Gene therapy for arrhythmogenic cardiomyopathy

Abstract

The present disclosure provides nucleic acids (including AAV expression cassettes), AAV vectors, and compositions for use in methods for treating and / or delaying the onset of diseases associated with mutations in genes such as PKP2, arrhythmogenic cardiomyopathy. Also provided herein are methods for treating and / or delaying the onset of arrhythmogenic cardiomyopathy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 383,639, filed November 14, 2022, and U.S. Provisional Application No. 63 / 311,840, filed February 18, 2022, the entire contents of each of which are incorporated herein by reference for all purposes.

[0002] Incorporating a sequence listing The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Computer-readable copy of the Sequence Listing (STRD_026_02WO_SeqList_ST26.xml, date of recording: February 6, 2023, file size: approximately 44,684 bytes). [Background technology]

[0003] Arrhythmogenic cardiomyopathy is a rare familial disorder that usually manifests in adulthood and can cause ventricular tachycardia (fast heartbeat) and sudden cardiac death in apparently healthy young people. The clinical hallmark of this disease is ventricular arrhythmias (abnormal heart rate) primarily arising from the right ventricle. The pathological hallmark of this disease is fibrofatty replacement of the right ventricular muscle. Symptoms commonly include a flapping or pounding sensation in the chest (palpitations), lightheadedness, and syncope (syncope). Over time, patients may experience shortness of breath and abnormal swelling of the legs or abdomen. Severe damage to the myocardium in the later stages of the disease can lead to heart failure.

[0004] Arrhythmogenic cardiomyopathy is often caused by mutations in genes encoding proteins such as plakophilin-2, which are part of desmosomes. Desmosomes are specialized adhesion protein complexes localized at intercellular junctions that facilitate adhesion between cardiac muscle cells, thereby maintaining the integrity of cardiac muscle tissue. Mutations in genes encoding desmosomal proteins impair desmosome function, leading to myocardial damage and replacement of cardiac tissue with fatty or fibrous tissue.

[0005] The standard of care for arrhythmogenic cardiomyopathy includes symptom management using medications (such as beta-blockers and amiodarone), implantable cardioverter-defibrillators (ICDs), and catheter ablation. Thus, there is a continuing need for disease-modifying therapeutic compositions and methods for treating arrhythmogenic cardiomyopathy. Summary of the Invention

[0006] The present disclosure provides a nucleic acid molecule comprising an adeno-associated virus (AAV) expression cassette, the AAV expression cassette comprising, from 5' to 3', (i) a 5' AAV inverted terminal repeat (ITR), (ii) a promoter, (iii) an arrhythmogenic cardiomyopathy-associated transgene, and (iv) a 3' AAV ITR. In some embodiments, the promoter is capable of expressing the transgene in cardiac cells. In some embodiments, the promoter comprises a cardiac troponin T (TNNT2) promoter, e.g., a TNNT2 promoter comprising the nucleic acid sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto. In some embodiments, the transgene encodes plakophilin-2, e.g., the transgene comprises the nucleic acid sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto. In some embodiments, the AAV expression cassette comprises the nucleic acid sequence SEQ ID NO: 12, or a sequence at least 90% identical thereto.

[0007] The present disclosure further provides a plasmid comprising any one of the nucleic acid molecules disclosed herein, and a cell comprising any one of the nucleic acid molecules or plasmids disclosed herein. Furthermore, the present disclosure provides a method for producing a recombinant AAV vector, comprising contacting an AAV producer cell with any one of the nucleic acid molecules or plasmids disclosed herein. The present disclosure also provides a recombinant AAV vector produced by any one of the methods for producing a recombinant AAV vector disclosed herein. In some embodiments, the recombinant AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the recombinant AAV vector is a self-complementary AAV (scAAV). In some embodiments, the AAV vector comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV.

[0008] In some embodiments, the AAV vector comprises a capsid protein having one or more substitutions or mutations compared to the wild-type AAV capsid protein. For example, in some embodiments, the AAV vector comprises a capsid protein comprising (i) the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto, or (ii) the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto, or (iii) the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15. The disclosure further provides a composition comprising any one of the nucleic acids, any one of the plasmids, any one of the cells, or any one of the recombinant AAV vectors disclosed herein and a pharmaceutically acceptable carrier.

[0009] The present disclosure also provides a method for expressing an arrhythmogenic cardiomyopathy-associated transgene in a cell, the method comprising contacting the cell with any one of the nucleic acids, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein, thereby expressing the arrhythmogenic cardiomyopathy-associated transgene in the cell.

[0010] The present disclosure also provides a method for expressing an arrhythmogenic cardiomyopathy-associated transgene in a tissue, the method comprising contacting the tissue with any one of the nucleic acids, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein, thereby expressing the arrhythmogenic cardiomyopathy-associated transgene in the tissue. In some embodiments, the tissue comprises at least one cell and at least one desmosomal junction.

[0011] In some embodiments, the cell is a cardiac cell, an endothelial cell, a skin cell, a bladder cell, or a gastrointestinal mucosal cell. In some embodiments, the cell is a cardiac cell. In some embodiments, the contacting step is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting step is performed in vivo in a subject in need thereof. In some embodiments, the contacting step comprises administering a therapeutically effective amount of a nucleic acid molecule, a plasmid, a recombinant AAV vector, or a composition to the subject. In some embodiments, the subject is suffering from or at risk of developing an arrhythmogenic cardiomyopathy.

[0012] The present disclosure also provides a method for treating arrhythmogenic cardiomyopathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the nucleic acids, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein, thereby treating the arrhythmogenic cardiomyopathy in the subject. In some embodiments, the arrhythmogenic cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy. In some embodiments, the arrhythmogenic cardiomyopathy is associated with, promoted by, or caused by a genetic mutation. In some embodiments, the genetic mutation comprises a mutation in the PKP2 gene. In some embodiments, the mutation in the PKP2 gene results in PKP2 haploinsufficiency.

[0013] In some embodiments, the method includes reducing the severity of arrhythmogenic cardiomyopathy: delaying its onset or progression; and / or eliminating its symptoms. In some embodiments, the symptoms of arrhythmogenic cardiomyopathy include (a) recurrent ventricular tachycardia, (b) syncope, (c) sudden death, or (d) any combination thereof. In some embodiments, the arrhythmogenic cardiomyopathy is associated with (a) decreased mechanical stability between the subject's cardiomyocytes, (b) disruption of gap junctions in the subject's cardiac tissue, (c) decreased sodium current in the subject's cardiac tissue, (d) fibrosis of the right ventricular muscle, or (e) any combination thereof.

[0014] In some embodiments, the method comprises increasing mechanical stability between cardiac muscle cells in the subject. In some embodiments, the method comprises improving gap junction function in cardiac tissue of the subject. In some embodiments, the method comprises increasing sodium current in cardiac tissue of the subject. In some embodiments, the method comprises reducing fibrosis of right ventricular myocardium in the subject.

[0015] In some embodiments, the subject is a human subject. In some embodiments, the nucleic acid molecule, plasmid, cell, recombinant AAV vector, or composition is administered to the subject's cardiac tissue. In some embodiments, the nucleic acid molecule, plasmid, cell, recombinant AAV vector, or composition is administered to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. In some embodiments, the nucleic acid molecule, plasmid, cell, recombinant AAV vector, or composition is administered to the subject via intravenous administration, intra-arterial administration, intra-aortic administration, direct cardiac injection, coronary artery perfusion, or any combination thereof.

[0016] These and other embodiments are addressed in more detail in the detailed description that follows.

