Plakophilin-2 (PKP2) gene therapy using AAV vectors

Abstract

For example, gene therapy for PKP2 (plakophilin-2) using an adeno-associated virus (AAV) vector is provided herein. The promoter of the vector may be the MHCK7 promoter or the cardiac troponin T (HTNNT2) promoter. The capsid may be AAV9 or AAVrh.74, and may contain the AAV9 or AAVrh74 capsid protein or a functional variant thereof. Other promoters or capsids may be used. Treatment methods, such as intravenous, intracoronary, intracarotid, or intracardiac administration of rAAV vectors, as well as other compositions and methods, are also provided. TIFF2026502465000046.tif34168

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 437,616, filed January 6, 2023, U.S. Provisional Patent Application No. 63 / 460,557, filed April 19, 2023, and U.S. Provisional Patent Application No. 63 / 525,426, filed July 7, 2023, which are incorporated by reference herein in their entireties.

[0002] Reference to the Electronic Sequence Listing The contents of the electronic sequence listing (ROPA_029_01WO_SeqList_ST26.xml; size: 232,839 bytes; and creation date: January 3, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Arrhythmogenic cardiomyopathy (ACM) is a form of adult-onset heart disease that affects an estimated 1 in 1,000 to 1 in 1,250 people. Because this cardiomyopathy frequently affects the right ventricular free wall, ACM has also been called arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic right ventricular dysplasia (ARVD). However, left-dominant and biventricular forms have also been observed, leading more recently to the use of the term "ACM" to encompass all forms of the disease. ACM manifests over time as destruction of the heart's muscular wall (myocardium), which increases the risk of abnormal heartbeats (arrhythmias) and sudden death during strenuous exercise in affected individuals. Individuals may also experience palpitations, lightheadedness, fainting (syncope), shortness of breath, and abnormal swelling of the legs or abdomen. Over time, ACM can lead to heart failure.

[0004] At least 13 genes have been linked to ACM, many of which are involved in the biogenesis of desmosomes, intracellular junctions that provide strong adhesion between cells. When desmosomes do not form properly, heart muscle cells can separate from one another and die. The right ventricle, in particular, can become weak and dilated, while fatty deposits and scar tissue can replace damaged heart muscle. Ultimately, these changes prevent effective cardiac pumping and disrupt the electrical signals that control heart rate, resulting in arrhythmias. Autosomal dominant plakophilin-2 (PKP2) cardiomyopathy (also known as ARVD or ARVC) is an inherited form of ACM in which mutations affecting PKP2 are detected.

[0005] Thus, there is an unmet need in the art to treat PKP2-associated diseases and disorders, including ACM. The compositions and methods disclosed herein address this need. Summary of the Invention

[0006] overview The present disclosure relates generally to gene therapy for diseases or disorders, such as cardiac diseases or disorders, using vectors that express PKP2 or functional variants thereof.

[0007] In one aspect, the disclosure provides a polynucleotide comprising a polynucleotide sequence encoding plakophilin-2 (PKP2) or a functional variant thereof operably linked to a promoter, the polynucleotide sequence comprising an expression cassette and, optionally, flanking adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0008] In some aspects, the promoter is a cardiac-specific promoter.

[0009] In some aspects, the promoter is a muscle-specific promoter.

[0010] In some aspects, the promoter is a cardiomyocyte-specific promoter.

[0011] In some embodiments, the promoter is the myosin heavy chain creatine kinase 7 (MHCK7) promoter.

[0012] In some embodiments, the MHCK7 promoter shares 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% or 100% identity with SEQ ID NO:31.

[0013] In some embodiments, the promoter is the cardiac troponin T (hTNNT2) promoter.

[0014] In some embodiments, the hTNNT2 promoter shares 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% or 100% identity with SEQ ID NO:32.

[0015] In some embodiments, the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene, and optionally, the hTNNT2 promoter and exon 1 together share 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% or 100% identity with SEQ ID NO:32.

[0016] In some embodiments, the promoter is a ubiquitous promoter, optionally a CMV promoter or a CAG promoter.

[0017] In some embodiments, the expression cassette comprises a polyA signal.

[0018] In some embodiments, the polyA signal is human growth hormone (hGH) polyA.

[0019] In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), optionally a WPRE(x) containing a mutation that abolishes potential synthesis of the Woodchuck Hepatitis Virus X protein.

[0020] In some embodiments, the plakophilin-2 (PKP2) or functional variant thereof is PKP2.

[0021] In some embodiments, the PKP2 is functional PKP2.

[0022] In some embodiments, the PKP2 is human PKP2.

[0023] In some embodiments, the PKP2 is PKP2 isoform A.

[0024] In some embodiments, PKP2 isoform A shares 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% or 100% identity with SEQ ID NO:1.

[0025] In some embodiments, the PKP2 is PKP2 isoform B.

[0026] In some embodiments, PKP2 shares 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% or 100% identity with SEQ ID NO:2.

[0027] In some embodiments, the polynucleotide sequence encoding PKP2 is a human PKP2 polynucleotide.

[0028] In some embodiments, the polynucleotide sequence encoding PKP2 shares 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% or 100% identity with SEQ ID NO:3.

[0029] In some embodiments, the polynucleotide sequence encoding PKP2 shares 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% or 100% identity with SEQ ID NO:4.

[0030] In some embodiments, the polynucleotide comprises at least about 4.0 kb, at least about 4.1 kb, at least about 4.2 kb, at least about 4.3 kb, at least about 4.4 kb, or at least about 4.5 kb.

