaggttctgaaacagacccgcgacctggaaacaaagaagcagatcaccggcctg ctctggaacctgagcagcaacgacaagctgaagaacctgatgatcacagaggccctgctgaccctg acagagaacatcatcatccctttcagcggctggcccgagggcgattaccctaaagctaatggcctgc tggacttcgacatcttctacaacgtgaccggctgcctgagaaacatgtctagcgctggcgccgatgg cagaaaggccatgagaagatgtgacggcctgatcgacagcctggtgcactatgtgcggggcacaat cgccgattaccagcctgatgataaggccaccgagaactgcgtgtgcatcctgcacaacctgagctac cagctggaagcagagctgcccgagaagtacagccagaacatctacatccagaaccggaacatcca gaccgacaacaacaagagcatcggctgcttcggcagccgcagccggaaagtgaaagaacagtaccaggacgtgcccatgcctgaggaaaagtctaaccccaaaggcgtggaatggctgtggcacagcatIPTS / 2.00262535.1cgtgatccggatgtacctgagcctgatcgccaagagcgtgcggaatacacccaagaggcatctctg ggcgccctgcagaatctgacagcaggatctggccctatgcctacctctgtggctcagaccgtggtgc agaaagagtctggcctgcagcacacccggaagatgctgcatgtgggagatcccagcgtgaagaaa accgccatcagcctgctgagaaacctgagccggaatctgtctctgcagaatgagatcgccaaagag acactgcccgacctggtgtctatcatccctgacaccgtgcctagcaccgacctgctgattgagacaac agccagcgcctgctacaccctgaacaacatcattcagaactcctaccagaacgcccgcgatctgctg aacacaggcggcatccagaaaatcatggccatctctgccggcgacgcctacgcctctaacaaggcc tctaaagccgccagcgtgctgctgtattctctgtgggcccataccgagctgcaccatgcctataagaa ggcccagttcaaaaagaccgacttcgtgaacagccggaccgccaaggcctaccactctctgaaaga ttaataagcttggatccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatg ttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttc attttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgt ggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagct cctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccg ctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcct ttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtccctcggc cctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgag atctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccct ggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgt cattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcag gcatgctggggactggggactcgagttaagggcgaattcccgataaggatcttcctagagcatggct acgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccact ccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggcttt gcccgggcggcctcagtgagcgagcgagcgcgcagAAV ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcg 6 Human cccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct PKP2a tgtagttaatgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgccc Expression ttaacatctcagcatcatggttggatgtttccacctggctacataagcaagctttacacaaggtgtaattt Cassette gcctaaatagtggtccattctattggggtgggagcaattgcttccaggactcacatccatatggctccc (PKP2 acttagccatgtggcctgctgacaaagggtggcggaactgtcactactctgttgtccacgctttcagtc promoter, ctttggtttcctcttcactccctggacgctcatgtaaaaagggaggccatatacctgtgcattgtgtgtct codon aagcattcagtgtgtgtctaaaggcagaagggtgtgggtaggaaaacaaagacgagggaagctgc optimized) gttctccaaacacttcagacttgagtaagtggggttttgcagcaattgagtgatttgagggaaagtgaa catacaaacccaagcaatcaaagggaatattatcttaataccagggatacatgtttttctttctgcctctta agtccaaagaggcaaatcaggacaagtggctttggttgtaaactttaaggtcaaggatcctttctgttg agcttagctctcaagttctcagtagtcaactgcggtgaaacataattaatagcacgataaatacaagttg tggaagattcgattgaaagttggaggccctctccgtggatctctctacaaagagcctgtaataaagag gacttaatcaacgttagcagggctatttaaaaagcatcgtctattaaaattcatttcttctctagagcctctt gttggagtttctctgtgtgggtgtgttcgtaagagaggaatgggttagcaagagtactgggtacaatttg tgtatccaagagaaaacagaagctctcaatgaggaagaacatatgtttctgggactgcatctgtgcaa aaagtacatagtcctgacgttgtactaagaaaaaaaacactctctttagaaagtcttttatttcacacgtta tcttcttggcacatttccctcatattgccctttccgcctgaccaaatagccctttctcaccctcaggtccag gaaaaccaggaaacgtttccaacagtgcgacaaagcctgactaaccagacatactactcgctcggg gatcccggaggcaagcctcagtccaagaacaggagtgactctcgagggctcacctgcctgcaggg cagcccctccctgcatcgagcggaaatccatcctgtccagcgcggggcgtgggcagagcggggcgcggccccggcaggcggtatccgctgggactccgacaacgtgcgcgaccccaggcgaaccgcgIPTS / 2.00262535.1cccctctccccacctccccgcgggcgggtacaagtctccaggtgtccgcgcgctcagcgggtccg gcccgcccccgcccccgcccccgggcccgactgcgcgtgcccggccggagccgcgccccctcc tcagggaaggccgggcgtccggcccacgaggccgagctcccccccggcccgggcctctcaccg gcgcggggggcgggccaggggcggggccggactcgagcggggcggggctcgcgccagcgcc cccagctccgtggcggcttcgcccgcgagtccagaggcaggcgagcagctcggtcgcccccacc ggccccatggctgctcctggtgctcctgccgagtacggctacatcagaacagtgctgggccagcag atcctgggacagctggattctagctctctggccctgccttctgaggccaagctgaaactggccggca gttctggaagaggcggccagacagtgaagtccctgcggatccaagaacaggtgcagcagaccctg gccagaaagggcagatcttctgtcggcaacggcaacctgcacagaaccagctctgtgcccgagtac gtgtacaatctgcacctggtggaaaacgacttcgtcggcggcagatcccctgtgcctaagacctacg atatgctgaaggccggcaccaccgccacctatgaaggcagatggggaagaggcacagcccagta cagcagccagaaaagcgtggaagagagaagcctgcggcaccctctgcggagactggaaatcagc cctgatagcagcccagagagagcccactacacccacagcgactaccagtactcccagagatctcag gccggccacacactgcaccaccaagagtctagaagggccgctctgctggtgcctcctagatacgcc agatctgagatcgtgggcgtgtccagagccggcacaacaagcagacagagacacttcgacaccta ccaccggcagtatcagcacggcagcgtgtccgataccgtgttcgatagcatccccgccaatcctgct ctgctgacataccctagacctggcacctccagatccatgggcaatctgctggaaaaagagaactacc tgaccgccggactgaccgtgggacaagttcgacctctggttcctctgcagcccgtgacacagaaca gagccagcagaagcagctggcaccagtccagcttccacagcaccagaacactgagagaagctgg ccctagcgtggccgtggattctctggtagaagggctcacctgacagttggccaagcagctgcaggc ggaagcggaaatctgctgaccgagagaagcaccttcaccgacagccagctgggcaacgccgaca tggaaatgacactggaacgggccgtgtccatgctggaagccgatcacatgctgcccagcagaatta gcgccgctgccacctttatccagcacgagtgcttccagaagtctgaggcccggaagagagtgaacc agctgagaggcatcctgaagctgctgcagctcctgaaggtgcagaacgaggatgtgcagagggct gtgtgtggggccctgagaaatctggtgttcgaggacaacgacaacaagctggaagtggccgagctg aacggcgtgccaagactgctgcaggttctgaaacagacccgcgacctggaaacaaagaagcagat caccggcctgctctggaacctgagcagcaacgacaagctgaagaacctgatgatcacagaggccc tgctgaccctgacagagaacatcatcatccctttcagcggctggcccgagggcgattaccctaaagc taatggcctgctggacttcgacatcttctacaacgtgaccggctgcctgagaaacatgtctagcgctg gcgccgatggcagaaaggccatgagaagatgtgacggcctgatcgacagcctggtgcactatgtg cggggcacaatcgccgattaccagcctgatgataaggccaccgagaactgcgtgtgcatcctgcac aacctgagctaccagctggaagcagagctgcccgagaagtacagccagaacatctacatccagaa ccggaacatccagaccgacaacaacaagagcatcggctgcttcggcagccgcagccggaaagtg aaagaacagtaccaggacgtgcccatgcctgaggaaaagtctaaccccaaaggcgtggaatggct gtggcacagcatcgtgatccggatgtacctgagcctgatcgccaagagcgtgcggaattacaccca agaggcatctctgggcgccctgcagaatctgacagcaggatctggccctatgcctacctctgtggct cagaccgtggtgcagaaagagtctggcctgcagcacacccggaagatgctgcatgtgggagatcc cagcgtgaagaaaaccgccatcagcctgctgagaaacctgagccggaatctgtctctgcagaatga gatcgccaaagagacactgcccgacctggtgtctatcatccctgacaccgtgcctagcaccgacctg ctgatgagacaacagccagcgcctgctacaccctgaacaacatcattcagaactcctaccagaacg cccgcgatctgctgaacacaggcggcatccagaaaatcatggccatctctgccggcgacgcctacg cctctaacaaggcctctaaagccgccagcgtgctgctgtattctctgtgggcccataccgagctgcac catgcctataagaaggcccagttcaaaaagaccgacttcgtgaacagccggaccgccaaggcctac cactctctgaaagatgtcgacggatccggtaccgattacaaggacgacgatgacaagtgaagcttaa taaaagatctttattttcattagatctgtgtgttggttttttgtgtgctggggactcgagttaagggcgaattcccgataaggatctcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaIPTS / 2.00262535.1ggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcg accaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag AAV9 acggcggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttc 7 genome tgacagctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctg sequence aatctgattgagcaggcacccctgaccgtggccgagaagctgcagcgcgactttctgacggaatgg