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0018] [Figure 1] A schematic diagram of the AAV expression cassette of SEQ ID NO: 12 is shown, which contains the following elements: (i) the 5' AAV ITR of SEQ ID NO: 5, (ii) the cardiac troponin T (TNNT2) promoter of SEQ ID NO: 4, (iii) the human beta globin (hBG) intron of SEQ ID NO: 7, (iv) the human PKP2a transgene sequence of SEQ ID NO: 2, (v) the bovine growth hormone (bGH) polyA signal of SEQ ID NO: 8, and (vi) the 3' AAV ITR of SEQ ID NO: 6. [Figure 2] A map of the plasmid containing the AAV expression cassette shown in Figure 1 is shown. [Figure 3] (A) shows the fold change in PKP2 mRNA expression relative to the housekeeping gene UBC in normal human iPSC-derived cardiomyocytes that were untreated ("wild-type"), treated with an antisense oligonucleotide (ASO) against PKP2 to knock down expression ("PKP2 KD"), or knocked down and then treated with AAV.STRV47-hTNNT2.PKP2a at a 50K MOI. (B) shows the results of a Western blot of PKP2 protein in wild-type cells, untreated PKP2 KD cells, and PKP2 KD cells treated with AAV.STRV47-hTNNT2.PKP2a at a 50K MOI. (C) illustrates the quantification of the protein band intensity in (B). [Figure 4A] 1 shows the results of Western blot analysis of PKP2a protein expression in wild-type cardiomyocytes and CRISPR-induced PKP2 knockout ("PKP2-KO") iPSC cardiomyocytes that were either untreated or transduced with rAAV encoding hTNNT2.PKP2a (AAV.STRV5-hTNNT2.PKP2a). [Figure 4B]Graph showing quantification of PKP2a protein expression normalized to GAPDH in wild-type cardiomyocytes and PKP2-KO iPSC cardiomyocytes that were untreated or transduced with rAAV encoding hTNNT2.PKP2a (AAV.STRV5-hTNNT2.PKP2a). [Figure 4C] Shown are images from immunofluorescence microscopy of wild-type and PKP2-KO iPSC cardiomyocytes that were either untreated or transduced with rAAV encoding hTNNT2.PKP2a (AAV.STRV5-hTNNT2.PKP2a). [Figure 5] (A) Spontaneous calcium transients measured using FLIPR Calcium6 (SpectraMax® i3x) in wild-type iPSC cardiomyocytes transduced with control AAV.STRV5-Cbh.GFP. (B and C) Spontaneous calcium transients measured using FLIPR Calcium6 (SpectraMax® i3x) in PKP2-KO iPSC cardiomyocytes (C) and in PKP2-KO iPSC cardiomyocytes transduced with control AAV.STRV5-Cbh.GFP (B) demonstrate that loss of PKP2 function resulted in early late transients. D is a graph showing spontaneous calcium transients measured using a FLIPR Calcium6 (SpectraMax® i3x) in PKP2-KO iPSC cardiomyocytes transduced with AAV.STRV5-hTNNT2.PKP2a, demonstrating the absence of early late transients after transduction with AAV.STRV5-hTNNT2.PKP2a. [Figure 6](A) is a graph showing vector copy number (VCN) in cardiac tissue of WT mice 21 days after intravenous injection of AAV.STRV47-hTNNT2.PKP2a (5e13 vg / kg) via the tail vein. (B) is a graph showing PKP2 mRNA levels in cardiac tissue of WT mice 21 days after intravenous injection of AAV.STRV47-hTNNT2.PKP2a (5e13 vg / kg) via the tail vein. (C) is a Western blot showing human PKP2 protein in extracts of cardiac tissue of WT mice 21 days after intravenous injection of AAV.STRV47-hTNNT2.PKP2a (5e13 vg / kg) via the tail vein. (D) illustrates quantification of protein band intensity in Figure 6C. [Figure 7] 1 shows the design for testing treatment of a tamoxifen-inducible cardiac-specific PKP2 knockout mouse model with AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a. [Figure 8A] The combined overall survival of Cohort 1 and Cohort 2 for the indicated groups of mice over time is shown. See also Table B. [Figure 8B] Figure 1 shows the survival rate of untreated PKP2-cKO mice (n=5), mice with normal PKP2 function—i.e., Pkp2 wt / wt), Cre+ ("WT,Cre+"; n=5) and Pkp2 fl / fl;Cre- (FL,Cre-; n=4) controls, and PKP2-cKO mice treated with AAV.STRV5-hTTNT2.PKP2a (n=7), calculated using Kaplan-Meier curves using GraphPad Prism. The study was terminated 54 days post-tamoxifen injection (dpi). [Figure 9A] Graph showing vector copy number (VCN) in liver and heart tissues of PKP2 cardiac knockout mice 70 days after intravenous injection (5e13 vg / kg) of AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a via the tail vein. [Figure 9B]10 is a graph showing PKP2 mRNA levels in liver and heart tissues of PKP2 cardiac knockout mice 70 days after intravenous injection via the tail vein of AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a (5e13 vg / kg). [Figure 10] (A) Western blot showing human PKP2 protein bands in cardiac tissue extracts from individual Group 2 mice (PKP2 wt / wt, Cre+), Group 3 mice (PKP2 fl / fl, Cre-), Group 1 mice (PKP2 fl / fl; Cre+), and treated PKP2 fl / fl, Cre+ mice 70 days after intravenous injection via the tail vein (5e13 vg / kg) of AAV.STRV5-hTNNT2.PKP2a (Group 4) or AAV.STRV84-hTNNT2.PKP2a (Group 5). (B) Total protein loading control. See also Table E in Example 6. [Figure 11] (A-E) Images showing PKP2 localization by immunohistochemistry in PKP2 wt / wt, Cre+ mice (A), PKP2 fl / fl;Cre-, tamoxifen-treated mice (B), PKP2 fl / fl, Cre+ and tamoxifen-treated mice (C), PKP2 fl / fl, Cre+ and tamoxifen-treated mice, and mice treated with 5e13 vg / kg AAV.STRV5-hTNNT2.PKP2a vector (D), or 5e13 vg / kg AAV.STRV84-hTNNT2.PKP2a vector (E). [Figure 12](A-B) Line graphs show the results of biweekly echocardiography quantifying left ventricular ejection fraction (LVEF) (A) and systolic right ventricular area (RV area-S) (B) in control mice with wild-type PKP2 function (WT; Cre+ mice and floxed; Cre- mice), PKP2-cKO mice, and PKP2-cKO mice treated with AAV.STRV5-hTNNT2.PKP2a. (C-D) Bar graphs show a comparison of LVEF (C) and RV area-S (D) in control mice with wild-type PKP2 function (WT; Cre+ mice and floxed; Cre- mice), PKP2-cKO mice, and PKP2-cKO mice treated with AAV.STRV5-hTNNT2.PKP2a at 42 dpi. *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001. [Figure 13]A shows Western blots of cardiac tissue extracts from WT (lane 2), antisense oligonucleotide knockdown in iPSC-derived cardiomyocytes ("-control") (lane 3), PKP2-cKO mice ("-control") (lane 4), cardiomyocyte lysates transfected with AAV.STRV84-hTNNT2.PKP2a ("+control") (lanes 5-13), and PKP2-cardiac knockout mice treated with AAV.STRV5-hTNNT2.PKP2a ("treated") (lanes 14-20). The PKP2 protein band is highlighted by an arrow (expected human size 92-97 kDa, expected mouse PKP2 size 88 kDa). Blue Coomassie indicates total protein loaded in each lane. (B) Quantification of PKP2 protein bands in Western blots generated from mouse heart tissue from PKP2 cardiac knockout mice ("-control"); WT, Cre+ mice ("+control"); FL, Cre- mice ("+control"); and PKP2-cardiac knockout mice treated with AAV.STRV5-hTNNT2.PKP2a ("hTNNT2.PKP2a-treated"). This shows that treatment of PKP2-cardiac knockout mice with AAV.STRV5-hTNNT2.PKP2a restores cardiac PKP2 levels. (C) Vector copy number / µg DNA in heart and liver tissue from PKP2-cKO mice treated with AAV.STRV5-hTNNT2.PKP2a, as measured by qPCR. D is a graph of PKP2a mRNA transcript levels in heart and liver tissues of PKP2-cKO mice treated with AAV.STRV5-hTNNT2.PKP2a, measured by RT-qPCR, showing tissue-specific expression in the heart. ****p<0.0001. [Figure 14]A-D are representative histological images from the right ventricle of hearts from PKP2-cKO mice treated with AAV.STRV84-hTNNT2.PKP2a (D) or AAV.STRV5-hTTNT2.PKP2a (C) compared with control (PKP2-cardiac knockout mice, A) and control (FL, Cre-mice, B) animals. Masson's trichrome images (top row) show collagen in blue. Corresponding H&E images are shown (bottom row). Images demonstrate reduction of RV fibrosis with hTNNT2.PKP2a treatment. [Figure 15] Quantification of fibrosis is shown in Figures 14A-14D. Quantification in Figure 14A is shown in the -control (PKP2-cKO) column. Quantification in Figure 14B is shown in the +control (FL, Cre-) column. Quantification in Figure 14C is shown in the AAV.STRV5-hTNNT2.PKP2a column. Quantification in Figure 14D is shown in the AAV.STRV84-hTNNT2.PKP2a column. +control (wt, Cre+) is an additional +control showing the results of quantification of fibrosis staining in wild-type animals. [Figure 16] Figures A-B show the correlation between dose and vector copy number in the heart or liver for murine PKP2 (mPKP2 (SEQ ID NO: 20)) (A) and human PKP2 (hPKP2 (SEQ ID NO: 2)) (B) transgenes in AAV particles containing a capsid protein having the amino acid sequence of SEQ ID NO: 15. Vector copy number in the liver is approximately 1-2 logs higher than in the heart for both transgenes. [Figure 17] The correlation between dose and mRNA expression of mouse PKP2 (A) and human PKP2 (B) transgenes in both the heart and liver is shown. [Figure 18]Western blot showing mouse PKP2 protein bands in cardiac tissue extracts from mice treated with mPKP2: WT (group 1); fl / fl Cre- (group 2); fl / fl Cre+ (group 3); ultra-low dose (group 4); low dose (group 5); medium dose (group 6); and high dose (group 7). The PKP2a protein band is highlighted with an arrow (expected human size 92-97 kDa, expected mouse PKP2 size 88 kDa). Blue Coomassie indicates total protein loaded in each lane. [Figure 19] Western blot showing human-mouse PKP2 protein bands in cardiac tissue extracts from mice treated with hPKP2: WT (group 1); fl / fl Cre- (group 2); fl / fl Cre+ (group 3); low dose (group 8); and high dose (group 9). PKP2a protein bands are highlighted with arrows (expected human size 92-97 kDa, expected mouse PKP2 size 88 kDa). Blue Coomassie indicates total protein loaded in each lane. [Figure 20] Western blots showing mouse PKP2 protein bands in left lobe liver tissue extracts from mice treated with mPKP2: WT (group 1); fl / fl Cre- (group 2); fl / fl Cre+ (group 3); very low dose (group 4); low dose (group 5); medium dose (group 6); and high dose (group 7). The PKP2a protein band is highlighted with an arrow (expected human size 92-97 kDa, expected mouse PKP2 size 88 kDa). Blue Coomassie indicates total protein loaded in each lane. [Figure 21] Western blot showing human PKP2 protein bands in left lobe liver tissue extracts from mice treated with hPKP2: WT (group 1); fl / fl Cre- (group 2); fl / fl Cre+ (group 3); low dose (group 8); and high dose (group 9). The PKP2a protein band is highlighted with an arrow (expected human size 92-97 kDa, expected mouse PKP2 size 88 kDa). Blue Coomassie indicates total protein loaded in each lane. [Figure 22]Increased survival of ARVC mice (PKP2 fl / fl; Cre+) injected with increasing doses of STRV84-murine PKP2 (A) or STRV84-human PKP2 (B) is shown. [Figure 23A] 1 shows the increase in ejection fraction rescue of ARVC mice compared with doses of mPKP2 (very low, low, medium, and high). [Figure 23B] 1 shows the increased recovery of LV volume in ARVC mice compared with mPKP2 doses (very low, low, medium, and high). [Figure 23C] 1 shows the increased recovery of stroke volume in ARVC mice compared with doses of mPKP2 (very low, low, medium, and high). [Figure 23D] Figure 1 shows the increase in recovery of ARVC mice compared to mPKP2 doses (very low, low, medium, and high) in percent fractional shortening. [Figure 23E] 1 shows the increased recovery of cardiac output in ARVC mice compared with mPKP2 doses (very low, low, medium, and high). [Figure 23F] 1 shows increased recovery in left ventricular outflow tract velocity time integral (LVOT VTI) in ARVC mice compared with doses of mPKP2 (very low, low, medium, and high). [Figure 24A] 1 shows the increase in ejection fraction rescue of ARVC mice compared to low and high doses of mPKP2. [Figure 24B] 1 shows increased recovery in LV volume in ARVC mice compared with mPKP2 doses (low and high). [Figure 24C] 1 shows the increased recovery of stroke volume in ARVC mice compared with low and high doses of mPKP2. [Figure 24D] Figure 1 shows the increase in recovery of ARVC mice compared to mPKP2 doses (low and high) in percent fractional shortening. [Figure 24E] 1 shows increased recovery in cardiac output in ARVC mice compared with mPKP2 doses (low and high). [Figure 24F]1 shows increased recovery in LVOT VTI in ARVC mice compared with mPKP2 doses (low and high). DETAILED DESCRIPTION OF THE INVENTION

[0019] Approximately 40-60% of patients with arrhythmogenic cardiomyopathy have mutations in genes encoding desmosomal proteins, with autosomal dominant mutations in PKP2 (encoding plakophilin-2) being the most common (approximately 50-70%). The estimated prevalence of arrhythmogenic cardiomyopathy associated with PKP2 haploinsufficiency is approximately 1:6,000 to 1:25,000. At the cellular level, reduction or elimination of PKP2 function leads to decreased mechanical stability between cardiomyocytes, disruption of gap junctions (such as those involving connexin 43), and reduced sodium currents. These cellular effects lead to fibrosis of the right ventricular myocardium, which can result in recurrent ventricular tachycardia, syncope, and sudden death.

[0020] PKP2 encodes plakophilin-2, one of three plakophilins expressed in both cardiac progenitor cells and differentiated cardiac myocytes. Plakophilin-2 is also expressed in other cell types with desmosomal connections, such as endothelial cells. Plakophilin-2 is a structural protein of desmosomes, connecting the intermediate filament network of cardiac cells with the intercellular cadherin proteins, desmocollin and desmoglein. Plakophilin-2 also plays a role in recruiting and stabilizing other desmosomal proteins, which contributes to the integrity and function of desmosomes in cardiac cells and, in turn, to the integrity and function of the myocardium.

[0021] The present disclosure provides nucleic acids (including AAV expression cassettes), AAV vectors, and compositions for use in methods for treating and / or delaying the onset of diseases associated with mutations in genes associated with arrhythmogenic cardiomyopathy, such as PKP2. Also provided herein are methods for treating and / or delaying the onset of arrhythmogenic cardiomyopathy.

[0022] definition The following terms are used in the description and appended claims.

[0023] The singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0024] Furthermore, as used herein, the term "about," when referring to a measurable value, such as a quantity such as the length of a polynucleotide or polypeptide sequence, a dose, a time, or a temperature, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0025] Also, as used herein, "and / or" refers to and includes any and all possible combinations of the associated listed items, as well as exclusive combinations when interpreted in the alternative ("or").

[0026] The term "wild-type" is a term of art understood by those skilled in the art and refers to a naturally occurring, typical form of an organism, strain, gene, protein, or characteristic, as distinguished from a mutant or variant form. For example, a wild-type protein is a typical form of a protein that occurs in nature.

[0027] The term "mutant protein" is a term of art understood by those skilled in the art and refers to a protein that is distinguished from the wild-type form of the protein based on the presence of an amino acid modification, such as, for example, an amino acid substitution, insertion, and / or deletion. The term "mutant gene" is a term of art understood by those skilled in the art and refers to a gene that is distinguished from the wild-type form of the gene based on the presence of a nucleic acid modification, such as, for example, a nucleic acid substitution, insertion, and / or deletion. In some embodiments, a mutant gene encodes a mutant protein.

[0028] A "nucleic acid" or "polynucleotide" is a sequence of nucleotide bases, e.g., RNA, DNA, or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides). In some embodiments, nucleic acids of the present disclosure are either single-stranded or double-stranded DNA sequences. Nucleic acids can be 1 to 1,000, 1,000 to 10,000, 10,000 to 100,000, 100,000 to 1 million, or over 1 million nucleotides in length. Nucleic acids generally contain phosphodiester bonds but optionally include nucleic acid analogs, which can have alternative backbones, including, for example, phosphoroamide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidite linkages, and peptide nucleic acid backbones and linkages. Other nucleic acid analogs include those with cationic, nonionic, and non-ribose backbones. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acid. These modifications of the ribose-phosphate backbone may facilitate the addition of labels and increase the stability and half-life of such molecules in physiological environments. The nucleic acids of the present disclosure may be linear or circular (e.g., plasmids).

[0029] As used herein, the term "promoter" refers to one or more nucleic acid control sequences that direct the transcription of an operably linked nucleic acid. A promoter may include a nucleic acid sequence near the start site of transcription, such as a TATA element. A promoter may also include cis-acting polynucleotide sequences that can be bound by transcription factors.

[0030] A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to the functional linkage between a nucleic acid expression control sequence (such as a promoter or an array of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0031] An "AAV expression cassette" is a nucleic acid that is packaged into a recombinant AAV vector and contains sequences encoding one or more transgenes. When the AAV vector contacts a target cell, the transgenes are expressed by the target cell.

[0032] As used herein, the terms "viral vector," "viral vector," or "gene delivery vector" refer to a viral particle that functions as a nucleic acid delivery vehicle and contains a nucleic acid (e.g., an AAV expression cassette) packaged within the virion. Exemplary viral vectors of the present disclosure include adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and retroviral vectors.

[0033] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV1, AAV2, AAV3 (including 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh8, AAVrh10, AAVrh74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other currently known or later discovered AAV. See Table 1.

[0034] [Table 1-1] [Table 1-2]

[0035] The terms "viral producer cells," "viral producer cell lines," or "viral producer cells" refer to cells used to produce viral vectors. HEK293 and 239T cells are common viral producer cell lines. Table 2 below lists exemplary viral producer cell lines for various viral vectors.

[0036] [Table 2]

[0037] "HEK293" refers to a cell line originally derived from human embryonic kidney cells grown in tissue culture. The HEK293 cell line grows easily in culture and is commonly used for virus production. As used herein, "HEK293" can also refer to one or more mutant HEK293 cell lines, i.e., cell lines derived from the original HEK293 cell line that further contain one or more genetic alterations. Many mutant HEK293 lines have been developed and optimized for one or more specific applications. For example, the 293T cell line contains the SV40 large T antigen, which allows episomal replication of transfected plasmids containing the SV40 origin of replication, leading to increased expression of the desired gene product.

[0038] "Sf9" refers to an insect cell line that is a clonal isolate derived from the parent Spodoptera frugiperda cell line IPLB-Sf-21-AE. Sf9 cells can be grown in the absence of serum and can be cultured adherently or in suspension.

[0039] "Transfection reagent" refers to a composition that facilitates the transfer of nucleic acids into cells. Some transfection reagents commonly used in the art contain nucleic acids and one or more lipids that bind to the cell surface (e.g., Lipofectamine™).