[0031] In some embodiments, the polynucleotide comprises up to about 4.1 kb, up to about 4.2 kb, up to about 4.3 kb, up to about 4.4 kb, up to about 4.5 kb, or up to about 4.6 kb.

[0032] In some embodiments, the polynucleotide comprises between 4.0 kb and 4.6 kb, between 4.0 kb and 4.5 kb, or between 4.0 kb and 4.4 kb, or the polynucleotide comprises between 4.0 kb and 4.3 kb, between 4.0 kb and 4.2 kb, or between 4.0 kb and 4.1 kb.

[0033] In some embodiments, the PKP2 or functional variant thereof comprises at least 800 or at least 830 amino acids.

[0034] In some embodiments, the polynucleotide shares 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%, or 100% identity with any one of SEQ ID NOs:8-15, 89-96, and 97-102.

[0035] In some embodiments, the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs).

[0036] In some embodiments, the ITR is an AAV2 ITR, and / or the ITR shares 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%, or 100% identity with any one of SEQ ID NOs:20-26.

[0037] In another aspect, the present disclosure provides a gene therapy vector comprising a polynucleotide of any one of the preceding aspects.

[0038] In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.

[0039] In some embodiments, the rAAV vector is AAV9 or a functional variant thereof.

[0040] In some embodiments, the rAAV vector comprises a capsid protein that shares at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NO:77.

[0041] In some embodiments, the rAAV vector is AAVrh.10 or a functional variant thereof.

[0042] In some embodiments, the rAAV vector comprises a capsid protein that shares at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NO:79.

[0043] In some embodiments, the rAAV vector is AAV6 or a functional variant thereof.

[0044] In some embodiments, the rAAV vector comprises a capsid protein that shares at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NO:78.

[0045] In some embodiments, the rAAV vector is AAVrh.74 or a functional variant thereof.

[0046] In some embodiments, the rAAV vector comprises a capsid protein that shares at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NOs:81-83.

[0047] In another aspect, the present disclosure provides a method of treating and / or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a vector according to any one of the preceding embodiments.

[0048] In some aspects, the disease or disorder is a cardiac disorder.

[0049] In some aspects, the disease or disorder is cardiomyopathy.

[0050] In some embodiments, the cardiomyopathy is arrhythmogenic cardiomyopathy (ACM).

[0051] In some aspects, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic right ventricular dysplasia (ARVD).

[0052] In some aspects, the cardiomyopathy is hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0053] In some aspects, the disease or disorder is characterized by fibrofatty infiltration of the myocardium.

[0054] In some aspects, the disease or disorder is heart failure.

[0055] In some embodiments, the subject is a mammal.

[0056] In some embodiments, the subject is a primate.

[0057] In some embodiments, the subject is a human.

[0058] In some embodiments, the subject has a PKP2 gene mutation.

[0059] In some embodiments, the mutation is a PKP2 gene "stop-gain" variant mutation.

[0060] In some embodiments, the subject has an implantable cardioverter defibrillator (ICD).

[0061] In some embodiments, the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheter.

[0062] In some embodiments, the administration increases PKP2 expression by at least about 5%.

[0063] In some embodiments, the administration increases PKP2 expression by at least about 30%.

[0064] In some embodiments, the administration increases PKP2 expression by at least about 70%.

[0065] In some embodiments, the administration increases PKP2 expression by about 5% to about 10%.

[0066] In some embodiments, the administration increases PKP2 expression by about 30% to about 50%.

[0067] In some embodiments, the administration increases PKP2 expression by about 50% to about 70%.

[0068] In some embodiments, the administration increases PKP2 expression by about 70% to about 100%.

[0069] In certain embodiments, the increased PKP2 expression is in the heart or cardiac tissue of a subject to which the vector has been administered. In certain embodiments, the increased PKP2 expression is in the left ventricle. In certain embodiments, the increased PKP2 expression is in the right ventricle.

[0070] In some embodiments, the methods treat and / or prevent a disease or disorder.

[0071] In some aspects, the methods comprise administering an effective amount of a vector.

[0072] In some embodiments, the disease or disorder is associated with or caused by loss of function of PKP2 in the subject.

[0073] In some embodiments, the disease or disorder is associated with or caused by a gain of function of PKP2 in the subject.

[0074] In some embodiments, the subject has a mutation causing an amino acid substitution selected from Arg490Trp, Asp26Asn, Thr50_Val51SerfsX60, Arg79X, Tyr86X, Gln133X, Val406SerfsX3, Tyr616X, Trp676X, Cys796Arg, Cys796E, Tyr807X, Glu62Lys, S688P, Trp848X, Y86X, V406X, Y616X, W848X, and Y807X compared to a human PKP2 gene encoding a human PKP2 having the sequence of SEQ ID NO:2.

[0075] In some aspects, the methods comprise administering an effective amount of a pharmaceutical composition comprising the vector.