cgccgtgtgagtaaggccccggaggcccttttctttgtgcaatttgagaagggagagagctacttcca catgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattc gcgaaaaactgattcagagaattaccgcgggatcgagccgactttgccaaactggttcgcggtcac aaagaccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattactt gctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgtt tgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggag cagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggt acatggagctggtcgggtggctcgtggacaaggggattacctcggagaagcagtggatccaggag gaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggctgccttgga caatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgt ggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggc ttccgtctttctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctg caactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggtgcgtaa actggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggg gaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtg gaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaac atgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatg ttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaaga ctttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggag ccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagtt gcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggtaccaaaacaaatgt tctcgtcacgtgggcatgaatctgatgctgtttccctgcagacaatgcgagagaatgaatcagaattca aatatctgcttcactcacggacagaaagactgtttagagtgctttcccgtgtcagaatctcaacccgttt ctgtcgtcaaaaaggcgtatcagaaactgtgctacattcatcatatcatgggaaaggtgccagacgctt gcactgcctgcgatctggtcaatgtggatttggatgactgcatctttgaacaataaatgacttaaaccag gtatggctgccgatggttatcttccagattggctcgaggacaaccttagtgaaggaattcgcgagtggt gggctttgaaacctggagcccctcaacccaaggcaaatcaacaacatcaagacaacgctcgaggtc ttgtgcttccgggttacaaataccttggacccggcaacggactcgacaagggggagccggtcaacg cagcagacgcggcggccctcgagcacgacaaggcctacgaccagcagctcaaggccggagaca acccgtacctcaagtacaaccacgccgacgccgagttccaggagcggctcaaagaagatacgtctt tgggggcaacctcgggcgagcagtcttccaggccaaaaagaggcttcttgaacctcttggtctggtt gaggaagcggctaagacggctcctggaaagaagaggcctgtagagcagtctcctcaggaaccgg actcctccgcgggtattggcaaatcgggtgcacagcccgctaaaaagagactcaatttcggtcagac tggcgacacagagtcagtcccagaccctcaaccaatcggagaacctcccgcagccccctcaggtgt gggatctcttacaatggcttcaggtggtggcgcaccagtggcagacaataacgaaggtgccgatgg agtgggtagttcctcgggaaattggcattgcgattcccaatggctgggggacagagtcatcaccacc agcacccgaacctgggccctgcccacctacaacaatcacctctacaagcaaatctccaacagcacat ctggaggatcttcaaatgacaacgcctacttcggctacagcaccccctgggggtattttgactcaaca gatccactgccacttctcaccacgtgactggcagcgactcatcaacaacaactggggattccggcct aagcgactcaacttcaagctcttcaacattcaggtcaaagaggttacggacaacaatggagtcaagaccatcgccaataaccttaccagcacggtccaggtcttcacggactcagactatcagctcccgtacgtgIPTS / 2.00262535.1ctcgggtcggctcacgagggctgcctcccgccgttcccagcggacgttttcatgattcctcagtacgg gtatctgacgcttaatgatggaagccaggccgtgggtcgttcgtccttttactgcctggaatatttcccg tcgcaaatgctaagaacgggtaacaacttccagttcagctacgagtttgagaacgtacctttccatagc agctacgctcacagccaaagcctggaccgactaatgaatccactcatcgaccaatacttgtactatctc tcaaagactattaacggttctggacagaatcaacaaacgctaaaattcagtgtggccggacccagca acatggctgtccagggaagaaactacatacctggacccagctaccgacaacaacgtgtctcaacca ctgtgactcaaaacaacaacagcgaatttgcttggcctggagcttcttcttgggctctcaatggacgta atagcttgatgaatcctggacctgctatggccagccacaaagaaggagaggaccgtttctttcctttgt ctggatctttaatttttggcaaacaaggaactggaagagacaacgtggatgcggacaaagtcatgata accaacgaagaagaaattaaaactactaacccggtagcaacggagtcctatggacaagtggccaca aaccaccagagtgcccaagcacaggcgcagaccggctgggttcaaaaccaaggaatacttccggg tatggtttggcaggacagagatgtgtacctgcaaggacccatttgggccaaaattcctcacacggac ggcaactttcacccttctccgctgatgggagggtttggaatgaagcacccgcctcctcagatcctcatc aaaaacacacctgtacctgcggatcctccaacggccttcaacaaggacaagctgaactctttcatcac ccagtattctactggccaagtcagcgtggagatcgagtgggagctgcagaaggaaaacagcaagc gctggaacccggagatccagtacacttccaactattacaagtctaataatgttgaatttgctgttaatact gaaggtgtatatagtgaaccccgccccatggcaccagatacctgactcgtaatctgtaa PKP2 MAAPGAPAEYGY1RTVLGQQILGQLDSSSLALPSEAKLKLAGSS 8 Protein GRGGQTVKSLRIQEQVQQTLARKGRSSVGNGNLHRTSSVPEYV YNLHLVENDFVGGRSPVPKTYDMLKAGTTATYEGRWGRGTA QYSSQKSVEERSLRHPLRRLEISPDSSPERAHYTHSDYQYSQRS QAGHTLHHQESRRAALLVPPRYARSEIVGVSRAGTTSRQRHFD TYHRQYQHGSVSDTVFDSIPANPALLTYPRPGTSRSMGNLLEKE NYLT AGLT VGQ VRPLVPLQP VTQNRASRS SWHQ S SFHSTRTLR EAGPS VA VD S S GRRAHLT VGQ A A AGGSGNLLTERSTFTDSQ LG NADMEMTLERAVSMLEADHMLPSRISAAATFlQHECFQKSEAR KRVNQLRGILKLLQLLKVQNEDVQRAVCGALRNLVFEDNDNK LE V AELN GVPRLLQ VLKQT RDLETKKQ1TGL LWNL S SNDKLKN LMITEALLTLTENIIIPFSGWPEGDYPKANGLLDFDIEYNVTGCL RNMSSAGADGRKAMRRCDGLIDSLVHYVTIGTIADYQPDDKAT ENCVCILHNLSYQLEAELPEKYSQNIYIQNRNIQTDNNKSIGCFG SRSRKVKEQYQDVPMPEEKSNPKGVEWLWHSIVIRMYLSLIAK SVRNYTQEASLGALQNLTAGSGPMPTSVAQTVVQKESGLQHTR KMLHVGDPSVKKTAISLLRNLSRNLSLQNEIAKETLPDLVSIIPD TVPSTDLLIETTASACYTLNNIIQNSYQNARDLLNTGGIQKIMAI SAGDAYASNKASKAASVLLYSLWAHTELHHAYKKAQFKKTDF VNSRTAKAYHSLKD WPRE tcaacctctggattacaaaattgtgaaagattgactggtattcttaactatgttgctccttttacgctatgt 9 ggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaa tcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgt ttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttccgggactttcgcttt ccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcg gctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgt gttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgIPTS / 2.00262535.1hGH poly A cctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaa 10 signal ggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattc tattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcat gctggggactggggaWPRE - tcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgt 11 hGH poly A ggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaasignal tcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtcassette ttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgcttt ccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcg gctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgt gttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggacctt ccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgcctt ctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccac tgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactggg gactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcatg gcgggttaatcattaactacaAAV9 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDN 12 capsid ARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQamino acid LKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRsequence LLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAK KRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPV ADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYN NHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPR DWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLT STVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLN DGSQ A VGRS SF YCLEYFPSQM LRTGNNFQF S YEFEN VPFHS S Y A HSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSN MAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALN GRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDA DKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQ NQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFG MKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNLViral Vectors