[0040] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant across the entire window of alignment of components, e.g., nucleotides or amino acids. The "percent identity" for an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The "percent identity" is the percent identity multiplied by 100. The degree of identity (homology) between two sequences can be determined using computer programs and mathematical algorithms. The percent identity can be calculated using the alignment program Clustal Omega, available at www.ebi.ac.uk / Tools / msa / clustalo, using default parameters. See Sievers et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.” (2011, October 11) Molecular systems biology 7:539.

[0041] As used herein, "treatment" or "treating" or "alleviating" or "ameliorating" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement or effect in one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, compositions can be administered to subjects at risk of developing a particular disease, condition, or symptom, or to subjects who report one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0042] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a vertebrate, such as a mammal. A mammal can be, for example, a mouse, rat, rabbit, cat, dog, pig, sheep, horse, non-human primate (e.g., cynomolgus monkey, chimpanzee), or human. Also encompassed are subject tissues, cells, or derivatives thereof obtained in vivo or cultured in vitro. A human subject can be an adult, teenager, child (2-14 years), infant (1-24 months), or newborn (up to 1 month). In some embodiments, the adult is about 65 years of age or older, or an elderly person about 60 years of age or older. In some embodiments, the subject is a pregnant woman or a woman contemplating pregnancy.

[0043] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to achieve a result, e.g., to bring about a beneficial or desired result. A therapeutically effective amount may vary depending on one or more of the subject and condition being treated, the subject's weight and age, the severity of the condition, the mode of administration, etc., which can be readily determined by one of ordinary skill in the art. The specific dose may vary depending on one or more of the particular agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system employed.

[0044] As used herein, the term "gene therapy" refers to the process of introducing genetic material into cells to compensate for an abnormal gene or to make a therapeutic protein.

[0045] As used herein, "left ventricular ejection fraction" refers to a measurement expressed as a percentage of the amount of blood pumped by the left ventricle with each contraction. For example, an ejection fraction of 60% means that 60% of the total amount of blood in the left ventricle is pushed out with each heartbeat. Left ventricular ejection fraction can be calculated based on the results of tests such as an echocardiogram, a MUGA scan, a CAT scan, cardiac catheterization, and a nuclear stress test.

[0046] As used herein, the term "right ventricular area" refers to measuring right ventricular area using long axis B-mode echocardiography, which measures a two-dimensional plane of the heart oriented along its long axis.

[0047] AAV expression cassette The present disclosure provides nucleic acid sequences comprising one or more adeno-associated virus (AAV) expression cassettes. In some embodiments, the AAV expression cassette comprises a 5' inverted terminal repeat (ITR), a promoter, a transgene, and a 3' ITR. In some embodiments, the transgene is an arrhythmogenic cardiomyopathy-associated gene. In some embodiments, the AAV expression cassette comprises a Kozak sequence, a polyadenylation sequence, and / or a stuffer sequence.

[0048] In some embodiments, the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 12, or a sequence at least 70% identical thereto (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical thereto (including all values ​​and subranges therebetween)). In some embodiments, the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 12.

[0049] (i) inverted terminal repeats Inverted terminal repeat (ITR) sequences mediate AAV proviral integration and packaging of AAV DNA into virions. ITRs are involved in various activities in the AAV life cycle. For example, ITR sequences, which can form hairpin structures, play a role in excision from plasmids, vector genome replication, and integration and rescue from the host cell genome.

[0050] The AAV expression cassette of the present disclosure can include a 5' ITR and a 3' ITR. The ITR sequences can be about 110 to about 160 nucleotides in length, e.g., 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, or 160 nucleotides in length. In some embodiments, the ITR sequences can be about 141 nucleotides in length. In some embodiments, the 5' ITR is the same length as the 3' ITR. In some embodiments, the 5' ITR and the 3' ITR have different lengths. In some embodiments, the 5' ITR is longer than the 3' ITR, and in other embodiments, the 3' ITR is longer than the 5' ITR.

[0051] The ITRs can be isolated or derived from the genome of any AAV, such as those listed in Table 1. In some embodiments, at least one of the 5' and 3' ITRs can be isolated or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, at least one of the 5' and 3' ITRs can be a wild-type or mutant ITR isolated or derived from a member of another parvovirus species other than AAV. For example, in some embodiments, the ITRs can be a wild-type or mutant ITR isolated or derived from bocavirus or parvovirus B19.

[0052] In some embodiments, the ITRs comprise a modification to facilitate scAAV production. In some embodiments, the modification to facilitate scAAV production is the deletion of a terminal resolution sequence (TRS) from the ITR. In some embodiments, the 5' ITR is a wild-type ITR and the 3' ITR is a mutant ITR lacking a terminal resolution sequence. In some embodiments, the 3' ITR is a wild-type ITR and the 5' ITR is a mutant ITR lacking a terminal resolution sequence. In some embodiments, the terminal resolution sequence is absent from both the 5' ITR and the 3' ITR. In other embodiments, the modification to facilitate scAAV production is the replacement of an ITR with a different hairpin-forming sequence, for example, a short hairpin (sh) RNA-forming sequence.

[0053] In some embodiments, the 5' ITR comprises the sequence of SEQ ID NO:5, or a sequence at least 70% identical thereto (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween). In some embodiments, the 3' ITR comprises the sequence of SEQ ID NO:6, or a sequence at least 70% identical thereto (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween). In some embodiments, the 5' ITR comprises the sequence of SEQ ID NO:5, and the 3' ITR comprises the sequence of SEQ ID NO:6.

[0054] In some embodiments, the AAV expression cassette includes one or more "surrogate" ITRs, i.e., non-ITR sequences that perform the same function as the ITRs. See, for example, Xie, J. et al., Mol. Ther., 25(6):1363-1374 (2017). In some embodiments, the ITRs in the AAV expression cassette are replaced by surrogate ITRs. In some embodiments, the surrogate ITRs include hairpin-forming sequences. In some embodiments, the surrogate ITRs are shRNA-forming sequences.

[0055] (ii) Promoter In some embodiments, the AAV expression cassettes described herein comprise a promoter. In some embodiments, the promoter is a synthetic promoter. In some embodiments, the promoter may comprise a nucleic acid sequence derived from an endogenous promoter and / or enhancer.

[0056] In some embodiments, the promoter comprises a nucleic acid sequence derived from one or more promoters commonly used in the art for gene expression. For example, in some embodiments, the promoter further comprises a nucleic acid sequence derived from a CMV promoter, an SV40 early promoter, an SV40 late promoter, a metallothionein promoter, a mouse mammary tumor virus (MMTV) promoter, a Rous sarcoma virus (RSV) promoter, a polyhedrin promoter, a chicken beta-actin (CBA) promoter, a dihydrofolate reductase (DHFR) promoter, and a phosphoglycerol kinase (PGK) promoter. In some embodiments, the promoter comprises a nucleic acid sequence derived from a chicken beta-actin (CBA) promoter, an EF-1 alpha promoter, or an EF-1 alpha short promoter.

[0057] In some embodiments, the promoter is capable of expressing a transgene in cardiac cells. In some embodiments, the promoter is a cell-specific promoter, such as a cardiac cell-specific promoter. As used herein, "cell-specific promoter" refers to a promoter that can express a transgene at a higher level in a particular cell (e.g., a cardiac cell) compared to a control cell (e.g., a non-cardiac cell). Thus, in some embodiments, the AAV expression cassettes disclosed herein include a promoter that expresses a transgene at a higher level in cardiac cells than the expression level of the transgene by the promoter in non-cardiac cells. In some embodiments, the promoter causes expression of the transgene in cardiac cells at a level that is at least about 1.2-fold (e.g., about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold, including all values ​​and subranges therebetween) higher than the level of expression of the transgene caused by the promoter in non-cardiac cells.

[0058] In some embodiments, the promoter may comprise a nucleic acid sequence derived from an endogenous promoter and / or enhancer, e.g., an endogenous promoter and / or enhancer of a gene that is expressed at a higher level in cardiac tissue compared to non-cardiac tissue. In some embodiments, the promoter is the promoter of the TNNT2 gene encoding cardiac troponin T, herein referred to as the cardiac troponin T (TNNT2) promoter. In some embodiments, the TNNT2 promoter comprises a nucleic acid sequence derived from (i) a human TNNT2 promoter, (ii) a chicken TNNT2 promoter, (iii) a mouse TNNT2 promoter, or (iv) any combination thereof. In some embodiments, the TNNT2 promoter comprises a human TNNT2 promoter. In some embodiments, the promoter comprises the sequence of SEQ ID NO:4, or a sequence at least 70% identical thereto (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween).

[0059] In some embodiments, the AAV expression cassettes described herein further comprise an enhancer. The enhancer can be, for example, a CMV enhancer. In some embodiments, the enhancer comprises a sequence of SEQ ID NO: 16, or a sequence at least 70% identical thereto (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical thereto, including all values ​​and subranges therebetween).

[0060] In some embodiments, the promoter further comprises a nucleic acid sequence derived from any one or more of the following promoters: HMG-COA reductase promoter, sterol regulatory element 1 (SRE-1), phosphoenolpyruvate carboxykinase (PEPCK) promoter, human C-reactive protein (CRP) promoter, human glucokinase promoter, cholesterol 7-alpha hydroxylase (CYP-7) promoter, beta-galactosidase alpha-2,6 sialyltransferase promoter, insulin-like growth factor binding protein (IGFBP-1) promoter, aldolase B promoter, human transferrin promoter, type I collagen promoter, prostatic acid phosphatase (PAP) promoter, prostate secretory protein 94 (PSP94) promoter, prostate specific antigen complex promoter, human glandular kallikrein gene promoter (hgt-1), myocardium thrombin gene promoter (MHGT-1), myocardium thrombin gene promoter (MYC ... Cell-specific enhancer-binding factor MEF-2, muscle creatine kinase promoter, pancreatitis-associated protein promoter (PAP), elastase 1 transcriptional enhancer, pancreas-specific amylase and elastase enhancer promoter, pancreatic cholesterol esterase gene promoter, uteroglobin promoter, cholesterol side-chain cleavage (SCC) promoter, gamma-gamma enolase (neuron-specific enolase, NSE) promoter, neurofilament heavy chain (NF-H) promoter, human CGL-1 / granzyme B promoter, terminal deoxytransferase (TdT), lambda 5, VpreB, and lck (lymphocyte-specific tyrosine protein kinase p561ck) promoters, human CD2 promoter and its 3' transcriptional enhancer, human NK and T cell-specific activation (NKG5) promoter, pp60c-src tyrosine kinase promoter, organ-specific neoantigen (OSN), mw40 kDa (p40) promoter, colon-specific antigen P promoter, human alpha-lactalbumin promoter, phosphoenolpyruvate carboxykinase (PEPCK) promoter, HER2 / neu promoter, casein promoter, IgG promoter, chorionic embryonic antigen promoter, elastase promoter, porphobilinogen deaminase promoter, insulin promoter, growth hormone factor promoter, tyrosine hydroxylase promoter, albumin promoter, alpha-fetoprotein promoter, acetyl-choline receptor promoter, alcohol dehydrogenase promoter, alpha or beta globin promoter, T cell receptor promoter, osteocalcin promoter, IL-2 promoter, IL-2 receptor promoter, whey (wap) promoter, and MHC class II promoter. In some embodiments, the AAV expression cassettes disclosed herein further comprise nucleic acid sequences derived from any one or more of the promoters, enhancers, and / or other sequences described in U.S. Pat. No. 8,708,948 B2, U.S. Pat. No. 9,138,596 B2, U.S. Pat. No. 10,286,085 B2, and U.S. Pat. No. US8,538,520 B2, the contents of each of which are incorporated herein by reference in their entirety.

[0061] (iii) Arrhythmogenic cardiomyopathy-associated genes As used herein, "arrhythmogenic cardiomyopathy-associated gene" refers to any gene in a subject with arrhythmogenic cardiomyopathy that can be targeted by gene therapy to alleviate at least one symptom of arrhythmogenic cardiomyopathy. In some embodiments, the level of a protein encoded by the arrhythmogenic cardiomyopathy-associated gene is reduced or undetectable in a subject with arrhythmogenic cardiomyopathy. In some embodiments, the arrhythmogenic cardiomyopathy-associated gene encodes a protein that contributes to normal cardiac function. In some embodiments, the arrhythmogenic cardiomyopathy-associated gene encodes a protein that contributes to normal cardiac function. In some embodiments, the arrhythmogenic cardiomyopathy-associated gene encodes a protein that contributes to the normal function of desmosomes in cells that contain desmosomes, such as cardiac cells and epithelial cells.

[0062] In some embodiments, a mutation in an arrhythmogenic cardiomyopathy-associated gene (e.g., the PKP2 gene) is present in a subject with arrhythmogenic cardiomyopathy. In some embodiments, loss of function or haploinsufficiency of an arrhythmogenic cardiomyopathy-associated gene (e.g., the PKP2 gene) is present in a subject with arrhythmogenic cardiomyopathy. In some embodiments, a mutation in an arrhythmogenic cardiomyopathy-associated gene or loss of function of an arrhythmogenic cardiomyopathy-associated gene is associated with, promotes, or causes arrhythmogenic cardiomyopathy. The type of mutation in an arrhythmogenic cardiomyopathy-associated gene (e.g., the PKP2 gene) is not limited and can be an insertion, deletion, duplication, and / or substitution. In some embodiments, the mutation in the PKP2 gene is any PKP2 mutation identified in patients with arrhythmogenic cardiomyopathy. For example, the mutation in the PKP2 gene is selected from one or more of the PKP2 gene mutations described in Gerull B, et al. Mutations in the desmosomal protein plakophilin-2 are common in arrhythmogenic right ventricular cardiomyopathy, Nat Genet. 2004 Nov;36(11):1162-4, which is incorporated by reference in its entirety for all purposes.