[0076] In some embodiments, the method comprises administering to about 1×10 11 Vector genome / kg ~ approx. 1 x 10 13 administering to the subject about 1 x 10 vector genomes / kg of vector; 12 Vector genome / kg ~ approx. 1 x 10 14 administering to a subject vector genomes / kg of vector, or about 1 x 10 13 Vector genome / kg ~ approx. 1 x 10 15 In some embodiments, the vector is AAV9, optionally comprising a sequence having at least 90% or at least 95% identity to any one of SEQ ID NOs: 12, 8, 13, 9, 97, 100, 89, 93, 90, or 94, and the method comprises administering about 1 x 10 vector genomes / kg of vector to the subject. 13 ~Approx. 1×10 14 genome vector / kg, e.g., approximately 1 x 10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , about 5×10 13 , about 6×10 13, about 7×10 13 , about 8×10 13 , about 9×10 13 , or approximately 1 × 10 14 In some embodiments, the vector is AAV.rh.74, optionally comprising a sequence having at least 90% or at least 95% identity to any one of SEQ ID NOs: 12, 8, 13, 9, 97, 100, 89, 93, 90, or 94, and the method comprises administering about 5×10 13 ~Approx. 5×10 14 genome vector / kg, e.g., approximately 5 x 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 vg / kg, approx. 2×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, or approximately 5 × 10 14 In some embodiments, the vector is administered intravenously, systemically, or locally to the heart.

[0077] In another aspect, the present disclosure provides a pharmaceutical composition comprising the vector of any one of the preceding embodiments.

[0078] In another aspect, the present disclosure provides a kit comprising the vector of any one of the preceding embodiments or the pharmaceutical composition of the preceding embodiments, and optionally instructions for use.

[0079] In another aspect, the present disclosure provides for the use of a vector of any one of the preceding aspects in the treatment of a disease or disorder, optionally according to a method of any one of the preceding aspects.

[0080] In another aspect, the present disclosure provides a vector of any one of the preceding aspects for use in treating a disease or disorder, optionally according to a method of any one of the preceding aspects.

[0081] In another aspect, the disclosure provides a polynucleotide comprising a polynucleotide sequence sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 12-15, 89-92, and 97-99, or any one of SEQ ID NOs: 8-11, 93-96, and 100-102.

[0082] In some embodiments, the promoter is the MHCK7 promoter.

[0083] In some embodiments, the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:31.

[0084] In some embodiments, the PKP2 is human PKP2.

[0085] In some embodiments, the PKP2 is PKP2 isoform A.

[0086] In some embodiments, PKP2 isoform A shares at least 80%, 90%, 95%, 99% or 100% identity with SEQ ID NO:1.

[0087] In another aspect, the present disclosure provides a gene therapy vector comprising a polynucleotide of any one of the preceding aspects.

[0088] In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.

[0089] In some embodiments, the rAAV vector is an AAV9 vector.

[0090] In some embodiments, the rAAV vector is an AAVrh.74 vector.

[0091] In another aspect, the present disclosure provides a method for treating and / or preventing cardiac damage in a subject identified as having a PKP2 gene mutation, the method comprising administering to the subject a vector according to any one of the preceding embodiments.

[0092] In some aspects, the cardiac disorder is a cardiomyopathy, optionally arrhythmogenic cardiomyopathy (ACM), hypertrophic cardiomyopathy, or dilated cardiomyopathy.

[0093] In some aspects, the cardiac disorder is heart failure.

[0094] In some embodiments, the subject is a mammal.

[0095] In some embodiments, the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheter.

[0096] In some embodiments, the methods prevent or reduce a decrease in left ventricular ejection fraction percent (LVEF%), optionally by at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or at least or about 100%, compared to the decrease observed in untreated subjects identified as having a PKP2 gene mutation.

[0097] In some embodiments, the methods prevent or reduce a decrease in left ventricular fractional shortening percent (FS%) by, optionally, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or at least or about 100%, as compared to the decrease observed in untreated subjects identified as having a PKP2 genetic mutation.

[0098] In some embodiments, the method prevents or reduces the increase in right ventricular area in square millimeters of RV area (mm2) by, optionally, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or at least or about 100%, as compared to the increase observed in untreated subjects identified as having a PKP2 gene mutation.

[0099] In some embodiments, the methods prevent or reduce a decrease in right ventricular velocity time integral in millimeters per second RV VTI (mm / sec) by, optionally, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or at least or about 100% as compared to the decrease observed in untreated subjects identified as having a PKP2 genetic mutation.

[0100] In some embodiments, the methods prevent or reduce an increase in left ventricular fibrosis or right ventricular fibrosis, optionally by at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, or at least or about 100%, as compared to the increase observed in untreated subjects identified as having a PKP2 gene mutation.

[0101] Various other aspects and embodiments are disclosed in the following detailed description. The present disclosure is limited only by the appended claims. [Brief explanation of the drawings]