[0113] 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 (eg, 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--61- IPTS / 2.00262535.186, 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 vims (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 vims, and vectors derived from retroviruses such as Rous Sarcoma Vims, Harvey Sarcoma Vims, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma vims, and mammary tumor vims); and the like. Numerous suitable expression vectors are known 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.

[0114] 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.

[0115] 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.-62- IPTS / 2.00262535.1

[0116] 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. ”

[0117] 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.

[0118] 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.

[0119] Examples of viral polypeptides capable of ai ding entiy 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 Vims (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-l 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.

[0120] 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 vims. 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-63- IPTS / 2.00262535.1support 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.

[0121] 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, p-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 known 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 lenti viral 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.

[0122] 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).-64- IPTS / 2.00262535.1

[0123] 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.

[0124] 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 target-specific 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.

[0125] Lentiviral vectors are known 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.-65- IPTS / 2.00262535.1

[0126] 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.).

[0127] 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,

[0128] 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 known. 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. Then, 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-66- IPTS / 2.00262535.1cap 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 nonconsensus 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).

[0129] 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, rep-cap) 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.-67- IPTS / 2.00262535.1

[0130] 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 known 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.

[0131] In some embodiments, the AAV expression vector is pseudotyped to enhance targeting. To promote gene transfer and sustain expression in cardiomyocytes, AAV6, AAV8, and AAV9, are contemplated for use. In some cases, the AAV2 genome is packaged into the capsid of producing pseudotyped vectors AA V2 / 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 Then 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.

[0132] 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?\AV capsid (or in particular an AAV9 capsid), one or more substitutions are contemplated to increase infectivity towards cells in the myocardium,-68- IPTS / 200262535.1epicardium, 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

[0133] 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 lenti viral proteins, e.g., gag, pol, env, vpr, vpu, vpx, vif, tat, rev, ornef. 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-deiived 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.

[0134] 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. Packaging cells are often used to package vims 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--69- IPTS / 200262535.1293 cell line, RetroPack PT67 cell line, and Retro- X Universal Packaging System (all available from Clontech).

[0135] 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 exemplar}' embodiments, the retroviral construct comprises blasticidin (e.g., pMXs-IB), puromycin (e.g., pMXs-puro, pBabePuro), or neomycin (e.g., pMXs-neo). Morgenstern et al. (1990) Nucleic Acids Research 18:3587-3596. Promoters and Enhancers

[0136] 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).

[0137] 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.

[0138] 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-70- IPTS / 2.00262535.1containing Tet-operator elements; and cardi c-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,

[0139] 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, AES, 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.

[0140] 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 nucleotide 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).

[0141] In some embodiments, the vectors of the disclosure include one or more poly A signals. Illustrative poly A signals useful in the vectors of the disclosure include the short poly A 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).IPTS / 2.00262535.1Gene Therapy Vector Compositions

[0142] 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.

[0143] For example, in some embodiments, about 1x102-1x1010vector genomes (vg) are be administered. In some embodiments, the dose be at least about 102vg, about 103vg, about 104vg, about 105vg, about 106vg, about 107vg, about 108vg, about 109vg, about 1010vg, or more vector genomes. In some embodiments, the dose be about 102vg, about 103vg, about 104vg, about 105vg, about 106vg, about 107vg, about 108vg, about 109vg, about 1010vg, or more vector genomes.

[0144] Daily doses of the compounds vary as well. Such daily doses often range, for example, from at least about 102vg / day, about 103vg / day, about 104vg / day, about 10’ vg / day, about 106vg / day, about 107vg / day, about 108vg / day, about 109vg / day, about 1010vg / day, or more vector genomes per day

[0145] 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 1x108and about 1x1015GC 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 1x108and about 1x1015GC, between about 1x108and about 1x1015GC, between about 1x109and about 1x1014GC, between about 1x1010and about 1x1013GC, between about 1x1011and about 1x1012GC, or between about 1x1012and about 1x1013GC of vector. In some embodiments, the subject is treated by administering between about IxlO8and about lxl0’°GC, between about 1x109and about 1x1011GC, between about 1x1010and about 1x1012GC, between about 1x1011and about 1x1013GC, between about 1x1012and about 1x1014GC, or between about 1x1013and about 1x1015GC of vector. In some embodiments, the subject is treated by administering at least 1x108, at least about 1x109, at-72- IPTS / 2.00262535.1least about 1x1010, at least about 1x1011, at least about 1x1012, at least about 1x1013, or at least about 1x1015GC of vector. In some embodiments, the subject is treated by administering at most 1x108, at most about 1x109, at most about 1x1010, at most about 1x1011, at most about 1x1012, at most about 1x1013, or at most about 1x1015GC of vector. In some embodiments, the subject (e.g., a human) is treated by administering between about 1x108and about 1x1015GC / 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 1x108and about 1x1015GC / kg, between about 1x108and about 1x1015GC / kg, between about 1x109and about 1x1014GC / kg, between about 1x1010and about 1x1013GC / kg, between about 1x1011and about 1x1012GC / kg, or between about 1x1012and about 1x1015GC / kg of vector. In some embodiments, the subject is treated by administering between about 1x108and about 1x1010GC / kg, between about 1x109and about 1x1011GC / kg, between about 1x1010and about 1x1012GC / kg, between about 1x1011and about 1x1013GC / kg, between about 1x1012and about 1x1014GC / kg, or between about 1x1013and about 1x1015GC / kg of vector. In some embodiments, the subject is treated by administering at least 1x108, at least about 1x109, at least about 1x1010, at least about 1x1011, at least about 1x1012, at least about 1x1013, or at least about 1x1015GC / kg of vector. In some embodiments, the subject is treated by administering at most 1x108, at most about 1x109, at most about 1x1010, at most about 1x1011, at most about 1x1012, at most about 1x1013, or at most about 1x1015GC / 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.

[0146] 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 known to 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.IPTS / 2.00262535.1

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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 (Lotensin), Captopril (Capoten), Enalapril (Vasotec), Fosinopril (Monopril), Lisinopril (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-74- IPTS / 2.00262535.1(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.

[0151] Additional agents are sometimes included such as antibacterial agents, antimicrobial agents, anti-viral 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.

[0152] 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 known to 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.Certain Definitions

[0153] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0154] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described-75- IPTS / 2.00262535.1herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein.

[0155] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Thus, for example, reference to “a cardiomyocyte” includes a plurality of cardiomyocytes,

[0156] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C ” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0157] The term “about” means a range within 10% of a given value. 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 known in the art.

[0158] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or-76- IPTS / 2.00262535.1'■‘containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0159] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0160] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1,7, 1 8, and 1.9. With respect to subranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0161] 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. For example, in some cases, the term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia (U. S. P.) or other generally recognized pharmacopeia for use in animals, including humans.

[0162] 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,-77- IPTS / 2.00262535.1the 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).

[0163] The term “optional” or “optionally” denotes that a subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.

[0164] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0165] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1

[0166] The “percent sequence identity” between a reference amino acid sequence and a query amino sequence (i.e., the amino sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference amino acid sequence) is determined by optimally aligning the sequences using the Needleman- Wunsch alignment algorithm with a gap existence penalty of 11 and a gap extension penalty of 1 and comparing the sequences The number of exact matches, divided by the total number of positions in the alignment (which corresponds with the number of amino acids in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query amino acid sequence and the reference amino acid sequence (i e., percent sequence identity = (# of exact matches / (total # of positions in alignment)* 100). An alignment using the Needleman-Wunsch alignment algorithm (with a gap existence penalty of 11 and a gap extension penalty of 1) can be generated using the “Global Align” BLAST program available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.-78- IPTS / 2.00262535.1

[0167] The “percent sequence identity” between a reference nucleic acid sequence and a query nucleic acid sequence (i.e., the nucleic acid sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference nucleic acid sequence) is determined by optimally aligning the sequences using the Needleman- Wunsch alignment algorithm (with match / mismatch scores of 2,-3, a gap existence penalty of 5, and a gap extension penalty of 2) and comparing the aligned nucleic acids. The number of exact matches divided by the total number of nucleotides in the alignment (which corresponds with the number of nucleotides in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query nucleic acid sequence and the reference nucleic acid sequence (i.e., percent sequence identity = (# of exact matches) / (total # of nucleotides in the alignment)* 100). An alignment using the Needleman-Wunsch alignment algorithm (with match / mismatch scores of 2,-3, a gap existence penalty of 5, and a gap extension penalty of 2) can be generated using the “Global Align” BLAST program available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.

[0168] 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, infiltrative, 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),

[0169] 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-79- IPTS / 2.00262535.1Higgins 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.

[0170] 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.

[0171] “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.

[0172] 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-80- IPTS / 200262535.1coronary 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.

[0175] 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.

[0174] 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).

[0175] 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,

[0176] 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).-81- IPTS / 2.00262535.1

[0177] “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.

[0178] 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.

[0179] 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.

[0180] 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”).IPTS / 2.00262535.1

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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,-83- IPTS / 2.00262535.1an adeno-associated virus (AAV) vector, an adenovirus, a retrovirus, an integrating lentiviral vector (LVV), or a non-integrating LVV. 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.

[0185] 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).EXAMPLESExample 1: AAV9: PKP2 largely maintained key mRNA signatures of cardiac energy metabolism that were perturbed in Pkp2-cKO ARVC mouse heart.