[0063] The present disclosure provides an AAV expression cassette comprising an arrhythmogenic cardiomyopathy-associated gene. In some embodiments, the AAV expression cassette comprises an arrhythmogenic cardiomyopathy-associated gene encoding a protein comprising a therapeutic (e.g., medical or veterinary) or immunogenic (e.g., vaccine) polypeptide. In some embodiments, the AAV expression cassette comprises a mammalian arrhythmogenic cardiomyopathy-associated gene. In some embodiments, the AAV expression cassette comprises a human arrhythmogenic cardiomyopathy-associated gene. In some embodiments, the AAV expression cassette comprises an arrhythmogenic cardiomyopathy-associated gene encoding plakophilin-2.

[0064] In some embodiments, the transgene encodes human plakophilin-2. In some embodiments, the transgene encodes human plakophilin-2 isoform 2a. In some embodiments, human plakophilin-2 isoform 2a comprises an amino acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween) to SEQ ID NO: 1. In some embodiments, human plakophilin-2 isoform 2a comprises the amino acid sequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto.

[0065] In some embodiments, the transgene encodes the RNA transcript variant 2a of the PKP2 gene. In some embodiments, the transgene comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO: 2 (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween). In some embodiments, the transgene comprises the nucleic acid sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto.

[0066] In some embodiments, the transgene encodes isoform 2b of human plakophilin-2. In some embodiments, isoform 2b of human plakophilin-2 comprises an amino acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween) to SEQ ID NO:3. In some embodiments, isoform 2b of human plakophilin-2 comprises the amino acid sequence of SEQ ID NO:3, or a sequence at least 90% identical thereto. In some embodiments, the transgene encodes the RNA transcript variant 2b of the PKP2 gene. In some embodiments, the PKP2 gene is a human PKP2 gene.

[0067] In some embodiments, the AAV expression cassette comprises a Kozak sequence. A Kozak sequence is a nucleic acid sequence that functions as a protein translation initiation site in many eukaryotic mRNA transcripts. In some embodiments, the Kozak sequence overlaps the start codon. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween) to the nucleic acid sequence of SEQ ID NO: 10 or acagccacc. In some embodiments, the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 10, or a sequence at least 90% identical thereto, or the nucleic acid sequence of acagccacc, or a sequence at least 90% identical thereto.

[0068] (iv) polyadenylation (polyA) signal A polyadenylation signal is a nucleotide sequence found in nearly all mammalian genes that controls the addition of a stretch of approximately 200 adenosine residues (a poly(A) tail) to the 3' end of a gene transcript. The poly(A) tail contributes to mRNA stability, and mRNA lacking a poly(A) tail is rapidly degraded. There is also evidence that the presence of a poly(A) tail positively contributes to mRNA translatability by influencing translation initiation.

[0069] In some embodiments, the AAV expression cassette of the present disclosure comprises a polyadenylation signal, which may be selected from the polyadenylation signals of simian virus 40 (SV40), rabbit beta globin (rBG), α-globin, β-globin, human collagen, human growth hormone (hGH), polyoma virus, human growth hormone (hGH), and bovine growth hormone (bGH).

[0070] In some embodiments, the AAV expression cassette comprises a bGH polyadenylation signal. In some embodiments, the bGH polyadenylation signal comprises a nucleic acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween) to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the bGH polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 8, or a sequence at least 90% identical thereto.

[0071] In some embodiments, the polyadenylation signal is an SV40 polyadenylation signal. In some embodiments, the polyadenylation signal is an rBG polyadenylation signal. In some embodiments, the polyadenylation signal comprises the sequence of SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the polyadenylation signal comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

[0072] (v) Stuffer array AAV vectors typically accommodate DNA insertions with a defined size range of about 4 kb to about 5.2 kb, or slightly larger. Therefore, for shorter sequences, it may be necessary to include additional nucleic acid in the insert to achieve the required length acceptable to the AAV vector. Thus, in some embodiments, the AAV expression cassettes of the present disclosure may include a stuffer sequence. The stuffer sequence can be, for example, a sequence between 1 and 10, 10 and 20, 20 and 30, 30 and 40, 40 and 50, 50 and 60, 60 and 75, 75 and 100, 100 and 150, 150 and 200, 200 and 250, 250 and 300, 300 and 400, 400 and 500, 500 and 750, 750 and 1,000, 1,000 and 1,500, 1,500 and 2,000, 2,000 and 2,500, 2,500 and 3,000, 3,000 and 3,500, 3,500 and 4,000, 4,000 and 4,500, or 4,500 and 5,000, or more nucleotides in length. The stuffer sequence can be placed at any desired location within the cassette so as not to interfere with the function or activity of the vector.

[0073] In some embodiments, the AAV cassette comprises at least one stuffer sequence. In some embodiments, the stuffer sequence comprises a nucleic acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween) to the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the stuffer sequence comprises the nucleic acid sequence of SEQ ID NO:9, or a sequence at least 90% identical thereto. In some embodiments, the stuffer sequence comprises the nucleic acid sequence of SEQ ID NO:9, or a portion thereof. In some embodiments, the stuffer sequence comprises a portion (e.g., a 500 nucleotide long portion) of the nucleic acid sequence of SEQ ID NO:9, or a sequence at least 90% identical thereto.

[0074] (vi) intron sequences In some embodiments, the AAV expression cassettes of the present disclosure may include intron sequences, hi some embodiments, the inclusion of the intron sequences enhances expression compared to expression in the absence of the intron sequences.

[0075] In some embodiments, the intron sequence is a hybrid or chimeric sequence. In some embodiments, the intron sequence is isolated or derived from one or more intron sequences of SV40, β-globin, chicken beta-actin, minute virus of mice (MVM), Factor IX, and / or human IgG (heavy or light chain). In some embodiments, the intron sequence is chimeric. In some embodiments, the intron sequence comprises a nucleic acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween) to the nucleic acid sequence of SEQ ID NO:7. In some embodiments, the intron sequence comprises the sequence of SEQ ID NO:7, or a sequence at least 90% identical thereto. In some embodiments, the intron sequence comprises the sequence of SEQ ID NO:7.

[0076] AAV production methods The AAV expression cassettes described herein can be incorporated into vectors (e.g., plasmids or bacmids) using standard molecular biology techniques. The present disclosure provides vectors comprising any one of the AAV expression cassettes described herein. The vector (e.g., plasmid or bacmid) can further comprise one or more genetic elements used during AAV production, including, for example, the AAV rep and cap genes, and helper virus protein sequences.

[0077] The AAV expression cassettes described herein, and vectors (e.g., plasmids) containing the AAV expression cassettes, can be used to generate recombinant AAV vectors.

[0078] The present disclosure provides methods for producing a recombinant AAV vector, comprising contacting an AAV producer cell (e.g., a HEK293 cell) with an AAV expression cassette or vector (e.g., a plasmid) of the present disclosure. The present disclosure further provides a cell comprising any one of the AAV expression cassettes or vectors disclosed herein. In some embodiments, the method further comprises contacting the AAV producer cell with one or more additional plasmids encoding, for example, AAV rep and cap genes and helper virus protein sequences. In some embodiments, the method for producing a recombinant AAV vector comprises contacting an AAV producer cell (e.g., an insect cell such as an Sf9 cell) with at least one insect cell-compatible vector comprising an AAV expression cassette of the present disclosure. An "insect cell-compatible vector" is any compound or formulation (biological or chemical) that facilitates transformation or transfection of an insect cell with a nucleic acid. In some embodiments, the insect cell-compatible vector is a baculovirus vector. In some embodiments, the method further comprises maintaining the insect cell under conditions such that AAV is produced.

[0079] The present disclosure provides a recombinant AAV vector produced using any one of the methods disclosed herein. The recombinant AAV vector produced can be of any serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the recombinant AAV vector produced can contain one or more amino acid modifications (e.g., substitutions and / or deletions) compared to a native AAV capsid. For example, the recombinant AAV vector can be a modified AAV vector derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the recombinant AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the recombinant AAV vector is a self-complementary AAV (scAAV).

[0080] In some embodiments, the AAV vector comprises a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the AAV vector comprises a capsid protein having one or more substitutions or mutations compared to the wild-type AAV capsid protein. The recombinant AAV vectors disclosed herein can be used to transduce a transgene sequence into a target cell, for example, by contacting the recombinant AAV vector with the target cell.

[0081] In some embodiments, the AAV vector comprises a capsid protein comprising an amino acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween) to SEQ ID NO: 13. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 13.

[0082] In some embodiments, the AAV vector comprises a capsid protein comprising an amino acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween) to SEQ ID NO: 14. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 14.

[0083] In some embodiments, the AAV vector comprises a capsid protein comprising an amino acid sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity, including all values ​​and subranges therebetween) to SEQ ID NO: 15. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto. In some embodiments, the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15.

[0084] In some embodiments, the AAV vector comprises a capsid protein comprising: (i) the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto; or (ii) the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto; or (iii) the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto.

[0085] Methods of Expression and Treatment The present disclosure provides compositions comprising any one of the nucleic acids, AAV expression cassettes, plasmids, cells, or recombinant AAV vectors disclosed herein. In some embodiments, the compositions disclosed herein comprise at least one pharmaceutically acceptable carrier, excipient, and / or vehicle, such as a solvent, buffer, solution, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent. In some embodiments, the pharmaceutically acceptable carrier, excipient, and / or vehicle may comprise saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. In some embodiments, pharmaceutically acceptable carriers, excipients, and / or vehicles include phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), or suitable mixtures thereof. In some embodiments, the compositions disclosed herein further contain minor amounts of emulsifying or wetting agents, or pH buffering agents.

[0086] In some embodiments, the compositions disclosed herein further comprise other conventional pharmaceutical ingredients such as preservatives or chemical stabilizers, for example, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, or albumin. In some embodiments, the compositions disclosed herein may further comprise antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, or thimerosal, isotonic agents such as sugars or sodium chloride, and / or agents that delay absorption such as aluminum monostearate and gelatin.

[0087] The present disclosure provides a method for expressing an arrhythmogenic cardiomyopathy-associated transgene in a cell, the method comprising contacting the cell with any one of the nucleic acid molecules, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein, thereby expressing the arrhythmogenic cardiomyopathy-associated transgene in the cell.

[0088] The present disclosure provides a method for expressing an arrhythmogenic cardiomyopathy-associated transgene in a tissue, the method comprising contacting the tissue with any one of the nucleic acid molecules, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein, thereby expressing the arrhythmogenic cardiomyopathy-associated transgene in the tissue. In some embodiments, the tissue comprises at least one cell and at least one desmosomal junction.

[0089] In some embodiments, the cell is a cardiac cell, an endothelial cell, a skin cell, a bladder cell, or a gastrointestinal mucosal cell. In some embodiments, the cell is a cardiac cell. In some embodiments, the cell is a dividing cell, e.g., a cultured cell in cell culture. In some embodiments, the cell is a non-dividing cell. In some embodiments, an arrhythmogenic cardiomyopathy-associated gene is delivered to a cell in vitro to produce an arrhythmogenic cardiomyopathy-associated polypeptide, e.g., for in vitro or ex vivo gene therapy.

[0090] In some embodiments, the contacting step is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting step is performed in vivo in a subject in need thereof. In some embodiments, the contacting step comprises administering a therapeutically effective amount of a nucleic acid molecule, plasmid, recombinant AAV vector, or composition to the subject. In some embodiments, the subject is suffering from or at risk of developing arrhythmogenic cardiomyopathy.

[0091] The present disclosure provides a method for treating arrhythmogenic cardiomyopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein, thereby treating the arrhythmogenic cardiomyopathy in the subject. In some embodiments, the subject is suffering from or at risk of developing arrhythmogenic cardiomyopathy. In some embodiments, the arrhythmogenic cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy. In some embodiments, the arrhythmogenic cardiomyopathy is associated with, precipitated by, or caused by a genetic alteration. In some embodiments, the genetic alteration comprises one or more genetic alterations to the PKP2 gene (e.g., one or more deletions, insertions, duplications, and / or substitutions) compared to the wild-type PKP2 gene and / or a corresponding alteration in the expression and / or activity of the PKP2 protein compared to the wild-type PKP2 protein. In some embodiments, the mutation in the PKP2 gene results in PKP2 haploinsufficiency. In some embodiments, the subject at risk for developing arrhythmogenic cardiomyopathy is a newborn identified as carrying a mutation in the PKP2 gene. In some embodiments, the arrhythmogenic cardiomyopathy-associated gene (e.g., PKP2) is targeted by gene therapy to increase its expression and / or function.

[0092] In some embodiments, the method includes reducing the severity of arrhythmogenic cardiomyopathy; delaying its onset or progression; and / or eliminating its symptoms. In some embodiments, the symptoms of arrhythmogenic cardiomyopathy include (a) recurrent ventricular tachycardia, (b) syncope, (c) sudden death, or (d) any combination thereof. In some embodiments, the arrhythmogenic cardiomyopathy is associated with (a) decreased mechanical stability between the subject's cardiomyocytes, (b) disruption of gap junctions in the subject's cardiac tissue, (c) decreased sodium current in the subject's cardiac tissue, (d) fibrosis of the right ventricular myocardium, or (e) any combination thereof.