[0102] [Figure 1]1A-1B show diagrams illustrating non-limiting examples of vector genomes. FIG. 1A shows a version without an SV40 intron. The complete polynucleotide sequence of one embodiment of the vector genome is SEQ ID NO:12, and the associated expression cassette is SEQ ID NO:8. FIG. 1B shows a version with an SV40 intron. The complete polynucleotide sequence of one embodiment of this vector genome is SEQ ID NO:89, and the associated expression cassette is SEQ ID NO:93. The MHCK7 promoter described herein is designated "enhancer / MHCK7" in the figures. [Figure 2] Figure 2 shows a diagram illustrating a non-limiting example of a vector genome. Although not shown, the hTNNT2 element also includes exon 1. The complete polynucleotide sequence of one embodiment of the vector genome is SEQ ID NO:13, and the associated expression cassette is SEQ ID NO:9. The complete polynucleotide sequence of another embodiment of the vector genome is SEQ ID NO:97, and the associated expression cassette is SEQ ID NO:100. [Figure 3] Figure 3 shows a diagram illustrating non-limiting examples of vector genomes. The complete polynucleotide sequence of one embodiment of the vector genome is SEQ ID NO:14, and the associated expression cassette is SEQ ID NO:10. The complete polynucleotide sequence of another embodiment of the vector genome is SEQ ID NO:98, and the associated expression cassette is SEQ ID NO:101. The MHCK7 promoter described herein is designated "enhancer / MHCK7" in the diagram. [Figure 4] Figure 4 shows a diagram illustrating non-limiting examples of vector genomes. The complete polynucleotide sequence of one embodiment of the vector genome is SEQ ID NO: 15, and the associated expression cassette is SEQ ID NO: 11. The complete polynucleotide sequence of another embodiment of the vector genome is SEQ ID NO: 99, and the associated expression cassette is SEQ ID NO: 102. [Figure 5]Figures 5A-5B show PKP2 protein expression in CHO-Lec2 cells. Figure 5A shows Western blots (WB) of PKP2 (upper panel) or the loading control, GAPDH (lower panel). Figure 5B shows a bar graph of the Western blot. The AAV vector serotype (AAV9 or AAVrh.74) and promoter (MHCK7 or hTnT) are indicated (AA9 indicates AAV9, rh.74 indicates AAVrh.74). Controls included GFP vector (CON-GFP) and no transduction (no Tdxn). [Figure 6] Figure 6 shows the left ventricular ejection fraction percent (LVEF%) of normal mice (control) or PKP2 knockout mice (cKO PKP2) given intravenous formulation control (FB), or mice given intravenous AAV vectors delivering the indicated human PKP2a. All AAV doses were 3E13 vg / kg. LVEF was determined using echocardiography in control-FB and PKP2-cKO mice 28 days after tamoxifen induction and 56 days after injection with FB, AAV9-MHCK7-PKP2a (3E13 vg / kg), AAV9-hTnT-PKP2a (3E13 vg / kg), AAVrh.74-MHCK7-PKP2a (3E13 vg / kg), or AAVrh.74-hTnT-PKP2a (3E13 vg / kg). Statistical analysis was performed using one-way ANOVA followed by Dunnett's post-hoc comparisons compared with the PKP2-cKO-FB control group (****p<0.0001). Data are plotted as mean ± SEM with number per group (N). [Figure 7]Figure 7 shows the percent left ventricular fractional shortening (FS%) in normal mice (control) or untreated PKP2 knockout mice (cKO PKP2) receiving intravenous formulation control (FB), or in mice receiving the indicated intravenous AAV vectors delivering human PKP2a. All AAV doses were 3E13 vg / kg. LVFS was determined by echocardiography 28 days after tamoxifen induction and 56 days after injection of AAV (AAV9-MHCK7-PKP2a, AAV9-hTnT-PKP2a, AAVrh.74-MHCK7-PKP2a, or AAVrh.74-hTnT-PKP2a at a dose of 3E13 vg / kg) or FB control. Statistical analysis was performed using one-way ANOVA with Dunnett's post-hoc comparisons (*p ≤ 0.05, ****p < 0.0001) versus the PKP2-cKO-FB control group. Data were plotted as mean ± SEM with number per group (N). [Figure 8] Figure 8 shows right ventricular area in millimeters squared (RV area (mm2)) in normal mice (control) or untreated PKP2 knockout mice (cKO PKP2) given intravenous formulation control (FB), or mice given the indicated intravenous AAV vectors delivering human PKP2a (right bar). All AAV doses were 3E13 vg / kg. RV area was determined by echocardiography 28 days after treatment with tamoxifen and 56 days after injection of AAV (AAV9-MHCK7-PKP2a, AAV9-hTnT-PKP2a, AAVrh.74-MHCK7-PKP2a, or AAVrh.74-hTnT-PKP2a at a dose of 3E13 vg / kg) or FB control. Statistical analysis was performed using one-way ANOVA followed by Dunnett's post-hoc comparisons against the PKP2-cKO-FB control group (*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, ****p < 0.0001). Data are presented as mean ± SEM with number per group (N). [Figure 9]Figure 9 shows right ventricular velocity time integral in millimeters per second (RV VTI (mm / sec)) for normal mice (control) or untreated PKP2 knockout mice (cKO PKP2) receiving intravenous formulation control (FB), or mice receiving the indicated intravenous AAV vectors delivering human PKP2a. All AAV doses were 3E13 vg / kg. RV-VTI was calculated from echocardiography. RV-VTI was measured 28 days after treatment with tamoxifen and 56 days after injection of either FB control or AAV (AAV9-MHCK7-PKP2a, AAV9-hTnT-PKP2a, AAVrh.74-MHCK7-PKP2a, or AAVrh.74-hTnT-PKP2a at a dose of 3E13 vg / kg). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc multiple comparisons test compared to PKP2-cKO-FB controls (*p ≤ 0.05, **p ≤ 0.01). Data are presented as mean ± SEM with number per group (N). [Figure 10]Figures 10A–10B show the extent of fibrosis in the left and right ventricles, based on quantification of percent collagen after cardiac trichrome histological staining, in normal mice (control) or untreated PKP2 knockout mice (cKO PKP2) receiving intravenous formulation control (FB), or mice receiving the indicated intravenous AAV vectors delivering human PKP2a. All AAV doses were 3E13 vg / kg. Control animals lacking the conditional PKP2 gene knockout (Cre Neg group) were found to have very little collagen, whereas control PKP2 knockout animals receiving formulation buffer (CKO FB group) were found to have significantly greater percentages of collagen in both the left and right ventricles. AAV-mediated PKP2 overexpression robustly attenuated collagen to varying degrees in all AAV-injected groups (n = 4 for all groups; p values ​​reflect results from one-way ANOVA and Bonferroni post-hoc analyses). Figure 10A is a bar graph of percent fibrosis in the left ventricle. Figure 10B is a bar graph of percent fibrosis in the right ventricle. Percent fibrosis was assessed in the cardiac left and right ventricles by trichrome staining performed on control and PKP2-cKO mice 28 days after tamoxifen treatment and 56 days after FB or AAV (AAV9-MHCK7-PKP2a, AAV9-hTnT-PKP2a, AAVrh.74-MHCK7-PKP2a, or AAVrh.74-hTnT-PKP2a at a dose of 3E13 vg / kg). [Figure 11]Figures 11A-11B show PKP2 protein expression in mouse hearts. All AAV doses were 3E13 vg / kg. Figure 11A shows Western blots (WB) of PKP2 (upper panel) or the loading control, GAPDH (lower panel). Figure 11B shows a bar graph of the Western blot. The AAV vector serotype (AAV9 or rh.74) and promoter (MHCK7 or hTnT) are indicated. Gel images (FIG. 11A) and quantification of PKP2 normalized to GAPDH (FIG. 11B, quantified from the simultaneously acquired upper gel in FIG. 11A) detected in the heart 28 days after tamoxifen injection and 56 days after AAV (AAV9-MHCK7-PKP2a, AAV9-hTnT-PKP2a, AAVrh.74-MHCK7-PKP2a, or AAVrh.74-hTnT-PKP2a at a dose of 3E13 vg / kg). Data are presented as mean ± SEM with number per group (N). [Figure 12]Figures 12A-12B show LVEF and FS in mice. Mice were dosed with AAV vector or formulation control (FB), and 4 weeks later, the mice were treated with tamoxifen. Figure 12A shows the percent left ventricular ejection fraction (LVEF%) at baseline (left bar) and after 28 days of tamoxifen treatment. The AAV9 dose was 1E13vg / kg, and the AAVrh.74 dose was 6E13vg / kg. LVEF was measured from echocardiograms in PKP2-cKO mice obtained 28 days after tamoxifen injection and 56 days after injection with FB, AAV9-hTnT-PKP2a (1E13vg / kg), or AAVrh.74-hTnT-PKP2a (6E13vg / kg). Statistical analysis was performed using repeated measures two-way ANOVA (***p≦0.001, ****p<0.0001). Data are plotted as mean ± SEM with number per group (N); dpi TAM = days after tamoxifen injection. Figure 12B shows percent left ventricular fractional shortening (FS%) at baseline (left bar) and after 28 days of tamoxifen treatment. The AAV9 dose was 1E13vg / kg, and the AAVrh.74 dose was 6E13vg / kg. FS was measured from echocardiograms in PKP2-cKO mice obtained 28 days after tamoxifen injection and 56 days after injection of FB, AAV9-hTnT-PKP2a (1E13vg / kg), or AAVrh.74-hTnT-PKP2a (6E13vg / kg). Statistical analysis was performed using two-way repeated measures ANOVA (***p≦0.001, ****p<0.0001). Data were plotted as mean ± SEM with number per group (N); dpi TAM = days after tamoxifen injection. [Figure 13]Figures 13A-13B show RV area and RV VTI in mice. Mice were dosed with AAV vector or formulation control (FB), and 4 weeks later, the mice were treated with tamoxifen. Figure 13A shows right ventricular area in square millimeters (RV area (mm)) at baseline (left bar) and after 28 days of tamoxifen treatment. RV area was measured from echocardiograms obtained 28 days after tamoxifen and 56 days after intravenous injection of FB, AAV9-hTnT-PKP2a (1E13 vg / kg), or AAVrh.74-hTnT-PKP2a (6E13 vg / kg). Statistical analysis was performed using two-way ANOVA (**p≦0.01, ***p≦0.001). Data are plotted as mean ± SEM with number per group (N); dpi TAM = days after tamoxifen injection. Figure 13B shows right ventricular velocity time integral in millimeters per second (RV VTI (mm / sec)) at baseline (left bar) and after 28 days of tamoxifen treatment. RV-VTI was measured from echocardiograms obtained after 28 days of tamoxifen treatment and 56 days after injection of FB (control), AAV9-hTnT-PKP2a (1E13 vg / kg), or AAVrh.74-hTnT-PKP2a (6E13 vg / kg). Statistical analysis was performed using two-way mixed effects ANOVA followed by Tukey's post hoc multiple comparison test (*p≦0.05). Data were plotted as mean ± SEM with number per group (N); dpi TAM = days after tamoxifen injection. [Figure 14]Figures 14A-14B show the extent of fibrosis in the left and right ventricles based on quantification of percent collagen after cardiac trichrome histological staining. The extent of fibrosis was assessed by trichrome staining in the left and right ventricles of control mice injected with FB and PKP2-cKO mice injected with