[0186] The study shows that a single dose of AAV9: PKP2 treatment in Pkp2-cKO ARVC mice after overt cardiomyopathy halted disease progression and extended median lifespan by > 50 weeks post induction of Pkp2 deletion. RNA sequencing was carried out for heart tissues collected from either the therapeutic mode of treatment (virus injection after overt structural change) or the preventive mode of treatment (virus injection before overt structural change) at 51 weeks post tamoxifen induction of Pkp2 deletion (as shown in FIG. 1A). When comparing vehicle-treated Pkp2-cKO animals vs WT, the top two negatively enriched gene sets identified by Gene Set Enrichment Analysis (GSEA) were mitochondrial dysfunction and cardiac muscle contraction (FIG. EC), suggesting that PKP2 deficiency led to impaired oxidative phosphorylation and contractile functions. Transcriptional changes in key enzymes involved in both oxidative and glycolytic metabolic pathways were observed in heart failure and cardiomyopathies. To investigate a connection between PKP2 and cardiac energy metabolism, mRNA changes was categorized in major cardiac metabolic pathways of lipid homeostasis and glycolysis / glucose oxidation with highlighted enzymes as known points of regulation (as shown in FIG. IB). Glucose metabolism generates ATP from cytoplasmic glycolysis and the mitochondrial oxidation of the pyruvate derived from glycolysis. Fatty acid homeostasis was further categorized to gene classes of FAO, TAG dynamics, and fatty acid synthesis (as shown in FIG. 1C). In addition, mRNA changes in master regulators of-84- IPTS / 2.00262535.1cardiac metabolism, members of the peroxisome proliferator-activated receptor (PPAR) family, were included (as shown in FIG. 1C).

[0187] mRNA signatures suggested depressed FAO and glycolvsis / glucose oxidation and increased faty acid synthesis in both right and left ventricles of Pkp2-cKO mouse heart (as shown in FIGS. 1C and ID). In addition, mRNA signatures of TAG dynamics were greatly perturbed with respect to storage and mobilization via lipolysis. PPARy mRNA was significantly upregulated in contrast to decreased PPARa mRNA (FIGs. 1C and ID). PPARy activation were associated with promoting fatty acid uptake, TAG formation, and storage in lipid droplets. Both preventive and therapeutic modes of AAV9: PKP2 treatment at 51 weeks post tamoxifen induction of Pkp2 deletion showed significant long-term reversal of the metabolic remodeling with the preventive mode supporting the most complete reversal among age-matched animals (as shown in FIGS. 1C and ID). Similar conclusions were drawn based on a short-term 10-week study where metabolic reversal revealed a dose-dependent response to PKP2 expression level, suggesting a direct connection between PKP2 function and the cardiac energy metabolism (as shown in FIGS. 2A-2C) Example 2: Multiomics identified metabolic remodeling in Pkp2-cKO cardiac tissue that was reversed by AAV9: PKP2.

[0188] Pkp2-cKO mouse model was used to assess the feasibility and the efficacy of AAV9: PKP2 on reversing the metabolic remodeling. Nontargeted polar metabolomics using FIA-TOF mass spectrometry were conducted on cardiac tissues collected from either the therapeutic or the preventive mode of treatment (therapeutic mode in FIG. 3A and Preventive mode in FIG. 4A). Multi omics, integrated nontargeted polar metabolomics and RNA sequencing data were used to enhance confidence of detection (FIG. 3B and FIG. 4B) as significantly changed steady-state metabolites in nodes and genes in rods were clustered and color-coded based on metabolic pathways Metabolites and associated enzymes were highlighted for FAO and glycolysis and glucose oxidation (FIG. 3B). The significance and fold change of a set of metabolites identified by the multiomics were categorized in nine metabolic pathways and represented in heatmap (FIG. 3C) and the corresponding sequencing reads of connecting genes highlighted on the cluster were summarized in box plots (FIG. 3B). Multiomics identified reduction of acylcarnitines in the pathway ‘'Fatty Acid 0- Oxidation’ in cKO mouse and reversed by AAV9: PKP2 treatment shown on the heatmap. The impaired FAO was further supported by mRNA reduction in Cptlb (carnitine palmitoyltransferase IB), Cact (Slc25a20 carnitine-acylcarnitine translocase), Crat (carnitine O-acetyltransferase), and-85- IPTS / 2.00262535.1Acsl1 (acyl-CoA synthetase long chain family member 1) and their reversal by AAV9: PKP2 (box plots, FIG.3B). ATGL mRNA (PNPLA2, adipose triglyceride lipase) was significantly down- regulated in cKO heart and reversed by AAV9: PKP2, suggesting a reduction in intracellular T AG-derived fatty acid mobilization (FIGS. IB, ID, and 3B).

[0189] Furthermore, in agreement with reduced glycolysis and glucose oxidation in human ARVC heart, the cKO heart showed increased L-lactate (FIG. 3C) and reduced mRNA of Pfkm (phosphofructokinase, muscle) and Ldhd (lactate dehydrogenase D) (FIG. 1C), and Mpc1 / 2 (mitochondrial pyruvate carrier 1 and 2), suggesting less pyruvate generated and / or less lactate was oxidized to pyruvate. Impaired TCA (tricarboxylic acid) cycle was demonstrated by reduced citrate, malate, and fumarate and increased AKG (a-ketoglutarate) with corresponding reduced mRNA of Idh2 (isocitrate dehydrogenase 2) and Bcatl (branched chain amino acid transaminase 1) (FIG. 3B).

[0190] Perturbed energy metabolism, particularly impaired FAO and increased de novo lipid synthesis, was further investigated by shotgun lipidomics to confirm increased TAGs that was normalized in AAV9: PKP2 treated cKO heart (FIG. 3D). TAG class average and TAG 54:2 species average reached statistical significance between cKO and CKO treated with AAV9: PKP2. Detailed analyses of all TAG species showed specific increases in TAGs with 50-56 carbon numbers and 2-5 double-bonds (FIG.3E) Lipidomics profiling of 685 plasma samples identified lipid species with a low carbon number and double-bond content, for example, TAG (54:2), to predict risk of cardiovascular disease in the prospective population-based Bruneck study.

[0191] Additional integrated omics were performed between metabolomics and RNA sequencing in response to a shorter-term 10-week treatment of AAV9: PKP2 in the preventive mode (FIGS. 4A-4D). The significantly changed metabolites identified in the preventive mode of treatment showed a partial overlap with the metabolites identified in therapeutic mode. This observation suggested that reversal of metabolic remodeling before and after overt structural changes by AAV9. PKP2 likely depends on the extent of the remodeling and the metabolic profiling at a given intervention time reflects a snapshot of metabolic changes.Example 3: Acute PKP2 silencing disrupted lipid homeostasis and led to increased intracellular lipid granules in iPSC-CMs.

[0192] A regulated balance between fatty acid uptake, TAG dynamics, and FAO ensures effective fatty acid energy metabolism and proper heart function. Intracellular fatty acid homeostasis, specifically fatty acid uptake, fatty acid storage, and fatty acid mobilization from the-86- IPTS / 2.00262535.1TAG pool in response to acute PKP2 silencing and other metabolic modulators in iPSC-CMs were examined (FIG. 5A).

[0193] First, nontargeted polar metabolomics showed trending reduced or reduced long- chain acylcarnitines, palmitoylcarnitine, oleoyl carnitine, and stearoylcamitine, in iPSC-CM in response to acute silencing of PKP2 (FIG. 5B). Correspondingly, a real-time fluorescence-based lipid uptake assay was used to analyze fatty acid uptake (FAU, green) kinetics and a fixed-cell based lipid droplet dye (LS610, red) to quantify lipid storage. After performing real-time FAU assay, iPSC-CMs were fixed and both green and red channels were imaged (FIG. 5C) and quantified by lipid granule counts (FIG. 5D). Acute silencing of PKP2 led to a right-shift upwards of the total numbers of lipid granules positively stained with LS610 (FIG. 5D) using Analysis of covariance (ANCOVA). Real-time kinetic readouts on the maximum FAU rate normalized to cell counts did not respond to acute PKP2 silencing (FIG. 5E, the left panel). In addition, short-term treatment of iPSC- CMs with small molecules such as isoproterenol (non-selective P-adrenergic agonist), dobutamine (Pl -adrenergic agonist), and danicamtiv (cardiac myosin activator) did not alleviate increased lipid granules in response to acute silencing of PKP2 as compared to siNeg control in the same treatment (FIG. 5E, the right panel).

[0194] The nature of these increased lipid granules in the presence of PKP2 depletion was further investigated by small-molecule metabolic modulators. Inhibition of the oxidation of long-chain fatty acids within mitochondria by etomoxir, an irreversible inhibitor of CPT1, exacerbated intracellular lipid granules by 30% relative to the DMSO-treated siPKP2 cells (FIG. 5F). The extent of increased lipid granules in response to acute silencing of PKP2 was comparable to acute silencing of ACADVL, encoding very long-chain specific acyl-CoA dehydrogenase that catalyzes the first step of FAO in mitochondria (FIG. 5G). Increased lipid granules observed by either depleting PKP2 or ACADVL can be alleviated by glucose starvation up to 45% in siPKP2 cells, putatively via increasing fatty acid mobilization from TAG storage to fuel FAO (FIG. 5G). Moderate concentration of phenformin inhibited mitochondrial complex 1 and did not exacerbate lipid granules (FIG. 5G). These lipid granules were revealed to be TAGs by nature as treatment of a highly selective DGAT1 (diacylglycerol acyltransf erase 1) inhibitor, AZD7687, reduced these lipid granules in a dose-dependent manners with almost 70% at the highest dose of 5 pM (FIG. 5H). Therefore, increased TAGs observed in Pkp2-cKO mouse heart tissue was recapitulated as increased intracellular TAGs in human iPSC-CMs in response to acute PKP2 depletion.-87- IPTS / 2.00262535.1Example 4: Acute PKP2 silencing decreased oxidative and glycolytic metabolism and limited fuel switch in human iPSC-CMs.