[0093] In some embodiments, the method comprises increasing mechanical stability between cardiac muscle cells in the subject. In some embodiments, the method comprises improving gap junction function in cardiac tissue of the subject. In some embodiments, the method comprises increasing sodium current in cardiac tissue of the subject. In some embodiments, the method comprises reducing fibrosis of right ventricular myocardium in the subject.

[0094] In some embodiments, the method comprises increasing the left ventricular ejection fraction of the heart compared to a control subject with arrhythmogenic cardiomyopathy, the control subject not being administered a therapeutically effective dose. In some embodiments, the method comprises increasing the left ventricular ejection fraction of the heart compared to the subject's left ventricular ejection fraction before administration of the therapeutically effective dose. In some embodiments, the method comprises increasing the left ventricular ejection fraction of the heart to a value within a range of about 30% to about 80%, e.g., about 35%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80%, including subranges and values ​​therebetween. In some embodiments, the method comprises increasing the left ventricular ejection fraction of the heart to about 60%.

[0095] In some embodiments, the method comprises increasing the left ventricular area of ​​the heart compared to a control subject having arrhythmogenic cardiomyopathy, wherein the control subject has not been administered a therapeutically effective amount. In some embodiments, the method comprises decreasing the right ventricular area of ​​the heart compared to the right ventricular area of ​​the heart of the subject prior to administration of the therapeutically effective amount.

[0096] In some embodiments, the method comprises increasing survival time of the subject compared to a control subject having arrhythmogenic cardiomyopathy, wherein the control subject has not been administered a therapeutically effective amount. In some embodiments, the method comprises increasing survival time of the subject compared to the expected survival time of the subject before administration of the therapeutically effective amount. In some embodiments, the method includes extending the survival of a subject by a value in a range of about 3 months to about 50 years (e.g., about 6 months, about 1 year, about 5 years, about 10 years, about 15 years, about 20 years, about 25 years, about 30 years, about 35 years, about 40 years, about 45 years, about 50 years, including subranges and values ​​therebetween) compared to (i) a control subject having arrhythmogenic cardiomyopathy who is not administered a therapeutically effective amount, or (ii) the subject's expected survival before administration of the therapeutically effective amount. The dosage of a recombinant AAV vector administered to a subject depends on the mode of administration, the disease or condition being treated and / or prevented, the condition of the individual subject, the particular viral vector or capsid, the nucleic acid being delivered, etc., and can be determined routinely. Exemplary doses to achieve a therapeutic effect include at least about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 , about 10 14 , about 10 15 transducing units, optionally about 10 8 ~about 10 13 is the titer of transducing units.

[0097] In some embodiments, the method comprises increasing the cardiac ejection fraction compared to a control subject having an arrhythmogenic cardiomyopathy, wherein the control subject has not been administered a therapeutically effective amount, hi some embodiments, the method comprises increasing the ejection fraction of the subject compared to the subject's ejection fraction before administration of the therapeutically effective amount.

[0098] In some embodiments, the method comprises increasing cardiac stroke volume compared to a control subject with arrhythmogenic cardiomyopathy, wherein the control subject is not administered a therapeutically effective amount. In some embodiments, the method comprises increasing the subject's stroke volume compared to the subject's stroke volume before administration of the therapeutically effective amount.

[0099] In some embodiments, the method comprises increasing cardiac output of the heart compared to a control subject having arrhythmogenic cardiomyopathy, wherein the control subject has not been administered a therapeutically effective amount, hi some embodiments, the method comprises increasing cardiac output of the subject compared to the subject's cardiac output before administration of the therapeutically effective amount.

[0100] In some embodiments, the method comprises increasing the fractional shortening of a subject having arrhythmogenic cardiomyopathy compared to a control subject, wherein the control subject has not been administered a therapeutically effective amount. In some embodiments, the method comprises increasing the fractional shortening of a subject compared to the subject's fractional shortening before administration of the therapeutically effective amount.

[0101] In some embodiments, the method comprises increasing a left ventricular outflow tract velocity time integral compared to a control subject having arrhythmogenic cardiomyopathy, wherein the control subject has not been administered a therapeutically effective dose. In some embodiments, the method comprises increasing a left ventricular outflow tract velocity time integral in the subject compared to the subject's left ventricular outflow tract velocity time integral before administration of the therapeutically effective dose.

[0102] In some embodiments, the method includes reducing left ventricular volume in a subject having arrhythmogenic cardiomyopathy compared to a control subject, wherein the control subject has not been administered a therapeutically effective amount. In some embodiments, the method reduces left ventricular volume in the subject compared to the subject's left ventricular volume before administration of the therapeutically effective amount.

[0103] In certain embodiments, two or more administrations (e.g., two, three, four, or more administrations) can be used to achieve the desired level of gene expression over various intervals, such as, for example, daily, weekly, monthly, yearly, etc.

[0104] In some embodiments, the subject is a human subject. In some embodiments, the nucleic acid molecule, plasmid, cell, recombinant AAV vector, or composition is administered to the subject's cardiac tissue. In some embodiments, the nucleic acid molecule, plasmid, cell, recombinant AAV vector, or composition is administered to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. In some embodiments, the nucleic acid molecule, plasmid, cell, recombinant AAV vector, or composition is administered to the subject via intravenous administration, intra-arterial administration, intra-aortic administration, direct cardiac injection, coronary artery perfusion, or any combination thereof.

[0105] Other modes of administration include oral, transmucosal, intrathecal, transdermal, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular (including administration to skeletal muscle, diaphragm, and / or cardiac muscle), intradermal, intrathoracic, intracerebral, and intra-articular), intralymphatic, etc., and direct injection into a tissue or organ (e.g., into the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain). Delivery to the target tissue can also be achieved by delivering a depot containing the viral vector and / or capsid. In an exemplary embodiment, a depot containing the viral vector and / or capsid is implanted into skeletal muscle tissue, cardiac muscle tissue, and / or diaphragm muscle tissue, or the tissue can be contacted with a film or other matrix containing the viral vector and / or capsid.

[0106] In some embodiments, the methods disclosed herein may include administering to a subject a therapeutically effective amount of any one of the nucleic acids, AAV expression cassettes, plasmids, cells, recombinant AAV vectors, or compositions disclosed herein in combination with one or more secondary therapies targeting arrhythmogenic cardiomyopathy. In some embodiments, the methods disclosed herein for treating at least one symptom of and / or delaying the onset of arrhythmogenic cardiomyopathy in a subject may further include administering one or more secondary therapies targeting arrhythmogenic cardiomyopathy. In some embodiments, the secondary therapies include administration of a drug such as a beta-blocker or amiodarone, an implantable cardioverter-defibrillator (ICD), catheter ablation, or any combination thereof. Non-limiting examples of beta-blockers include acebutolol, atenolol, bisoprolol, metoprolol, nadolol, nebivolol, and propranolol.

[0107] The term "administered in combination," as used herein, is understood to include two (or more) different treatments delivered to a subject over the course of the subject's illness (e.g., arrhythmogenic cardiomyopathy), such that the effects of the treatments on the patient overlap at some point. In certain embodiments, there is an overlap in administration, such that the delivery of one treatment is still occurring when the delivery of a second treatment begins. This may be referred to herein as "simultaneous" or "concurrent" delivery. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins, which may be referred to as "sequential" delivery.

[0108] In some embodiments, the treatments are more effective due to combined administration. For example, the second treatment is more effective, or the same effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than when the second treatment is administered without the first treatment, or a similar situation is seen with the first treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive (synergistic).

[0109] All articles, publications, and patents cited herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which they are cited, as if each individual article, publication, or patent was specifically and individually indicated to be incorporated by reference. However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be taken as, an acknowledgment or any indication that they constitute available prior art or form part of the common general knowledge in any country in the world.

[0110] Unless the context indicates otherwise, it is expressly intended that the various features described herein can be used in any combination.

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0112] It is to be understood that the foregoing description and the following examples are intended to illustrate, but not limit, the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. [Example]

[0113] The following examples are included herein for illustrative purposes only and are not intended to be limiting.

[0114] Example 1: Preparation of AAV expression cassettes and recombinant AAV vectors in mammalian cells An AAV expression cassette was generated using standard cloning techniques, containing the following elements: (i) the 5' AAV ITR of SEQ ID NO:5, (ii) the cardiac troponin T (TNNT2) promoter of SEQ ID NO:4, (iii) the human beta globin (hBG) intron of SEQ ID NO:7, (iv) the human PKP2a transgene sequence of SEQ ID NO:2, (v) the bovine growth hormone (bGH) polyA tail of SEQ ID NO:8, and (vi) the 3' AAV ITR of SEQ ID NO:6. See Figure 1, which shows a schematic diagram of the AAV cassette. The AAV cassette comprises the sequence of SEQ ID NO:12.

[0115] The AAV expression cassette was incorporated into a plasmid (Figure 2) and transfected into virus-producing cells (e.g., HEK293) using an appropriate transfection reagent (e.g., Lipofectamine™) along with the Rep / Cap plasmid encoding the Rep and Cap genes and a helper plasmid containing various helper sequences (E4, E2a, and VA) required for AAV production. After incubation at 37°C for a predetermined period, AAV particles were either collected from the medium or the cells were lysed to release the AAV particles. The AAV particles were then purified, titered, and stored at -80°C for later use.

[0116] Using the methods described above, we generated AAV particles containing the disclosed AAV expression cassette of SEQ ID NO: 12 and a capsid protein having the amino acid sequence of SEQ ID NO: 13. These AAV particles are referred to herein as "AAV.STRV47-hTNNT2.PKP2a."

[0117] Similarly, an AAV particle (referred to herein as "AAV.STRV5-hTNNT2.PKP2a") was generated that contained the disclosed AAV expression cassette of SEQ ID NO:12 and a capsid protein having the amino acid sequence of SEQ ID NO:14.

[0118] Finally, AAV particles (referred to herein as "AAV.STRV5-hTNNT2.PKP2a") were generated that contained the disclosed AAV expression cassette of SEQ ID NO:12 and a capsid protein having the amino acid sequence of SEQ ID NO:15.

[0119] Example 2: Increased PKP2 mRNA and protein levels in cardiomyocytes following treatment with AAV.STRV47-hTNNT2.PKP2a Healthy normal human iPSC-derived cardiomyocytes were treated with AAV.STRV47-hTNNT2.PKP2a at a 50K MOI 3 days after plating. PKP2a mRNA and protein expression were assessed by RT-qPCR and Western blot, respectively, 7 days after treatment. PKP2 was knocked down (KD) using 1 μM antisense oligonucleotide (ASO) against PKP2 as a control. For mRNA expression, RNA was collected using a Cell-Paired CT kit according to the manufacturer's protocol (Invitrogen, catalog no. A35377). RT-qPCR was performed to measure mRNA expression using TaqMan probes for PKP2 and the housekeeping gene UBC (Hs00428040_m1 and Hs00824723_m1, respectively).

[0120] As shown in Figure 3, the results indicate that PKP2 mRNA expression is reduced upon knockdown of PKP2 using antisense oligonucleotides. However, PKP2 mRNA increased several-fold when cells were transduced with AAV.STRV47-hTNNT2.PKP2a compared to wild-type cells. These results indicate that treatment of cardiomyocytes with AAV.STRV47-hTNNT2.PKP2a results in increased expression of PKP2 mRNA.

[0121] For protein expression, plates were lysed with 300 μl of 1x RIPA plus protease inhibitors. Plates were scraped and then rocked in the cold for 15 minutes. Cell lysates were collected and centrifuged at 4°C for 10 minutes. The supernatant was then recovered. Protein lysates were diluted with 4x LiCOR sample buffer, and samples were loaded onto Tris / glycine 20-4% SDS-page gels and subjected to electrophoresis until the dye front reached the bottom of the gel. Gels were transferred to PDVF using the multi-MW setting on a BioRad Trans-Blot Turbo system according to the manufacturer's protocol. Membranes were blocked with LiCOR blocking buffer for 30 minutes at room temperature and then incubated with antibodies against PKP2 (American Research Products, Inc.), cTnT (Abcam ab45932), or beta-tubulin at a 1:1000 dilution.

[0122] As shown in Figure 3B and quantified in Figure 3C, the results indicate that PKP2 protein expression is reduced upon knockdown of PKP2 using antisense oligonucleotides. However, upon transduction of cells with AAV.STRV47-hTNNT2.PKP2a, PKP2 protein expression was clearly increased compared to wild-type cells. These results indicate that treatment of cardiomyocytes with AAV.STRV47-hTNNT2.PKP2a results in increased expression of PKP2 protein.

[0123] Example 3: AAV.STRV5-hTNNT2.PKP2a can rescue PKP2a expression levels in an iPSC PKP2-KO cardiomyocyte model Human PKP1 knockout iPSC cells were generated by CRISPR / Cas9-mediated homozygous insertion of a premature stop codon (S70X). These cells were then differentiated into cardiomyocytes. Methods for generating cardiomyocytes are described in Burridge et al., 2014, Nat Methods. 2014 Aug;11(8):855-60; and Lian et al., 2012, Proc Natl Acad Sci USA. 2012 Jul 3;109(27):E1848-57, the contents of each of which are incorporated herein by reference in their entirety. Successful differentiation resulted in spontaneously beating cells that expressed the cardiomyocyte marker TNNT2.