FB, AAV9-hTnT-PKP2a (1E13 vg / kg), or AAVrh.74-hTnT-PKP2a (6E13 vg / kg) 28 days after tamoxifen and 56 days after AAV injection. The percent fibrotic cardiac tissue in the left (Figure 14A) and right (Figure 14B) ventricles was quantified. Statistical analysis was performed using one-way ANOVA with Tukey's post hoc comparisons where appropriate. The figures show the extent of fibrosis in the left and right ventricles based on quantification of percent collagen after cardiac trichrome histological staining. AAV-mediated PKP2 overexpression robustly attenuated collagen to various degrees in all AAV-injected groups (n = 4 for all groups; p values ​​reflect results from one-way ANOVA with Bonferroni post-hoc analysis). Figure 14A shows a bar graph of percent collagen in the left ventricle. Figure 14B shows a bar graph of percent collagen in the right ventricle. [Figure 15]Figures 15A-15B show PKP2 protein expression in mouse hearts. Figure 15A shows Western blots (WB) of PKP2 (upper panel) or the loading control, GAPDH (lower panel). PKP2 protein was assessed by Western blot in hearts from control mice injected with FB, or PKP2-cKO mice injected with FB, AAV9-hTnT-PKP2a (1E13 vg / kg), or AAVrh.74-hTnT-PKP2a (6E13 vg / kg) 56 days after AAV injection and 28 days after tamoxifen injection. Figure 15B shows a bar graph of the Western blot. Quantification of PKP2 protein bands relative to GAPDH. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc analysis (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Data are plotted as mean ± SEM with number per group (N). AAV vector serotype (AAV9 or rh.74rh.74) and dose (1E13vg / kg or 6E13vg / kg) are indicated. [Figure 16] Figures 16A-C show diagrams illustrating the experimental timeline for days -28, +7, and +14 of the experiment. AAV was administered to PKP2-cKO mice 7 or 14 days after tamoxifen injection (Figure 16C). Echocardiograms were performed at baseline (for AAV-treated animals) and 28 days after tamoxifen (for all animals), as well as at the study endpoint, 5 months after tamoxifen (Figures 16A and 16B). [Figure 17]Figure 17 shows Kaplan-Meier survival curves demonstrating prevention of disease-related mortality after intravenous AAVrh.74-hTnT-PKP2a in the PKP2-cKO model of ACM. The Kaplan-Meier survival curves illustrate the long-term survival of PKP2-cKO mice after intravenous AAVrh.74-hTnT-PKP2a injection when administered on day +7 (6E13 vg / kg) or +14 (2E14 vg / kg) after tamoxifen. Survival curves from animals receiving intravenous AAV9-hTnT-PKP2a (3E13 vg / kg) after tamoxifen on day +7, as well as from FB-injected PKP2-cKO (PKP2-cKO-FB) and control (control-FB) mice, are also shown. Statistical analysis using the log-rank (Mantel-Cox) test revealed significant differences between the AAVrh.74-hTnT-PKP2a and AAV9-hTnT-PKP2a groups, as well as between both groups compared to the control (****p<0.0001). The top and bottom lines at 50 days correspond to AAVrh.74-hTnt-6E13 + 7D, AAVrh.74-hTnt-2E14 + 14D, and PKP2-cKO-FB. The top and bottom lines at 150 days correspond to AAVrh.74-hTnt-6E13 + 7D, AAVrh.74-hTnt-2E14 + 14D, and AAV9-hTnt-3E13 + 7D. [Figure 18]Figures 18A-18B show the left ventricular ejection fraction (LVEF%) and left ventricular fractional shortening (FS%) for the delayed AAV injection paradigm (days +7 and +14). Figure 18A shows the left ventricular ejection fraction (LVEF%) at 28 days and 5 months after tamoxifen treatment. LVEF was determined from echocardiography performed at 28 days and 5 months after tamoxifen injection in FB-injected control (control-FB) or PKP2-cKO mice injected with FB (PKP2-cKO-FB), AAVrh.74-hTnT-PKP2a (6E13 vg / kg, day +7 of tamoxifen and 2E14 vg / kg, day +14 of tamoxifen), and AAV9-hTnT-PKP2a (3E13 vg / kg, day +7 of tamoxifen). Statistical analysis was performed using one-way ANOVA followed by Dunnett's post hoc comparison. p values ​​reflect significance compared to PKP2-cKO-FB after 28 days of tamoxifen treatment (***p≦0.001, ****p≦0.0001). Data are presented as mean ± SEM with number per group (N). Figure 18B shows the percent left ventricular fractional shortening (FS%) after 28 days and 5 months of tamoxifen treatment. FS was calculated from echocardiography performed 28 days and 5 months after tamoxifen injection. Statistical analysis (one-way ANOVA) followed by Dunnett's post hoc comparison was performed. p values ​​reflect significance compared to FB-injected PKP2-cKO (PKP2-cKO-FB) after 28 days of tamoxifen treatment (*p≦0.05, **p≦0.01, ****p≦0.0001). Data are presented as mean ± SEM with number per group (N). Graph bars from left to right correspond to legends from top to bottom. [Figure 19]Figures 19A-19B show right ventricular area (mm) and right ventricular velocity time integral in millimeters per second (RV VTI (mm / sec)) for the AAV delayed injection paradigm (days +7 and +14). Figure 19A shows right ventricular area in millimeters squared (RV area (mm)) after 28 days and 5 months of treatment with tamoxifen. RV area was calculated from echocardiograms performed 28 days and 5 months after tamoxifen injection in FB-injected control mice (control-FB) or PKP2-cKO mice injected with FB (PKP2-cKO-FB), AAVrh.74-hTnT-PKP2a (6E13 vg / kg, +7 days after tamoxifen), AAVrh.74-hTnT-PKP2a (2E14 vg / kg, +14 days after