[0195] Both mouse heart tissue and human iPSC-CMs revealed impairment in cardiac energy metabolism, particularly disrupted lipid homeostasis, in response to PKP2 deficiency. The study was designed to investigate how impaired energy metabolism altered fuel choices, thus impacting contractile function of human iPSC-CMs (FIG. 6A).

[0196] Seahorse-based bioenergetic analysis was used to evaluate the glycolytic and oxidative state of iPSC-CM in response to acute PKP2 silencing. PKP2 silencing led to a significant reduction in the glycolysis-derived ATP production rate (“glycoATP”). Although the basal mitochondrial respiration-derived ATP production rate (“mitoATP”) was not affected, maximal oxygen consumption rate (Max OCR) was significantly decreased by 25%, confirming impaired glycolytic and oxidative metabolism due to PKP2 silencing (FIG. 6B). However, in the context of FAO inhibition by either etomoxir or ACADVL silencing (FIG. 6C), bioenergetic analysis showed a 13 or 11% reduction in mitoATP rate, respectively, and a 39 or 13% increase in glycoATP rate, respectively (FIG. 6C, the bar graph only showed induced ATP rate). In contrast to impaired oxidative and glycolytic metabolism due to PKP2 deficiency (FIG. 6B), FAO reduction by etomoxir and ACADVL silencing in iPSC-CMs having wild-type PKP2 (FIG. 6C) led to fuel switch to glycolysis.

[0197] Direct measurements of glucose and lactate in the culturing media further confirmed impaired glycolysis with 15% less glucose consumption and a concomitant trending reduction of lactate produced in response to PKP2 silencing as compared to the control, siNeg (at 0 pM phenformin, FIG. 6D). Increasing phenformin, an inhibitor of mitochondrial complex 1, increased glucose usage by tuning down mitochondrial oxidative phosphorylation, and was accompanied with reduced glucose, increased lactate, and enhanced glycolysis in the culturing media for both siNeg and siPKP2 cells, however, with consistently 15% less glucose consumed and 10% less lactate produced in the siPKP2 cells, (at 8-120 pM phenformin, FIG. 6D). PKP2 silenced cardiomyocytes showed no change in mitoATP and a lower glycoATP rate (at 0 pM phenformin, FIG. 6E) and in the presence of increasing phenformin, kept no change in mitoATP rates and a 30%-50% lower glycoATP rates than the control (at 8-120 pM phenformin, FIG. 6E). Phenformin titration showed that iPSC-CMs switched to glycolysis as oxidative phosphorylation was suppressed, and that glycolytic metabolism alone (>80pM phenformin) supported poor contractile function as measured-88- IPTS / 2.00262535.1by reduced beat rate and contraction velocity, both being worse in PKP2 silenced cardiomyocytes, >10% reduced beat rate and <25% reduced contraction velocity, than the siNeg control (FIG. 6F).

[0198] The polar metabolomics of cKO heart tissue (FIG. 3B and 3C) and PKP2-silenced iPSC-CMs (FIG. 6D) revealed opposite effects on lactate levels, with steady-state lactate levels increased in the cKO context but lowered in iPSC-CMs. This apparent discrepancy is consistent with reduction of oxidative mitochondrial metabolism of pyruvate in each system, likely reflecting the documented uptake of circulating lactate by cardiac tissue, a fuel source that is not available to the iPSC-CMs grown in culture. The reduced oxidative metabolism of circulating lactate possibly explained increased lactate levels in the cKO heart tissue whereas reduced glycolysis in iPSC-CMs was an intrinsic cellular response to PKP2 silencing. Thus, this discrepancy was seen to support the shared mechanism of impaired glycolysis and glucose oxidation between cKO mouse heart and human iPSC-CMs. At the cell level, our results also supported that residual glycolytic metabolism was not the optimal energy source to propel contractile function of cardiomyocytes and worsened when desmosome function was corrupted due to PKP2 depletion.Example 5: Isogenic human iPSC-CMs carrying a pathogenic PKP2 mutation showed impaired oxidative and glycolytic metabolism and desmosome-associated dysfunctions.

[0199] Acute depletion of PKP2 helped to understand a ‘rapid’ metabolic response that can be attributed to the metabolic flexibility observed in cardiomyocytes. To understand a longer-term or stable PKP2-associated metabolic alterations that leads to metabolic impairment, heterozygous, PKP2Het, and homozygous, PKP2Hom, isogenic iPSC-CMs carrying a pathogenic mutation c.2146G> C were generated. Differentiation of wild-type, PKP2Hetand PKP2Homisogenic cardiomyocytes was confirmed by cardiac troponin T expression (FIG. 8A). PKP2 and two other desmosome proteins, desmoplakin (DSP) and plakoglobin (PKG), were quantified based on signal intensity of immunofluorescence (FIG. 8A and FIG. 8B). Reduction in PKP2 protein level was associated with reduced DSP and JUP proteins, supporting the observation of a stoichiometric relationship between PKP2 with other desmosome components. Semi -quantitative Western blot confirmed reduced PKP2 protein level in the mutant lines that were less severe in the PKP2Hetline (FIG. 8C and 8D) Functional impact by PKP2 genotypes, PKP2Hetand PKP2Hom, was characterized by contractility (FIG. 8E), electrophysiological properties (FIG. 8F), and levels of long-chain acylcarnitines (FIG. 8G), confirming that PKP2 dosage effects between isogenic PKP2Hetand PKP2HomiPSC-CMs were associated with the majority of functional phenotypes that we-89- IPTS / 2.00262535.1characterized. To emphasize the reversibility of metabolic impairment and subsequent improvement of functional outcomes, for example, contractility, electrophysiological properties, and calcium transient, should be delineated in PKP2Homisogenic cells PKP2Hetcardiomyocytes, expressing some PKP2, could confound data interpretation when these structural functions are intrinsically connected to PKP2 function.

[0200] An about 50% increase in lipid granules were observed in PKP2Homisogenic mutant cells relative to the WT isogenic control (FIG, 9A). Nontargeted polar metabolomics confirmed the same observation seen in the acute PKP2 silencing, that long-chain acyl carnitines, palmitoylcarnitine, oleoylcarnitine, and stearoyl carnitine, were reduced by about 50% in isogenic PKP2Homcells (FIG. 9B). Bioenergetics analyses of isogenic mutant cells showed significantly impaired oxidative and glycolytic metabolism as demonstrated by 50% reductions in glycoATP and mitoATP rates and 50% reduction in and Max OCR (FIG. 9C). In addition, Max OCR was affected less by etomoxir treatment in PKP2Homisogenic cells, glycoATP rate increasing 2 folds in WT vs.1.2 fold in PKP2Homand Max OCR decreasing 70% in WT vs. 50% in PKP2Hom. These results suggested a limited capacity to switch to glycolysis as a result of PKP2 deficiency (FIG. 9C). Early acute silencing of PKP2 did not lead to decreased basal mitoATP rates (FIG. 6B). Measurement of basal mitoATP rates in PKP2Homisogenic cells showed inconsistency as compared to other mitochondrial and glycolytic bioenergetic parameters, Max OCR and glycoATP rates.

[0201] Perturbed electrophysiological properties of monolayers, determined by an extracellular recording of cardiac field potential using Maestro Pro Microelectrode array (MEA, Axion Biosystem), have been reported Compared to the WT isogenic control, PKP2Homisogenic cells showed 50% prolonged field potential duration and 50% depressed spike amplitude and slope and 40% depressed conduction velocity (FIG. 9D and FIG 8D). Contractile function of isogenic lines was characterized in either monolayer or 3D engineered heart tissue (EHT) platform. PKP2Homisogenic cells showed significant impaired contractility than that of WT cells as quantified by: 25% depressed contraction amplitude, 40% depressed contraction velocity, contraction and relaxation time, respectively, for monolayers, and 35% depressed twitch force, 50% depressed contraction and relaxation velocity, respectively, for 3D EHTs (FIG. 9E and FIG. 8E) In addition, calcium transients were also disrupted in the PKP2Homisogenic line compared to the WT isogenic control, as shown by 20% prolonged rise time from 50% to peak (tso-Peak) and 9% increased Ca2+transient width at 50% (CaD50), and a 30% depressed rise rate (FIG. 9F).-90- IPTS / 2.00262535.1

[0202] Since iPSC-CMs used predominantly fatty acids for energy production as shown earlier (FIG. 6B and FIG. 6C), selective FAO disruption by ACADVL silencing altered electrophysiological properties (FIG. 9G and FIG. 7A), contractility (FIG. 9G and FIG. 7B), and Ca2+transients (FIG. 9G and FIG. 7C) that were all functionally connected with cellular structure, supporting the notion that energy metabolism is tightly integrated with structural functions. So far, it was demonstrated that both cKO heart tissue (FIGS 1A-1D, 2A-2C, 3A-3E, and 4A-4D) and human PKP2 isogenic cardiomyocytes (FIG. 9 A, FIG. 9B, and FIG. 9C) showed impaired FAO, glycolysis, and glucose oxidation. Functionally, it was demonstrated how the impaired energy metabolism was manifested as depressed bioenergetics, consequently, supporting poor contractility in cardiomyocytes with compromised PKP2 function (FIG. 9D, FIG. 9E, and FIG. 9F). However, it was not confirmed whether reversing metabolic impairment can improve these structural functions that were compromised due to PKP2 mutation.Example 6: Enhancing oxidative and glycolytic metabolism partially rescued desmosoine-associated functions of PKP2HomiPSC-CMs.