[0124] Notably, transduction of iPSC PKP2-KO cardiomyocytes with AAV.STRV5-hTNNT2.PKP2a resulted in restoration of PKP2a expression to wild-type levels 10 days after transduction (Figures 4A and 4B). PKP2a expression was then examined by immunofluorescence, and precise plasma membrane localization of PKP2a expressed using AAV was observed in transduced iPSC PKP2-KO cardiomyocytes, similar to that of the wild-type (Figure 4C).

[0125] Knockout of PKP2a also affects the electrical phenotype of human iPSC cardiomyocytes. Calcium transients were measured in spontaneously beating confluent cardiomyocyte cultures. A transduction control (encoding GFP) was included to account for any effects of transduction. Calcium recordings in PKP2-KO cardiomyocytes showed early late transients (double peaks seen in Figures 5B and 5C), representing a type of calcium handling dysfunction that may underlie arrhythmias. Restoring PKP2a expression in iPSC PKP2-KO cardiomyocytes transduced with AAV.STRV5-hTNNT2.PKP2a resulted in normal calcium transients similar to those seen in WT iPSC cardiomyocytes (Figures 5A and 5D).

[0126] In summary, these data demonstrate that transduction of AAV.STRV5-hTNNT2.PKP2a restores PKP2 expression in iPSC PKP2-KO cardiomyocytes, resulting in a striking rescue of the electrical phenotype of cardiomyocytes lacking PKP2.

[0127] Example 4: Expression of PKP2 in wild-type mice treated with AAV.STRV47-hTNNT2.PKP2a Eleven-week-old wild-type mice were used for an in vivo pilot study in two groups (control and treatment, n=4 in both groups). See Table A. The treatment group received 5e13vg / kg AAV.STRV47-hTNNT2.PKP2a (total volume of 150μL for a total VG dose of 1.25e12) by intravenous injection via the tail vein. Twenty-one days after administration, expression of the human PKP2 gene in mouse cardiac tissue was assessed for both groups of mice using molecular techniques including qPCR, RT-qPCR, and Western blot.

[0128] [Table 3]

[0129] For DNA extraction, 50 mg of tissue was homogenized in a tube containing stainless steel beads with 300 μL of TE buffer and 30 μL of 20 mg / mL proteinase K solution. The sample was then incubated at 56°C for 20 minutes, and DNA was then extracted using the Maxwell RSC Tissue DNA Kit (Promega, catalog no. AS1610) on a Maxwell RSC48 instrument according to the manufacturer's protocol. DNA concentration and A260 / A280 were measured using a NanoDrop. qPCR was performed on a QS3 instrument (Thermo) using custom PKP2 primers and probe. Figure 6A shows that the PKP2a transgene is detectable in cardiac tissue from mice treated with the AAV.STRV47-hTNNT2.PKP2a vector.

[0130] For RNA extraction, 10–20 mg of tissue was placed in a tube containing 250 μL of homogenization buffer and stainless steel beads and homogenized using a bead blaster. The sample was incubated at 70°C for 2 minutes. RNA was extracted using the Maxwell RSC Tissue RNA Kit (Promega, catalog number AS1340) on a Maxwell RSC48 instrument according to the manufacturer's protocol. RNA concentration and A260 / A280 were measured using a NanoDrop. For RT-qPCR, Dnase I was added to the sample and incubated at 37°C for 2 minutes. Next, the sample was diluted 1:20, and a reverse transcriptase reaction was performed using a dNTP mix and SuperScript IV reverse transcriptase to generate cDNA. qPCR was performed on a QS3 instrument (Thermo) using custom PKP2 primers and probes and normalized to the housekeeping gene, mouse GAPDH. Figure 6B shows that PKP2a mRNA is detectable in cardiac tissue from mice treated with the AAV.STRV47-hTNNT2.PKP2a vector.

[0131] For protein extraction, 50-100 mg of tissue samples were homogenized in PBS and protease inhibitors (Boston Bioproducts, catalog number BP-475) using a bead blaster. As shown in Figure 6C, proteins were detected using an anti-PKP2 antibody (1:100) from American Research Products, Inc. (catalog number 03-651101) using a Jess automated Western blot system. Quantification, shown in Figure 6D, was performed using Jess software and normalized to the mean value of the PBS control. The results demonstrate that PKP2a protein is readily detectable in cardiac tissue from mice treated with the AAV.STRV47-hTNNT2.PKP2a vector.

[0132] Example 5: Expression of PKP2 using AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a results in extended survival in a cardiac-specific PKP2 knockout mouse model ("PKP2-cKO") A gene targeting construct was generated by introducing two loxP sites flanking mouse Pkp2 exons 2 and 3, followed by a neomycin resistance gene. The linearized construct was electroporated into mouse (C57BL / 6) embryonic stem (ES) cells, followed by neomycin selection of positive ES cell clones. Positive clones were injected into mouse blastocysts, which were then injected into foster mice. The resulting F1 heterozygous mice were mated with mice expressing flippase to excise the neomycin resistance gene. Mice homozygous for the Pkp2 targeting allele (Pkp2 fl / fl) were mated with mice expressing Cre recombinase fused to a mutant form of the human estrogen receptor ligand-binding domain under the control of a cardiomyocyte-specific promoter (αMHC-Cre-ER(T2)). Treatment of Pkp2 fl / fl, αMHC-Cre-ER(T2) mice with tamoxifen results in cardiac-specific deletion of Pkp2 exons 2 and 3, resulting in a substantial reduction in PKP2 protein expression. This tamoxifen-induced, cardiomyocyte-specific deletion of mouse PKP2 protein results in a model of ARVC referred to as the PKP2-cKO mouse model.

[0133] Figure 7 shows the design for testing treatment with AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a in a tamoxifen-induced cardiac-specific PKP2 knockout mouse model. Approximately 3-month-old mice were injected via the tail vein with either AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a at 5e13 vg / kg. Five groups (two positive controls: Groups 2 and 3 in Table B, one negative control: Group 1 in Table B, and two treatments: Groups 4 and 5 in Table B) were used to evaluate the ability to express the human PKP2 gene in mouse cardiac tissue compared to PKP2 levels in wild-type and knockout mice. See Table B.

[0134] [Table 4]

[0135] For cardiac-specific PKP2 deletion, mice were intraperitoneally injected with tamoxifen (0.1 mg / g body weight) for four consecutive days approximately 16 days after AAV injection, and were then observed for an additional 54 days to assess survival.

[0136] As shown in Figure 8A and Table C, positive control mice, either "PKP2 wt / wt;Cre+" or "PKP2 fl / fl;Cre-," exhibited the highest survival rate, with the majority of mice surviving beyond 52 days post-induction (dpi). In contrast, negative control mice with a cardiac-specific PKP2 knockout ("PKP2 fl / fl;Cre+") survived only to approximately 50 days. Surprisingly, treatment of cardiac-specific PKP2 knockout mice with either AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a resulted in rescue of the PKP2 knockout phenotype and a significant prolongation of survival.

[0137] In the second cohort of Table B, cardiac-specific PKP2 knockout animals were treated with AAV.STRV5-hTNNT2.PKP2a (see Table D below) as described above, with the study endpoint being 54 days or approximately 8 weeks after tamoxifen injection. The experimental design is shown in Table D below.

[0138] In the second cohort, all unmasked animals in the positive control group (Pkp2 fl / fl, Cre- and Pkp2 wt / wt, Cre+) survived to the scheduled necropsy endpoint, whereas all mice in the untreated PKP2-cKO (Pkp2 fl / fl; Cre+) negative control group reached the study facility criteria for early euthanasia before the study endpoint, consistent with the disease model phenotype. Notably, all unmasked PKP2-cKO mice treated with AAV.STRV5-hTNNT2.PKP2a survived to the scheduled necropsy endpoint, demonstrating treatment-induced survival (Figure 8B, Tables B and D).

[0139] [Table 5]

[0140] [Table 6]

[0141] Example 6: Expression of PKP2 using AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a in a cardiac-specific PKP2 knockout mouse model The presence of the PKP2a transgene and expression of PKP2 mRNA were evaluated in a cardiac-specific PKP2 knockout mouse model treated with an AAV vector containing the PKP2 gene. DNA and RNA extraction and analysis were performed as described in Example 4 above. As shown in Figure 9A, the results indicate that the PKP2a transgene is detectable in the cardiac tissue of cardiac-specific PKP2 knockout mice treated with the AAV.STRV5-hTNNT2.PKP2a vector or the AAV.STRV84-hTNNT2.PKP2a vector. Also, as shown in Figure 9B, the results indicate that PKP2a mRNA is detectable in the cardiac tissue of mice treated with the AAV.STRV5-hTNNT2.PKP2a vector or the AAV.STRV84-hTNNT2.PKP2a vector.

[0142] Treatment with AAV.STRV84-hTNNT2.PKP2a advantageously increased copy number in the heart by a log-fold compared to treatment with the AAV.STRV5-hTNNT2.PKP2a vector. Furthermore, treatment with AAV.STRV84-hTNNT2.PKP2a advantageously increased mRNA expression in the heart by a half-log-fold compared to treatment with the AAV.STRV5-hTNNT2.PKP2a vector. Liver copy number and mRNA expression were similar between treatments with the two AAV vectors. Notably, Figure 9B shows that expression from the TNNT2 promoter resulted in high PKP2 expression in the heart, but very low expression in the liver. This differential expression has an advantageous effect in minimizing off-target effects during treatment with the AAV vectors disclosed herein.

[0143] PKP2 protein expression was assessed in PKP2 cardiac knockout mice treated with the AAV.STRV5-hTNNT2.PKP2a vector or the AAV.STRV84-hTNNT2.PKP2a vector using the methods described above in Example 3. As shown in Figure 10A, along with total protein (loading control) in Figure 10B, PKP2 protein was detected using anti-PKP2a-b (1:100) from ARP (catalog no. 03-651101) using a Jess automated Western blot system. Quantification, shown in Table E, was performed using Jess software and normalized to the mean value of Group 2 (positive control).

[0144] As shown in Figures 10A and 10B and Table E, treatment of cardiac-specific PKP2 knockout mice with either AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a led to expression of PKP2 protein in cardiac tissue. Furthermore, treatment with AAV.STRV84-hTNNT2.PKP2a resulted in much higher PKP2 protein expression in the heart (1.71-fold compared to wild-type) compared to treatment with the AAV.STRV5-hTNNT2.PKP2a vector (0.52-fold compared to wild-type). This suggests that treatment with AAV particles comprising the disclosed AAV expression cassette of SEQ ID NO:12 and a capsid protein having the amino acid sequence of SEQ ID NO:15 is particularly effective in rescuing PKP2 levels and function in vivo.

[0145] [Table 7]

[0146] Example 7: Desmosomal localization of PKP2 in a cardiac-specific PKP2 knockout mouse model following treatment with AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a Immunohistochemical analysis of mouse cardiac tissue sections showed results consistent with the molecular analysis (Figures 11A–11E). Specifically, PKP2 wt / wt;Cre+ and PKP2 fl / fl;Cre-, tamoxifen-treated (+ control) mice exhibited widespread PKP2 signals throughout the heart (Figures 11A and 11B), clearly corresponding to the intercalated disc regions of cardiomyocyte-cell junctions and consistent with PKP2 localization to cardiac desmosomes, whereas cardiac-specific PKP2 knockout (PKP2fl / fl,Cre+, tamoxifen-treated; - control) animals exhibited minimal PKP2 signals in cardiac tissue sections (Figure 11C). Notably, after 10 weeks, PKP2-cKO animals treated with either AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a displayed cardiac PKP2a signal and localization throughout the heart consistent with that observed in positive control animals (Figures 11D and 14E), providing further evidence of vector-driven PKP2a expression and confirming the localization of PKP2a to the appropriate cellular regions.

[0147] Taken together, the results described herein demonstrate that treatment of mice lacking PKP2 function in cardiac tissue with an AAV vector disclosed herein containing PKP2a (e.g., AAV.STRV47-hTNNT2.PKP2a, AAV.STRV5-hTNNT2.PKP2a, or AAV.STRV84hTNNT2.PKP2a) not only results in expression of PKP2 mRNA and protein in cardiac tissue, but also promotes the correct localization of human PKP2 protein to desmosomes. These results also demonstrate that expression of human PKP2 from the AAV vectors disclosed herein results in a significant improvement in survival of animals lacking PKP2 function in cardiac tissue. Finally, expression of human PKP2 from the AAV vectors disclosed herein is advantageously higher in the heart compared to the liver.

[0148] Example 8: Cardiac structure and function are significantly improved upon treatment of cardiac-specific PKP2 knockout mice with AAV.STRV5-hTNNT2.PKP2a Echocardiographic analysis of cardiac function and structure in PKP2-cKO mice revealed reduced left ventricular ejection fraction and increased right ventricular area compared with control mice with wild-type PKP2 function (WT; Cre+ mice and floxed; Cre- mice). Surprisingly, AAV.STRV5-hTNNT2.PKP2a-treated PKP2-cKO mice maintained ejection fractions comparable to control animals and showed a delayed increase in right ventricular area (Figures 12A and 12B (arrows point to AAV.STRV5-hTNNT2.PKP2a-treated mice)). Even 42 days after tamoxifen induction, both cardiac parameters in AAV.STRV5-hTNNT2.PKP2a-treated mice were significantly improved compared with untreated PKP2-cKO mice, indicating that treatment prevented cardiac dysfunction and delayed diastolic progression (Figures 12C and 12D). In summary, these data demonstrate that AAV.STRV5-mediated expression of PKP2 prevents the decline in cardiac function and delays the onset and progression of cardiac dilatation.

[0149] Analysis of mouse cardiac tissue by Western blot showed minimal to no endogenous PKP2 expression in PKP2-cKO hearts, consistent with Cre-mediated deletion of Pkp2 exons 2 and 3. In contrast, PKP2-cKO mice treated with AAV.STRV5-hTNNT2.PKP2a showed robust human PKP2a expression in the heart at approximately 1.5-fold the endogenous PKP2 level (Figures 13A and 13B). Analysis of vector copy number (VCN) by qPCR revealed approximately 10 VCN per microgram of input DNA in cardiac tissue. 5 vector copy number of approximately 0.5 x 10 per microgram of DNA in liver tissue. 6 Further analysis of vector-induced PKP2a mRNA by RT-qPCR revealed a vector copy number of 10 per 10 ng of cDNA input into cardiac tissue. 4 ~10 5Despite the higher VCN in the liver compared to the heart, the copy number of PKP2a mRNA was approximately 500-fold lower in the liver, highlighting the cardiac-specific expression profile of the hTNNT2 promoter utilized in the cassette (Figures 13C and 13D). Together, these data confirm vector-induced expression of PKP2a in the mouse heart and demonstrate negligible expression in the liver.

[0150] Further analysis of cardiac tissue by Masson's trichrome staining revealed substantial collagen deposition in the right ventricle of untreated PKP2-cKO mice (Figure 14A), indicating pathological cardiac fibrosis. In contrast, mice with PKP2 function (+control; FL, Cre-) showed minimal collagen deposition in the right ventricle, indicating normal cardiac tissue. Surprisingly, PKP2-cKO mice treated with either AAV.STRV5-hTNNT2.PKP2a or AAV.STRV84-hTNNT2.PKP2a exhibited significantly less right ventricular (RV) fibrosis (Figures 14C and 14D). Figure 15 shows quantification of fibrosis in the two cohorts of animals (as seen in Figures 8A and 8B and Table B). Treatment with AAV.STRV84-hTNNT2.PKP2a reduced the level of fibrosis to nearly that of the positive control.

[0151] In summary, the above data demonstrate that expression of PKP2 in an AAV-mediated manner (e.g., using AAV.STRV5 or AAV.STRV84 vectors) not only rescues PKP2 protein levels in mouse models of cardiac-specific PKP2 knockout, but also restores PKP2 localization and function in these animals. AAV-mediated expression of PKP2 protein as described herein inhibits the formation of fibrotic regions in cardiac tissue, thus preserving cardiac function. Thus, the methods disclosed herein, including expression of PKP2 using AAV vectors such as AAV.STRV5 and AAV.STRV84, can alleviate pathologies associated with loss of PKP2 function, such as in arrhythmogenic cardiomyopathy.

[0152] Example 9: Dose finding and efficacy studies in the PKP2-cKO mouse model This was an 85-day dose-finding study using 79 mice. On day 1, mice were injected intravenously with the AAV transgene into the tail vein. On day 16, mice were induced intraperitoneally with tamoxifen. Mice were divided into nine groups, as shown in Table F. Group 1 was wild-type mice. Group 2 was PKP2 fl / fl Cre- mice. Group 3 was PKP2 fl / fl Cre+ mice. Group 4 was PKP2 fl / fl Cre+ mice administered 3e12 vg / kg of AAV.STRV84-hTNNT2.mPKP2 (murine PKP2). Group 5 was PKP2 fl / fl Cre+ mice administered 1e13 vg / kg of AAV.STRV84-hTNNT2.mPKP2. Group 6 is PKP2 fl / fl Cre+ mice administered 3e13 vg / kg of AAV.STRV84-hTNNT2.mPKP2. Group 7 is PKP2 fl / fl Cre+ mice administered 1e14 vg / kg of AAV.STRV84-mTNNT2.mPKP2. Group 8 is PKP2 fl / fl Cre+ mice administered 1e13 vg / kg of AAV.STRV84-hTNNT2.PKP2. Group 9 is PKP2 fl / fl Cre+ mice administered 1e14 vg / kg of AAV.STRV84-hTNNT2.PKP2. In summary, in this experiment, the ultra-low (UL) dose is 3e12, the low (L) dose is 1e13, the medium dose (M) is 3e13, and the high (H) dose is 1e14 vg / kg. For reference, the dose selected for the proof-of-concept (POC) study was 5e13vg / kg.

[0153] Mice were evaluated using echocardiography every two weeks, and cardiac and liver tissues of mice were analyzed for AAV vector biodistribution and PKP2 transgene expression, as described below.

[0154] [Table 8]

[0155] As can be seen in Figures 16A-16B, as the dose of human or mouse PKP2 administered increases, the vector copy number of PKP2 DNA increases. This applies to both the heart and liver of treated animals. Figure 16A relates to data from groups 1-7, and Figure 16B relates to data from groups 1-3 and 8-9.

[0156] Figures 17A-17B show a similar correlation between dose and PKP2 mRNA levels. However, PKP2 mRNA levels in the hearts of PKP2-treated animals continued to increase across the dose range, while liver levels were approximately 1e2 copies / 10 ng of cDNA in all groups. Also, mPKP2 and hPKP2 had similar mRNA levels at similar doses.

[0157] Figures 18-21 are Western blots showing the change in PKP2 levels based on treatment. The results for heart levels of mPKP2 (Figure 18) are shown in Table G. The results for heart levels of hPKP2 (Figure 19) are shown in Table H. The results for liver levels of mPKP2 (Figure 20) are shown in Table I. The results for liver levels of hPKP2 (Figure 21) are shown in Table J.

[0158] [Table 9]

[0159] [Table 10]

[0160] [Table 11]

[0161] [Table 12]

[0162] In conclusion, vector copy number and mRNA expression in the heart increased with increasing AAV-PKP2 dose. Liver vector copy number increased slightly with increasing dose, but mRNA levels were low and relatively similar across all dose groups. These results indicate that PKP2 protein levels increased with dose in the heart, while no overexpression was detected in the liver at any dose. Without being bound by theory, these results are likely driven by the liver-nontargeting profile of the STRV84 capsid and the use of a cardiac-specific promoter. Western blot analysis of the heart showed that PKP2 expression increased with increasing dose. Expression followed this pattern in both mouse and human transgene-treated groups. Western blot analysis of the liver showed similar expression levels across all groups tested, including mPKP2 and hPKP2-treated groups. Human versus mouse transgenes exhibit comparable protein expression in the liver.

[0163] Figures 22A-B show that administration of either the mouse transgene (Figure 22A) or the human transgene (Figure 22B) dose-dependently increased survival of mice lacking PKP2 function.

[0164] Figures 23A-F and 24A-F show the dose of human transgene at which animals show statistically improved health status relative to untreated control mice. Echocardiographic data provide a quantitative measure of cardiac structure and function. The graphs in these figures show the performance of unaffected healthy control hearts (two positive control groups (wt / wt, Cre+;NA and fl / fl, Cre-;NA)) as well as the effect of ARVC (untreated, negative control group (fl / fl, Cre+;NA)), providing a measure of the extent to which AAV treatment preserves cardiac structure and function. A correlation between dose and phenotypic rescue was observed for the following parameters: ejection fraction (Figures 23A and 24A), left ventricular (LV) volumes (Figures 23B and 24B), stroke volume (Figures 23C and 24C), fractional shortening (Figures 23D and 24D), cardiac output (Figures 23E and 24E), and left ventricular outflow tract velocity time integral (LVOT VTI) (Figures 23F and 24F). Note that all data are from the left side of the animals.

[0165] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof.