[0203] The reversibility of metabolic impairment and subsequent improvement of functional outcome was delineated by examining contractility, electrophysiological properties, and Ca2+transients in PKP2Homisogenic cells (FIGS. lOA-lOF). As mentioned earlier, PKP2Hetcardiomyocytes, expressing some PKP2, could confound the data interpretation when examining functions such as contractility, calcium transient, and electrophysiological properties that have well understood structural connection to PKP2 and desmosome.

[0204] A serial of metabolic enhancers or repressors, isoproterenol, AZD7687, etomoxir, bezafibrate, seladelpar, and phenformin, were tested to examine their ability to modify contractility of PKP2Homisogenic cells (FIG. 10A) As showed in FIGS. I A-1D, mRNAs of the master regulators of cardiac energy metabolism, PPAR family members, were significantly changed in cKO heart tissue and restored by AAV9: PKP2. Since the three PPAR members each displayed distinct mRNA changes, a pan-PPAR agonist, bezafibrate, was selected used for treating metabolic syndrome with activities in reducing TAG levels and increasing both glucose and fatty acid metabolism as a tool to enhance overall energy metabolism in iPSC-CMs. Although PPAR8 mRNA showed differential responses to the mode of AAV9: PKP2 treatment (FIG. ID and FIG. 2C) in Pkp2-cKO mouse heart, PPAR5 activation is documented in its association with increasing total oxidative metabolism and glycolysis in cardiomyocytes. A highly selective PPAR6 agonist,-91- IPTS / 2.00262535.1seladelpar, was selected to stimulate FAO and glycolysis and glucose oxidation. Isoproterenol, a known chronotropic agent, served as a positive control for modulating bioenergetics, contractile function, electrophysiology, and calcium transient. Compared to isoproterenol treatment, seladelpar significantly improved contraction velocity by 15% and showed a trending improvement (p = 0.06) in beat rate of the PKP2Hommonolayer at 48 hours of drug treatment, in contrast to no response to the pan-PPAR agonist bezafibrate (FIG. 10B) Compared to isoproterenol and seladelpar, AZD7687, etomoxir, or phenformin showed a general trend of suppressing beat rate in both WT and PKP2Homisogenic cells at 48 hours of drug treatment, suggesting metabolic modulation generates distinct functional outcome.

[0205] Limited bioenergetic stimulation was observed in WT or PKP2Homisogenic cells by isoproterenol or seladelpar using the standard Seahorse assays where the assay buffer contains BSA-palmitate as the only source for FAO. Statistical significance was reached when comparing bioenergetic responses within the genotypes at 48 hours of drug treatment. Isoproterenol stimulated basal glycoATP and mitoATP rates by about 20% as well as a 30% increase of Max OCR whereas seladelpar showed bioenergetic stimulation of 15% on Max OCR in PKP2Homisogenic cells (FIG.10C). Similar to the improved contractility observed on the monolayer, stimulated contractility was observed in 3D EHTs of PKP2Homisogenic cells by either isoproterenol or Seladelpar as demonstrated by 15% increased twitch force and 15%-20% contraction and relaxation velocity at either 1 hour or 48 hours of drug treatment (FIG. 10D) Seladelpar-mediated improvement on contractility was mainly observed on PKP2Homnot WT isogenic cells. In contrast to expected response by isoproterenol treatment, seladelpar had little or no effect on electrophysiological properties of PKP2Hommonolayers as characterized by spike amplitude and spike slope of field potential (FIG. 10E) and on calcium transient as characterized by t50-peak and rise rate of monolayers and by t50-peak, rise rate, and Ca2+transient width at 50% (CaD50) of 3D EHTs (FIG.10F).

[0206] Results herein show that promoting PPARδ activity or β-adrenergic response in PKP2Homcardiomyocytes partially increased glycolytic or mitochondrial bioenergetics accompanied with limited improvement in contractility, intriguingly, decoupled from electrophysiological properties and calcium transients in which little or no improvement was observed. These results suggest that desmosome-associated dysfunctions can be partially rescued by metabolic enhancers.. These results revealed a new observation on differential sensitivity among structural functions of-92- IPTS / 2.00262535.1cardiomyocytes to metabolic perturbation. Therefore, energy-responsive contractile function can be asynchronized or decoupled from electrophysiological properties and calcium transient, likely- presenting a higher risk when there is an increased metabolic demand by heart during exercise. Example 7: Increased PKP2 expression by TN-401 improved both bioenergetics and desmosome-associated dysfunctions in PKP2HomiPSC -CMs.

[0207] Monolayers or engineered heart tissues (EHTs) were transduced with TN-401 before the measurement on bioenergetics, desmosome protein expression, iPSC-CM functions in contractility, electrophysiological properties (field potential), and Ca2+transients (FIG. 11A). In response to 15 days of TN-401 treatment of isogenic WT and PKP2Homcells, vehicle / etomoxir (ETO)-induced Max OCR, basal mitoATP and glycoATP rates were quantified using the Seahorse MitoStress test (FIG. 11B). Basal glycolysis, compensatory glycolysis, and %PER from glycolysis were quantified using the Seahorse Glycolytic Rate assay (FIG. 11C). The Seahorse MitoStress test and Seahorse Glycolytic Rate test support that TN-401 treatment is sufficient to rescue glycolytic bioenergetics in PKP2Homcells to nearly WT levels. PKP2Ilombioenergetic parameters were normalized to the average WT value at each corresponding TN-401 treatment due to negative impact of the high MOI (Multiplicity of Infection) of TN-401 on the bioenergetics of both WT and PKP2Homisogenic cells (FIGS. 11D-11E). This normalization revealed a dose-dependent partial rescue of oxidative Max OCR of a 40% increase at 100k MOI relative to the 10k MOI or 60% increase relative to the untreated. This rescue was suppressed by etomoxir treatment, suggesting TN-401 mainly rescued FAO (FIG. 11B), suggesting a functional link between desmosomes and FAO Compared to partially stimulated basal glycoATP rate by pharmacological enhancers (FIG. 10C), TN-401 -mediated restoration of glycolytic bioenergetics suggests a functional link between desmosomes and glycolysis.

[0208] To fully restore essential roles dictated by mechanical and structural integrity of PKP2 and desmosome, TN-401 was administered to PKP2Homisogenic cells, restoring PKP2 expression (FIG. 11F), as determined using immunofluorescence-based detection and quantification of PKP2 and DSP expression in a dose-dependent fashion (FIG. 11G). At equal or more than 100k MOI of TN-401, DSP expression reached a plateau in PKP2Homisogenic cells (p > 0.22) Contractile function of PKP2Homisogenic cells was rescued in a dose-dependent manner by TN-401, measured as displacement and velocity on monolayers and both parameters reached plateau in PKP2Homisogenic cells at equal or more than 100k MOI (p > 0.31) (FIG. 11H). When comparing PKP2Hom-93- IPTS / 2.00262535.1cells treated with TN-401 to WT cells treated with TN-401, a single-dose rescue of contractility at 100k MOI, measured by twitch force and contraction and relaxation velocity, was demonstrated with 3D EHTs (all p > 0.92) (FIG. 111). A single-dose rescue was achieved with monolayers for electrophysiological properties measured by spike amplitude and spike slope (all p > 0.99) (a trending increase in conduction velocity between PKP2Homand PKP2Homtreated with TN-401); and with monolayers for calcium transient measured by time to peak, calcium transient area under the curve, and calcium transient width at 50% (CaD50) (all p > 0.38) (FIG. 11J)

[0209] In addition, a single dose of AAV9: PKP2 renormalized the metabolite profiles and the integrated metabo-transcriptional network of cKO mouse heart (FIGS 1A-1D, 2A-2C, 3A-3E, and 4A-4D). Modeling metabolic impairment of FAO and glycolysis / glucose oxidation using PKP2 isogenic iPSC-CMs enabled investigation of functional link between desmosome dysfunctions and bioenergetic changes. Results disclosed herein indicate that pharmacological enhancers of oxidative and glycolytic metabolism can improve bioenergetics and partially rescue some aspects of contractility with smaller or minimal improvement in electrophysiology (FIG. 10) In contrast, TN-401 dose-range studies demonstrated improvement not only in bioenergetics but also the rescue of desmosome-associated functions such as contractility and electrophysiology of PKP2-deficient iPSC-CMs, supporting that maintaining cardiomyocyte energy metabolism is an integral part of PKP2 and desmosomes (FIG. 12).

[0210] Neither promoting PPAR8 activity nor increasing PKP2 expression by TN-401 was sufficient to completely rescue mitochondrial bioenergetics, possibly suggesting immaturity of the iPSC-CMs in terms of fuel choices and the flexibility of the gene networks that regulate energy metabolism.

[0211] While preferred embodiments of the present disclosure 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 disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.-94- IPTS / 2.00262535.1

Claims

CLAIMS1. A method of measuring a response to plakophilin-2 (PKP2) gene therapy in an individual with arrhythmogenic right ventricular cardiomyopathy (ARVC), comprising: (a) administering the PKP2 gene therapy to the individual; and (b) measuring a metabolic enzyme messenger ribonucleic acid (mRNA) signature in a biological sample of the individual.

2. The method of claim 1, wherein the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and triacylglyceride (TAG) dynamics enzyme gene; a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene; and a cardiac contraction gene.

3. The method of claim 2, wherein the glycolysis and glucose oxidation enzyme gene comprises one or more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hk1, Slc16a3, Slc2a1, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slc16a1, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgk1, Mpc1, Mpc2, and PDHB.

4. The method of claim 2, wherein the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcatl, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fadsl, Fads2, Elovll, EIovl5, Acaca, Plinl, and Meat.

5. The method of claim 2, wherein the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH.

6. The method of claim 2, wherein the transcripti onal regulator of energy metabolism gene comprises one or more of PPAR6, PPARa, PPARv, PPARGCla, Rxra, Ncor2, Gnaq, Nr4al, and Prdml 6.

7. The method of claim 2, wherein the mitochondrial dynamics gene comprises one or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS 1.-95- IPTS / 2.00262535.

18. The method of claim 2, wherein the cardiac contraction gene comprises one or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1.

9. The method of any one of claims 1 to 8, further comprising measuring at least one metabolite in the biological sample of the individual.

10. The method of claim 9, wherein the at least one metabolite comprises acylcarnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate.

11. The method of claim 9, wherein the at least one metabolite comprises a lipid.

12. The method of claim 11, wherein the lipid comprises a triacylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds.

13. The method of claim 11 or claim 12, wherein the lipid comprises TAG (54:2).

14. The method of claim 9, wherein the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component.

15. The method of claim 14, wherein the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2; prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12,13-EpOME.16 The method of any one of claims 9 to 15, wherein the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, di acyl glycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin.

17. The method of any one of claims 9 to 16, wherein the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine.-96- IPTS / 200262535.

118. The method of any one of claims 1 to 17, wherein the PKP2 gene therapy comprises a gene therapy vector comprising a nucleic acid encoding a PKP2 polypeptide or a fragment thereof operatively linked to at least one promoter.

19. The method of claim 18, wherein the gene therapy vector comprises a viral vector.

20. The method of claim 19, 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, and a herpes vims.

21. The method of any one of claims 18 to 20, wherein the gene therapy vector is an adeno-associated virus.

22. The method of claim 20 or claim 21, wherein the adeno-associated virus is an AAV6, an AAV8, an AAV9, an AAVrh7, an AAVrhl 0, a derivative, or a pseudotype thereof.

23. The method of any one of claims 20 to 22, wherein the adeno-associated virus is an AAV9.

24. The method of claim 22 or claim 23, wherein the AAV9 comprises a genome comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 7.

25. The method of any one of claims 18 to 24, wherein the gene therapy vector targets cells in the myocardium, the epicardium, or both.

26. The method of any one of claims 18 to 25, wherein the promoter is a cardiac specific promoter.

27. The method of claim 26, wherein the cardiac specific promoter directs gene expression in the myocardium, the epicardium, or both.

28. The method of claim 26 or claim 27, wherein the cardiac specific promoter is a troponin promoter or an alpha-myosin heavy chain promoter.

29. The method of claim 28, wherein the troponin promoter has a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 3.

30. The method of any one of claims 18 to 25, wherein the promoter is a PKP2 promoter.-97- IPTS / 200262535.

131. The method of claim 30, wherein the PKP2 promoter has a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 4.32 The method of any one of claims 18 to 25, wherein the promoter is a constitutive promoter33. The method of claim 32, wherein the constitutive promoter is a beta-actin promoter.

34. The method of any one of claims 18 to 33, wherein the gene therapy vector further comprises a cardiac specific enhancer.

35. The method of claim 34, wherein the cardiac specific enhancer comprises a LMNA enhancer or a MYH7 enhancer.

36. The method of any one of claims 18 to 35, wherein the gene therapy vector further comprises a 3’ element.

37. The method of claim 36, wherein the 3’ element comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

38. The method of any one of claims 18 to 37, wherein the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 95% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.39 The method of any one of claims 18 to 38, wherein the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 95% identity to the sequence of SEQ ID NO. 2.

40. The method of any one of claims I 8 to 39, wherein the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 99% identity to the sequence of SEQ ID NO: 2.

41. The method of any one of claims 18 to 40, wherein the nucleic acid encoding the PKP2 polypeptide has the sequence of SEQ ID NO: 2.

42. The method of any one of claims 18 to 41, wherein the gene therapy vector comprises an expression cassette comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 5.-98- IPTS / 2.00262535.

143. The method of any one of claims 1 to 42, wherein the individual has at least one mutant copy of a PKP2 gene.44 The method of any one of claims 1 to 43, wherein the PKP2 gene therapy is administered intravenously, intracardially, pericardially, or intraarterially.

45. The method of any one of claims 1 to 44, wherein the mRNA signature or the at least one metabolite is measured about one week, about one month, about two months, about six months, or about one year after administering the PKP2 gene therapy to the individual46. The method of any one of claims 1 to 45, wherein the biological sample comprises cardiac tissue, blood, serum, or plasma.

47. A method of measuring a response to plakophilin-2 (PKP2) gene therapy in an individual with arrhythmogenic right ventricular cardiomyopathy (ARVC), comprising: (a) administering the PKP2 gene therapy to the individual; and (b) measuring at least one metabolite in a biological sample of the individual.

48. The method of claim 47, wherein the at least one metabolite comprises acylcarnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate.

49. The method of claim 47, wherein the at least one metabolite comprises a lipid,50. The method of claim 49, wherein the lipid comprises a triacylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds.

51. The method of claim 49 or claim 50, wherein the lipid comprises TAG (54:2).

52. The method of claim 47, wherein the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component.

53. The method of claim 52, wherein the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2, prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12,13-EpOME.-99- IPTS / 2.00262535.

154. The method of any one of claims 47 to 53, wherein the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin.

55. The method of any one of claims 47 to 54, wherein the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine.

56. The method of any one of claims 47 to 55, further comprising measuring at least one metabolic enzyme messenger ribonucleic acid (mRNA) signature in the biological sample of the subject.

57. The method of claim 56, wherein the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and tri acylglyceride (TAG) dynamics enzyme gene, a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene; and a cardiac contraction gene.

58. The method of claim 57, wherein the glycolysis and glucose oxidation gene comprises one or more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hkl, Slcl6a3, Slc2al, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slcl6al, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgkl, Mpc1, Mpc2, and PDHB.

59. The method of claim 57, wherein the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36, ACSL1, ACSL6, CPT1A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcat1, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fadsl, Fads2, Elovll, Elovl5, Acaca, Plinl, and Meat.

60. The method of claim 57, wherein the transcriptional regulator of energy metabolism gene comprises one or more of PPAR5, PPARa, PPARy, PPARGCla, Rxra, Ncor2, Gnaq, Nr4al, and Prdml6.-100- IPTS / 2.00262535.

161. The method of claim 57, wherein the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH.

62. The method of claim 57, wherein the mitochondrial dynamics gene comprises one or more of OPA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS 1.

63. The method of claim 57, wherein the cardiac contraction gene comprises one or more of ACTA1, MYH7, TNNT2, MYBPC3, TNN13K, MYFI6, MYL2, TNNI3, and TNNC1.

64. The method of any one of claims 47 to 63, wherein the PKP2 gene therapy comprises a gene therapy vector comprising a nucleic acid encoding a PKP2 polypeptide or a fragment thereof operatively linked to at least one promoter.

65. The method of claim 64, wherein the gene therapy vector comprises a viral vector66. The method of claim 65, 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, and a herpes virus.

67. The method of any one of claims 64 to 66, wherein the gene therapy vector is an adeno-associated virus.

68. The method of claim 66 or claim 67, wherein the adeno-associated virus is an AAV6, an AAV8, an AAV9, an AAVrh7, an AAVrhlO, a derivative, or pseudotype thereof.

69. The method of any one of claims 66 to 68, wherein the adeno-associated virus is an AAV9.

70. The method of claim 68 or claim 69, wherein the AAV9 comprises a genome comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 7.