[0166] Numbered Embodiments The following list of embodiments is included herein for illustrative purposes only and is not intended to be exhaustive or limiting. Claimed subject matter is expressly not limited to the following embodiments. Embodiment 1. A nucleic acid molecule comprising an adeno-associated virus (AAV) expression cassette, said AAV expression cassette comprising, 5' to 3': a. 5'AAV inverted terminal repeat (ITR) b. Promoter c. arrhythmogenic cardiomyopathy-associated transgenes, and d. 3' AAV ITR. Embodiment 2. The nucleic acid molecule of embodiment 1, wherein the promoter drives expression of the arrhythmogenic cardiomyopathy-associated transgene. Embodiment 3. The nucleic acid molecule of embodiment 1 or 2, wherein the promoter is capable of expressing the transgene in cardiac cells. Embodiment 4. The nucleic acid molecule of any one of embodiments 1 to 3, wherein the promoter comprises a cardiac troponin T (TNNT2) promoter. Embodiment 5. The nucleic acid molecule of embodiment 4, wherein the TNNT2 promoter comprises a nucleic acid sequence derived from (i) a human TNNT2 promoter, (ii) a chicken TNNT2 promoter, (iii) a mouse TNNT2 promoter, or (iv) any combination thereof. Embodiment 6. The nucleic acid molecule of embodiment 5, wherein the TNNT2 promoter comprises a human TNNT2 promoter. Embodiment 7. The nucleic acid molecule of any one of embodiments 4 to 6, wherein the TNNT2 promoter comprises the nucleic acid sequence of SEQ ID NO: 4, or a sequence at least 90% identical thereto. Embodiment 8. The nucleic acid molecule of any one of embodiments 1 to 7, wherein the transgene encodes plakophilin-2. Embodiment 9. The nucleic acid molecule of any one of embodiments 1 to 8, wherein the transgene encodes human plakophilin-2. Embodiment 10. The nucleic acid molecule of any one of embodiments 1 to 9, wherein the transgene encodes isoform 2a of human lacofilin-2. Embodiment 11. The nucleic acid molecule of embodiment 10, wherein the isoform 2a of human plakophilin-2 comprises the amino acid sequence of SEQ ID NO: 1, or a sequence at least 90% identical thereto. Embodiment 12. The nucleic acid molecule of any one of embodiments 1 to 11, wherein the transgene encodes the RNA transcript variant 2a of the PKP2 gene. Embodiment 13. The nucleic acid molecule of any one of embodiments 1 to 12, wherein the transgene comprises a PKP2 gene. Embodiment 14. The nucleic acid molecule of embodiment 13, wherein the PKP2 gene is a human PKP2 gene. Embodiment 15. The nucleic acid molecule of any one of embodiments 1 to 14, wherein the transgene comprises the nucleic acid sequence of SEQ ID NO: 2, or a sequence at least 90% identical thereto. Embodiment 16. The nucleic acid molecule of any one of embodiments 1 to 15, wherein at least one of the 5' ITR and the 3' ITR is about 110 to about 160 nucleotides in length. Embodiment 17. The nucleic acid molecule of any one of embodiments 1 to 16, wherein the 5' ITR is the same length as the 3' ITR. Embodiment 18. The nucleic acid molecule of any one of embodiments 1 to 17, wherein the 5' ITR and the 3' ITR have different lengths. Embodiment 19. The nucleic acid molecule of any one of embodiments 1 to 18, wherein the 5' ITR and the 3' ITR are each about 141 nucleotides in length. Embodiment 20. The nucleic acid molecule of any one of embodiments 1 to 19, wherein at least one of the 5' ITR and the 3' ITR is isolated from or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. Embodiment 21. The nucleic acid molecule of any one of embodiments 1 to 20, wherein the 5' ITR and the 3' ITR are each isolated from or derived from the genome of AAV2. Embodiment 22. The nucleic acid molecule of any one of embodiments 1 to 21, wherein the 5' ITR comprises the sequence of SEQ ID NO: 5. Embodiment 23. The nucleic acid molecule of any one of embodiments 1 to 22, wherein the 3' ITR comprises the sequence of SEQ ID NO:6. Embodiment 24. The nucleic acid molecule of any one of embodiments 1 to 23, wherein the AAV expression cassette comprises an intron. Embodiment 25. The nucleic acid molecule of embodiment 24, wherein the intron is derived from the human beta globin gene. Embodiment 26. The nucleic acid molecule of embodiment 24 or embodiment 25, wherein the intron comprises the nucleic acid sequence of SEQ ID NO: 7, or a sequence at least 90% identical thereto. Embodiment 27. The nucleic acid molecule of any one of embodiments 1 to 26, wherein the AAV expression cassette comprises a polyadenylation signal. Embodiment 28. The nucleic acid molecule of embodiment 27, wherein the polyadenylation signal is a polyadenylation signal isolated or derived from one or more of the following genes: simian virus 40 (SV40), rBG, α-globin, β-globin, human collagen, human growth hormone (hGH), polyomavirus, human growth hormone (hGH), or bovine growth hormone (bGH). Embodiment 29 The nucleic acid molecule of embodiment 27 or embodiment 28, wherein the AAV expression cassette comprises a bGH polyadenylation signal. Embodiment 30. The nucleic acid molecule of embodiment 29, wherein the bGH polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 8, or a sequence at least 90% identical thereto. Embodiment 31. The nucleic acid molecule of any one of embodiments 1 to 30, wherein the AAV expression cassette comprises at least one stuffer sequence. Embodiment 32. The nucleic acid molecule of embodiment 31, wherein the at least one stuffer sequence comprises the nucleic acid sequence of SEQ ID NO: 9, or a sequence at least 90% identical thereto. Embodiment 33. The nucleic acid molecule of embodiment 31, wherein the at least one stuffer sequence comprises the nucleic acid sequence of SEQ ID NO: 9, or a portion thereof. Embodiment 34. The nucleic acid molecule of any one of embodiments 1 to 33, wherein the AAV expression cassette comprises a Kozak sequence. Embodiment 35. The nucleic acid molecule of embodiment 34, wherein the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 10, or a sequence at least 90% identical thereto, or the nucleic acid sequence of acagccacc, or a sequence at least 90% identical thereto. Embodiment 36. The nucleic acid molecule of any one of embodiments 1 to 35, wherein the AAV expression cassette comprises an enhancer. Embodiment 37. The nucleic acid molecule of any one of embodiments 1 to 36, wherein the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 12, or a sequence at least 90% identical thereto. Embodiment 38. A plasmid comprising the nucleic acid molecule of any one of embodiments 1 to 37. Embodiment 39. A cell comprising a nucleic acid molecule according to any one of embodiments 1 to 37, or a plasmid according to embodiment 38. Embodiment 40. A method of producing a recombinant AAV vector, comprising contacting an AAV producer cell with a nucleic acid molecule of any one of embodiments 1 to 37 or a plasmid of embodiment 38. Embodiment 41. A recombinant AAV vector produced by the method described in embodiment 40. Embodiment 42. The recombinant AAV vector of embodiment 41, wherein the vector is a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, and bovine AAV. Embodiment 43. The recombinant AAV vector of embodiment 41 or embodiment 42, wherein the recombinant AAV vector is a single-stranded AAV (ssAAV). Embodiment 44. The recombinant AAV vector of embodiment 41 or embodiment 42, wherein the recombinant AAV vector is a self-complementary AAV (scAAV). Embodiment 45. The recombinant AAV vector of any one of embodiments 41 to 44, wherein the AAV vector comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV. Embodiment 46. A recombinant AAV vector described in any one of embodiments 41 to 45, wherein the AAV vector comprises a capsid protein having one or more substitutions or mutations compared to a wild-type AAV capsid protein. Embodiment 47. The recombinant AAV vector of any one of embodiments 41 to 46, wherein the AAV vector comprises a capsid protein comprising: a. (i) the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto; or b.(ii) the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto; or c.(iii) The amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto. Embodiment 48. The recombinant AAV vector of embodiment 47, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 13, or a sequence at least 90% identical thereto. Embodiment 49. The recombinant AAV vector of embodiment 48, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 13. Embodiment 50. The recombinant AAV vector of embodiment 47, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto. Embodiment 51. The recombinant AAV vector of embodiment 50, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 14. Embodiment 52. The recombinant AAV vector of embodiment 47, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15, or a sequence at least 90% identical thereto. Embodiment 53. The recombinant AAV vector of embodiment 52, wherein the AAV vector comprises a capsid protein comprising the amino acid sequence of SEQ ID NO: 15. Embodiment 54. A composition comprising: (a) a nucleic acid molecule described in any one of embodiments 1 to 37, a plasmid described in embodiment 38, a cell described in embodiment 39, or a recombinant AAV vector described in any one of embodiments 41 to 53, and (b) a pharmaceutically acceptable carrier. Embodiment 55. A method for expressing an arrhythmogenic cardiomyopathy-associated transgene in a tissue, comprising contacting the tissue with a nucleic acid molecule described in any one of embodiments 1 to 37, a plasmid described in embodiment 38, a recombinant AAV vector described in any one of embodiments 41 to 53, or a composition described in embodiment 54, thereby expressing the arrhythmogenic cardiomyopathy-associated transgene in the tissue. Embodiment 56. The method of embodiment 55, wherein the tissue comprises one or more cells and one or more desmosome junctions. Embodiment 57. The method of embodiment 55 or embodiment 56, wherein the one or more cells are cardiac cells, endothelial cells, skin cells, bladder cells, or gastrointestinal mucosal cells. Embodiment 58. The method of embodiment 57, wherein the one or more cells are cardiac cells. Embodiment 59. The method of any one of embodiments 55 to 58, wherein the contacting step is carried out in vitro, ex vivo, or in vivo. Embodiment 60 The method of embodiment 59, wherein said contacting step is performed in vivo in a subject in need thereof. Embodiment 61. The method of embodiment 60, wherein the contacting step comprises administering to the subject a therapeutically effective amount of the nucleic acid molecule, the plasmid, the recombinant AAV vector, or the composition. Embodiment 62 The method of embodiment 60 or embodiment 61, wherein the subject is suffering from or at risk of developing the arrhythmogenic cardiomyopathy. Embodiment 63. A method for treating arrhythmogenic cardiomyopathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a nucleic acid molecule described in any one of embodiments 1 to 37, a plasmid described in embodiment 38, a cell described in embodiment 39, a recombinant AAV vector described in any one of embodiments 41 to 53, or a composition described in embodiment 54, thereby treating the arrhythmogenic cardiomyopathy in the subject. Embodiment 64. The method of embodiment 63, wherein the subject is suffering from or at risk of developing the arrhythmogenic cardiomyopathy. Embodiment 65. The method of any one of embodiments 62 to 64, wherein the arrhythmogenic cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy. Embodiment 66. The method of any one of embodiments 62-65, wherein the arrhythmogenic cardiomyopathy is associated with, precipitated by, or caused by a genetic mutation. Embodiment 67. The method of embodiment 66, wherein the genetic mutation comprises a mutation in the PKP2 gene. Embodiment 68. The method of embodiment 67, wherein said mutation in said PKP2 gene results in PKP2 haploinsufficiency. Embodiment 69. The method of any one of embodiments 62 to 68, wherein the method comprises reducing the severity of the arrhythmogenic cardiomyopathy; delaying the onset or progression; and / or eliminating symptoms. Embodiment 70. The method of embodiment 69, wherein the symptoms of the arrhythmogenic cardiomyopathy include (a) recurrent ventricular tachycardia, (b) syncope, (c) sudden death, or (d) any combination thereof. Embodiment 71. The method of any one of embodiments 62 to 70, wherein the arrhythmogenic cardiomyopathy is accompanied by (a) decreased mechanical stability between the subject's cardiomyocytes, (b) disruption of gap junctions in the subject's cardiac tissue, (c) decreased sodium current in the subject's cardiac tissue, (d) fibrosis of the right ventricular myocardium, or (e) any combination thereof. Embodiment 72. The method of any one of embodiments 62 to 71, comprising increasing mechanical stability between cardiomyocytes in a control subject having arrhythmogenic cardiomyopathy, compared to the control subject not administered the therapeutically effective amount, or compared to the subject prior to administration of the therapeutically effective amount. Embodiment 73. The method of any one of embodiments 62-72, comprising improving gap junction function in cardiac tissue of a control subject having arrhythmogenic cardiomyopathy, compared to the control subject not administered the therapeutically effective amount, or compared to the subject prior to administration of the therapeutically effective amount. Embodiment 74. The method of any one of embodiments 62-73, comprising increasing sodium current in cardiac tissue of a control subject having arrhythmogenic cardiomyopathy compared to the control subject not administered the therapeutically effective amount, or compared to the subject prior to administration of the therapeutically effective amount. Embodiment 75. The method of any one of embodiments 62-74, comprising reducing fibrosis of the right ventricular myocardium in a control subject having arrhythmogenic cardiomyopathy, compared to the control subject not administered the therapeutically effective amount, or compared to the subject prior to administration of the therapeutically effective amount. Embodiment 76. The method of any one of embodiments 62 to 75, comprising increasing the cardiac left ventricular ejection fraction in a control subject having arrhythmogenic cardiomyopathy, compared to the control subject not administered the therapeutically effective amount, or compared to the subject prior to administration of the therapeutically effective amount. Embodiment 77. The method of any one of embodiments 62-76, comprising increasing the right ventricular area of ​​the heart in a control subject having arrhythmogenic cardiomyopathy, compared to the control subject not administered the therapeutically effective amount, or compared to the subject prior to administration of the therapeutically effective amount. Embodiment 78. The method of any one of embodiments 62 to 77, comprising extending survival of a control subject having arrhythmogenic cardiomyopathy compared to the control subject not administered the therapeutically effective amount, or compared to the subject prior to administration of the therapeutically effective amount. Embodiment 79. The method of any one of embodiments 60 to 78, wherein the subject is a human subject. Embodiment 80. The method of any one of embodiments 60 to 79, wherein the nucleic acid molecule, the plasmid, the cell, the recombinant AAV vector, or the composition is administered to cardiac tissue of the subject. Embodiment 81. The method of embodiment 80, wherein the nucleic acid molecule, the plasmid, the cell, the recombinant AAV vector, or the composition is administered to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. Embodiment 82. The method of any one of embodiments 60 to 81, wherein the nucleic acid molecule, the plasmid, the cell, the recombinant AAV vector, or the composition is administered to the subject via intravenous administration, intra-arterial administration, intra-aortic administration, direct cardiac injection, coronary artery perfusion, or any combination thereof.

Claims

1. 1. A nucleic acid molecule comprising an adeno-associated virus (AAV) expression cassette, the AAV expression cassette comprising, from 5′ to 3′: (i) 5'AAV inverted terminal repeat (ITR), (ii) a promoter; (iii) an arrhythmogenic cardiomyopathy-associated transgene encoding plakophilin-2 (PKP2) or plakophilin-2 (PKP2) isoform-2a, operably linked to the promoter; and (iv) 3'AAV ITR, The nucleic acid molecule comprising:

2. 2. The nucleic acid molecule of claim 1, wherein the TNNT2 promoter comprises a nucleic acid sequence derived from (i) a human TNNT2 promoter, (ii) a chicken TNNT2 promoter, (iii) a mouse TNNT2 promoter, or (iv) any combination thereof.

3. 2. The nucleic acid molecule of claim 1, wherein the transgene comprises the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence at least 90% identical to SEQ ID NO: 1 or SEQ ID NO:

2.

4. 2. The nucleic acid molecule of claim 1, wherein at least one of the 5' ITR and the 3' ITR is isolated or derived from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV.

5. The nucleic acid molecule of claim 1 , wherein the AAV expression cassette further comprises an intron, a polyadenylation signal, a stuffer sequence, a Kozak sequence, and / or an enhancer.

6. (a) the intron is an intron derived from the human beta globin gene; (b) the polyadenylation signal is a polyadenylation signal isolated from or derived from one or more of the following genes: simian virus 40 (SV40), rBG, α-globin, β-globin, human collagen, human growth hormone (hGH), polyomavirus, human growth hormone (hGH), or bovine growth hormone (bGH); and / or (c) the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 10, or a sequence at least 90% identical thereto, or the nucleic acid sequence of acagccacc, or a sequence at least 90% identical thereto; The nucleic acid molecule of claim 1.

7. 2. The nucleic acid molecule of claim 1, wherein the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 12, or a sequence at least 90% identical thereto.

8. A cell comprising the nucleic acid molecule of claim 1.

9. 10. A method for producing a recombinant AAV vector, comprising contacting an AAV producer cell with the nucleic acid molecule of claim 1.

10. A recombinant AAV vector produced by the method of claim 9.

11. 11. The recombinant AAV vector of claim 10, wherein the AAV vector is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, avian AAV, and bovine AAV; and / or the AAV vector is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

12. 11. The recombinant AAV vector of claim 10, wherein the AAV vector comprises a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrhlO, AAVrh32.33, AAVrh74, avian AAV, or bovine AAV; and wherein the AAV vector comprises a capsid protein having one or more substitutions or mutations compared to a wild-type AAV capsid protein.

13. A method for treating arrhythmogenic cardiomyopathy and / or delaying the onset of at least one symptom of arrhythmogenic cardiomyopathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition comprising the nucleic acid molecule of claim 1, thereby treating arrhythmogenic cardiomyopathy and / or delaying the onset of at least one symptom of arrhythmogenic cardiomyopathy in the subject, wherein the arrhythmogenic cardiomyopathy is associated with, precipitated by, or caused by a mutation in the PKP2 gene.

14. 14. The method of claim 13, wherein the symptoms of the arrhythmogenic cardiomyopathy include (a) recurrent ventricular tachycardia, (b) syncope, (c) sudden death, or (d) any combination thereof, and / or the arrhythmogenic cardiomyopathy is associated with (a) decreased mechanical stability between the subject's cardiomyocytes, (b) disruption of gap junctions in the subject's cardiac tissue, (c) decreased sodium current in the subject's cardiac tissue, (d) fibrosis of the right ventricular myocardium, or (e) any combination thereof.

15. 14. The method of claim 13, wherein the method comprises increasing mechanical stability between cardiac muscle cells in a control subject having arrhythmogenic cardiomyopathy compared to the control subject not administered the therapeutically effective amount or compared to the subject prior to administration of the therapeutically effective amount.

16. 14. The method of claim 13, wherein the method comprises increasing sodium current in cardiac tissue of a control subject having arrhythmogenic cardiomyopathy compared to the control subject not administered the therapeutically effective amount or compared to the subject prior to administration of the therapeutically effective amount.

17. 14. The method of claim 13, wherein the method comprises reducing fibrosis of the right ventricular myocardium in a control subject having arrhythmogenic cardiomyopathy compared to the control subject not administered the therapeutically effective amount or compared to the subject prior to administration of the therapeutically effective amount.

18. 14. The method of claim 13, wherein the method comprises increasing the left ventricular ejection fraction of the heart in a control subject having arrhythmogenic cardiomyopathy compared to the control subject not administered the therapeutically effective amount or compared to the subject prior to administration of the therapeutically effective amount.

19. 14. The method of claim 13, wherein the method comprises reducing the right ventricular area of ​​the heart in a control subject having arrhythmogenic cardiomyopathy compared to the control subject not administered the therapeutically effective amount or compared to the subject prior to administration of the therapeutically effective amount.

20. 14. The method of claim 13, wherein the method comprises extending survival of a control subject having arrhythmogenic cardiomyopathy compared to the control subject not administered the therapeutically effective amount, or compared to the expected survival of the subject prior to administration of the therapeutically effective amount.

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