71. The method of any one of claims 64 to 70, wherein the gene therapy vector targets cells in the myocardium, the epicardium, or both.

72. The method of any one of claims 64 to 71, wherein the promoter is a cardiac specific promoter.-101- IPTS / 2.00262535.

173. The method of claim 72, wherein the cardiac specific promoter directs gene expression in the myocardium, the epicardium, or both.74 The method of claim 72 or claim 73, wherein the cardiac specific promoter is a troponin promoter or an alpha-myosin heavy chain promoter.

75. The method of claim 74, wherein the troponin promoter has a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 3.

76. The method of any one of claims 64 to 71, wherein the promoter is a PKP2 promoter.

77. The method of claim 76, wherein the PKP2 promoter has a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO: 4.

78. The method of any one of claims 64 to 71, wherein the promoter is a constitutive promoter.

79. The method of claim 78, wherein the constitutive promoter is a beta-actin promoter.

80. The method of any one of claims 64 to 79, wherein the gene therapy vector further comprises a cardiac specific enhancer.

81. The method of claim 80, wherein the cardiac specific enhancer comprises a LMNA enhancer or a MYH7 enhancer.

82. The method of any one of claims 64 to 81, wherein the gene therapy vector further comprises a 3’ element.83 The method of claim 82, wherein the 3’ element comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a bovine growth hormone polyadenylation (bGH poly A) sequence, or a combination thereof.

84. The method of any one of claims 64 to 83, wherein the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 95% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.-102- IPTS / 2.00262535.

185. The method of any one of claims 64 to 84, wherein the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 95% identity to the sequence of SEQ ID NO: 2.86 The method of any one of claims 64 to 85, wherein the nucleic acid encoding the PKP2 polypeptide has a sequence having at least 99% identity to the sequence of SEQ ID NO: 2.

87. The method of any one of claims 64 to 86, wherein the nucleic acid encoding the PKP2 polypeptide has the sequence of SEQ ID NO: 2.

88. The method of any one of claims 64 to 87, wherein the gene therapy vector comprises an expression cassette comprising a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO:

589. The method of any one of claims 47 to 88, wherein the individual has at least one mutant copy of a PKP2 gene.

90. The method of any one of claims 47 to 89, wherein the gene therapy is administered intravenously, intracardially, pericardially, or intraarterially.

91. The method of any one of claims 47 to 90, wherein the mRNA signature or the at least one metabolite is measured about one week, about one month, about two months, about six months, or about one year after administering the PKP2 gene therapy to the individual92. The method of any one of claims 47 to 91, wherein the biological sample comprises cardiac tissue, blood, serum, or plasma.

93. A method of identifying an individual with arrhythmogenic right ventricular cardiomyopathy (ARVC), comprising measuring a metabolic enzyme messenger ribonucleic acid (mRNA) signature or at least one metabolite in a biological sample of the individual.

94. The method of claim 93, wherein the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and triacylglyceride (TAG) dynamics enzyme gene; a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene; and a cardiac contraction gene.-103- IPTS / 2.00262535.

195. The method of claim 94, wherein the glycolysis and glucose oxidation gene comprises one or more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hk1, Slc16a3, Slc2a1, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slc16a1, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, Idh2, Pfkm, Pgk1, Mpc1, Mpc2, and PDHB.

96. The method of claim 94, wherein the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36, ACSL1, ACSL6, CPT1 A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Bcatl, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fadsl, Fads2, Elovll, Elovl5, Acaca, Plin 1, and Meat.

97. The method of claim 94, wherein the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH.

98. The method of claim 94, wherein the transcriptional regulator of energy metabolism gene comprises one or more of PPAR8, PPARa, PPARy, PPARGCla, Rxra, Ncor2, Gnaq, Nr4al, and Prdml6.

99. The method of claim 94, wherein the mitochondrial dynamics gene comprises one or more of OP Al, MFN2, MFN1, MFF, MEDI2, NFE2L2, and FIS1.

100. The method of claim 94, wherein the cardiac contraction gene comprises one or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1.

101. The method of claim 94, wherein the at least one metabolite comprises acylcarnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate.

102. The method of claim 94, wherein the at least one metabolite comprises a lipid.

103. The method of claim 102, wherein the lipid comprises a triacylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds.

104. The method of claim 102 or claim 103, wherein the lipid comprises TAG (54:2).-104- IPTS / 2.00262535.1105. The method of claim 94, wherein the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component.

106. The method of claim 105, wherein the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2, prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12,13-EpOME.

107. The method of any one of claims 94 to 106, wherein the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidyl serine, or sphingomyelin.

108. The method of any one of claims 94 to 107, wherein the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine.

109. The method of any one of claims 94 to 108, wherein the biological sample comprises cardiac tissue, blood, serum, or plasma.

110. A kit for use in the method of any one of 1-109.

111. A kit for measuring a metabolic enzyme messenger ribonucleic acid (mRN A) signature and / or at least one metabolite in a biological sample of an individual with arrhythmogenic right ventricular cardiomyopathy (AKVC) receiving plakophilin-2 (PKP2) gene therapy.

112. The kit of claim 111, wherein the metabolic enzyme mRNA signature comprises mRNA levels of one or more of a glycolysis and glucose oxidation enzyme gene; a fatty acid synthesis and oxidation and triacylglyceride (TAG) dynamics enzyme gene; a pro-inflammatory eicosanoid signaling pathway gene; a transcriptional regulator of energy metabolism gene; a mitochondrial dynamics gene; and a cardiac contraction gene.113 The kit of claim 112, wherein the glycolysis and glucose oxidation enzyme gene comprises one or more of SLC2A1, SLC2A4, SLC16A1, SCL16A3, MPC1, MPC2, PDHA1, Hkl, Slcl6a3,-105- IPTS / 2.00262535.1Slc2al, Prkacb, Pdk4, Suclg2, Sdhd, Cs, Slcl6al, Pdhb, Hk2, Sdha, Slc2a4, Eno3, Ldhd, ldh2, Pfkm, Pgkl, Mpcl, Mpc2, and PDHB.

114. The kit of claim 112, wherein the fatty acid synthesis and oxidation and TAG dynamics enzyme gene comprises one or more of CD36, ACSL1, ACSL6, CPT1 A, CPT1B, CPT2, DGAT1, DGAT2, ACLY, PLIN2, Hmgcl, Hmgcs2, Acsl4, Acsl3, Acaa2, Acot2, Slc25a20, Hadh, Hadhb, Acadl, Hadha, Crat, Acadm, Fasn, Beat, Fabp4, Mgll, Ffar2, Abhd5, Cidea, Fabp3, Lpl, Pnpla2, G0s2, Bcat2, Fadsl, Fads2, Elovll, Elovl5, Acaca, Plinl, and Meat.

115. The kit of claim 112, wherein the pro-inflammatory eicosanoid signaling pathway gene comprises one or more of ACSL6, FADS2, CYP2U1, GSTK1, GSTA3, COX-1, COX-2, DHRS4, PTGES, HPGDS, TBXAS1, and sEH.

116. The kit of claim 112, wherein the transcriptional regulator of energy metabolism gene comprises one or more of PPAR8, PPARa, PPARy, PPARGC la, Rxra, Ncor2, Gnaq, Nr4al, and Prdml6.

117. The kit of claim 112, wherein the mitochondrial dynamics gene comprises one or more of 0PA1, MFN2, MFN1, MFF, MED12, NFE2L2, and FIS1.

118. The kit of claim 112, wherein the cardiac contraction gene comprises one or more of ACTA1, MYH7, TNNT2, MYBPC3, TNNI3K, MYH6, MYL2, TNNI3, and TNNC1.

119. The kit of claim 111, wherein the at least one metabolite comprises acyl carnitine, L-lactate, citrate, malate, fumarate, and / or a-ketoglutarate120. The kit of claim 111, wherein the at least one metabolite comprises a lipid.

121. The kit of claim 120, wherein the lipid comprises a tri acylglyceride (TAG) with 50-56 carbon numbers and 2-5 double bonds.

122. The kit of claim 120 or claim 121, wherein the lipid comprises TAG (54:2).

123. The kit of claim 111, wherein the at least one metabolite comprises a pro-inflammatory eicosanoid signaling pathway component.-106- IPTS / 200262535.1124. The kit of claim 123, wherein the pro-inflammatory eicosanoid signaling pathway component comprises one or more of prostaglandin A2; prostaglandin D2; prostaglandin E2; prostaglandin F2alpha; prostaglandin H2; malonic dialdehyde (MDA); 9,10-EpOME; or 12,13-EpOME125. The kit of claim 111, wherein the at least one metabolite comprises acylcarnitine, a cholesterol ester, a ceramide, cholesterol, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, diacylglycerol, a free fatty acid, hexosylceramide, lactosylceramide, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, or sphingomyelin.

126. The kit of claim 111, wherein the at least one metabolite comprises palmitoylcarnitine, oleoylcarnitine, or stearoylcarnitine.-107- IPTS / 2.00262535.1

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