tamoxifen), and AAV9-hTnT-PKP2a (3E13 vg / kg, +7 days after tamoxifen). Statistical analysis was performed using one-way ANOVA followed by Dunnett's post-hoc comparisons. p values ​​reflect significance compared to PKP2-cKO-FB mice after 28 days of tamoxifen (****p ≤ 0.0001). Data are presented as mean ± SEM with number per group (N). Figure 19B shows right ventricular velocity time integral in millimeters per second (RV VTI (mm / sec)) at 28 days and 5 months after tamoxifen treatment. RV-VTI was calculated from echocardiography performed at 28 days and 5 months after tamoxifen injection in PKP2-cKO mice injected with control-FB or FB (PKP2-cKO-FB), AAVrh.74-hTnT-PKP2a (6E13vg / kg, +7 days after tamoxifen), AAVrh.74-hTnT-PKP2a (2E14vg / kg, +14 days after tamoxifen), and AAV9-hTnT-PKP2a (3E13vg / kg, +7 days after tamoxifen). Statistical analysis was performed using one-way ANOVA followed by Dunnett's post-hoc comparisons for PKP2-cKO-FB after 28 days of tamoxifen (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Data are presented as mean ± SEM with number per group (N). Graph bars from left to right correspond to legends from top to bottom. [Figure 20]Figures 20A-20C show the reduction of arrhythmias after AAVrh.74-mediated PKP2a overexpression in PKP2-cKO mice using the AAV delayed injection paradigm (+14 days) animal model. AAVrh.74-hTnT-PKP2a was injected 14 days after tamoxifen-induced PKP2 knockout. Electrocardiography (ECG) data were collected 21 and 28 days after tamoxifen injection (7 and 14 days after AAV, respectively). Figure 20A shows the percentage of cases with >100 premature ventricular contractions (PVCs) in animals treated with AAVrh.74-hTnT-PKP2a compared with FB-treated PKP2-cKO controls. Figure 20B shows the level of ectopic beats, demonstrating a reduction in arrhythmia burden. (*p≦0.05, **p≦0.01). Data are presented as mean ± SEM with number per group (N). For each treatment, the left bar is 21 days after tamoxifen injection, and the right bar is 28 days after tamoxifen injection. Figure 20C shows electrocardiograms in control mice (FB) and AAVrh.74-PKP2a-treated mice 21 days after tamoxifen treatment. [Figure 21A]Figures 21A-21B show the extent of fibrosis after 5 months of tamoxifen in the left ventricle (Figure 21A) and right ventricle (Figure 21B) based on quantification of percent collagen after cardiac trichrome histological staining. AAV-mediated PKP2 overexpression robustly attenuated collagen to various degrees in all AAV-injected groups [n = 4 or 8 / group; p values ​​reflect results from one-way ANOVA with Bonferroni post-hoc analysis]. The extent of fibrosis was assessed 5 months after tamoxifen induction in the left and right ventricles of FB-injected control mice (CON-FB) or PKP2-cKO mice treated with AAV9-hTnT-PKP2a (3E13 vg / kg, +7 days after tamoxifen), AAVrh.74-hTnT-PKP2a (6E13 vg / kg, +7 days after tamoxifen), and AAVrh.74-hTnT-PKP2a (2E14 vg / kg, +14 days after tamoxifen). Quantification of the percent fibrotic cardiac tissue in the left ventricle (Figure 21A) and right ventricle (Figure 21B) was performed. Statistical analysis was performed using one-way ANOVA followed by Dunnett's post-hoc comparisons (*p ≤ 0.05, **p ≤ 0.01) versus FB-treated control mice 28 days after tamoxifen. Data are presented as mean±SEM with number per group (N). [Figure 21B] See legend to Figure 21A. [Figure 22]Figure 22 shows PKP2 protein expression in the mouse heart 5 months after injection of AAV (7 or 14 days after the tamoxifen paradigm). The bar graph shows the quantification of the Western blot. AAV vector serotype AAV rh.74rh.74 at a dose of 6E13 vg / kg (AAV 7 days after tamoxifen) or 2E14 vg / kg (AAV injection 14 days after tamoxifen). PKP2 protein was evaluated by Western blot in hearts from control mice administered FB (CON-FB) and AAVrh.74-hTnT-PKP2a (6E13 vg / kg and 2E14 vg / kg) 5 months after injection of AAV (7 days after tamoxifen). Quantification of the PKP2 protein band was shown relative to GAPDH. No significant difference between groups was revealed from the statistical analysis. Data were shown as mean ± SEM using the number per group (N). [Figure 23A] Figures 23A - 23C show dose-dependent detection of (Figure 23A) vector genome (ddPCR), (Figure 23B) transgene PKP2a mRNA (RT-ddPCR), and (Figure 23C) PKP2 protein expression (Western blot) in heart tissue from AAVrh.74-PKP2a-injected animals compared to controls. The red dotted line in Figure 23C indicates the endogenous expression level of PKP2 protein normalized to GAPDH in control-FB mice. Data were shown as mean ± SD. Left bar, control mice treated with formulation buffer (FB) (control); second bar from the left, PKP2-cKO mice treated with FB (PKP2-cKO); third bar from the left, PKP2-cKO mice treated with AAVrh.74-PKP2a 3×1013 vg / kg; right bar, PKP2-cKO mice treated with AAVrh.74-PKP2a 6×1013 vg / kg. The number of mice studied was indicated by the corresponding bar (N). Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc analysis. <LoQ: below the limit of quantification; FB: formulation buffer. [Figure 23B] See the description of Figure 23A. [Figure 23C] See the description of Figure 23A. [Figure 24]Figures 24A-24C show representative images of immunofluorescence staining for plakophilin 2 (PKP2; red) and nuclei (DAPI; blue) in hearts from (Figure 24A) control mice treated with formulation buffer (FB), (Figure 24B) PKP2-cKO mice treated with FB, and (Figure 24C) PKP2-cKO mice treated with AAVrh.74-PKP2a 6 x 10 vg / kg. Mice were treated with FB or AAVrh.74-PKP2a 28 days before tamoxifen injection. White arrowheads in panels A and C highlight the localization of PKP2 to the intercalated discs. FB: formulation buffer. Scale bar in left panel = 150 μm; scale bar in right panel = 50 μm. [Figure 25A] Figures 25A-25C show representative images of left ventricular (LV) and right ventricular (RV) echocardiography to assess contractility in control and PKP2-cKO mice treated with formulation buffer (FB) or AAVrh.74-PKP2a 6 x 10 vg / kg 28 days before tamoxifen injection (Figure 25A). Measurements were collected 28 days after tamoxifen injection. Figure 25B shows quantification of left ventricular ejection fraction (LVEF) measured by long-axis B-mode echocardiography. Figure 25C shows right ventricular (RV) area measured by modified long-axis B-mode echocardiography. The red dotted line indicates the mean recorded from FB-injected PKP2-cKO mice. Data are presented as mean ± SD. Left bar: control mice treated with FB; second bar from the left: PKP2-cKO mice treated with FB; third bar from the left: PKP2-cKO mice treated with AAVrh.74-PKP2a 3 × 10 vg / kg; right bar: PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 10 vg / kg. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc analysis. LVID: left ventricular diameter; LVEF: left ventricular ejection fraction; RV: right ventricle; FB: formulation buffer. [Figure 25B] See legend to Figure 25A. [Figure 25C] See legend to Figure 25A. [Figure 26A]Figures 26A-26C show cardiac fibrosis in AAVrh.74-PKP2a-treated PKP2-cKO mice. The left panel of Figure 26A shows representative images of Masson's trichrome staining of longitudinal cardiac sections from control and PKP2-cKO mice treated with formulation buffer (FB) and PKP2-cKO mice treated with AAVrh.74-PKP2a 28 days before tamoxifen injection. The right panel of Figure 26A shows a high-contrast mask of the same sections, highlighting collagen deposition in blue. Scale bar = 1 mm for all images. Hearts were removed 28 days after tamoxifen injection. Figure 26B shows quantification of the percent left ventricular fibrosis, and Figure 26C shows right ventricular fibrosis in hearts from four different groups. Data are presented as mean ± SD. Left bar, control mice treated with FB; second bar from the left, PKP2-cKO mice treated with FB; third bar from the left, PKP2-cKO mice treated with AAVrh.74-PKP2a 3x1013vg / kg; right bar, PKP2-cKO mice treated with AAVrh.74-PKP2a 6x1013vg / kg. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc analysis. FB: formulation buffer. [Figure 26B] See legend to Figure 26A. [Figure 26C] See legend to Figure 26A. [Figure 27] Figure 27 is a graph showing survival rates after injection of AAVrh.74-PKP2a into PKP2-cKO mice 7 or 14 days after tamoxifen. Kaplan-Meier curves illustrating long-term survival rates of PKP2-cKO mice after administration of AAVrh.74-PKP2a (6 x 10 vg / kg, 7 days after tamoxifen or 2 x 10 vg / kg, 14 days after tamoxifen) compared with control and PKP2-cKO mice injected with formulation buffer (FB). [Figure 28A]Figures 28A-28D show disease progression in PKP2-cKO mice upon treatment with AAVrh.74-PKP2a 7 or 14 days after tamoxifen. The upper panel of Figure 28A shows representative images of Masson's trichrome staining of longitudinal cardiac sections from control mice treated with formulation buffer (FB) and PKP2-cKO mice treated with AAVrh.74-PKP2a 2 x 10 vg / kg 14 days after tamoxifen. Hearts were removed 5 months after tamoxifen injection. The lower panel of Figure 28A shows a high-contrast mask of the same section, highlighting collagen deposition in blue. Scale bar = 1 mm for all images. Figure 28B shows quantification of the percent left ventricular fibrosis (left panel) and right ventricular fibrosis (right panel) by Masson's trichrome staining of longitudinal heart sections in control mice and PKP2-cKO mice treated with FB at 7 days (6 x 10 vg / kg) or 14 days (2 x 10 vg / kg) after tamoxifen. Hearts were removed 5 months after tamoxifen. The dotted red line indicates the level of fibrosis in PKP2-cKO mice injected with formulation buffer (FB). Figure 28C shows quantification of left ventricular ejection fraction (LVEF), and Figure 28D shows quantification of right ventricular area (RV area) over time in PKP2-cKO mice treated with AAVrh.74-PKP2a at 7 or 14 days after tamoxifen, using the doses indicated below the bars. Echocardiography was performed 28 days and 5 months after tamoxifen administration. Echocardiography for control and FB-treated PKP2-cKO mice was performed only after 28 days of tamoxifen administration. Data are shown as mean ± SD. The left bar represents control mice treated with FB; the second bar represents PKP2-cKO mice treated with FB; the third and fourth bars represent PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 10 vg / kg; and the two right bars represent PKP2-cKO mice treated with AAVrh.74-PKP2a 2 × 10 vg / kg. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc analysis. FB: formulation buffer. [Figure 28B] See legend to Figure 28A. [Figure 28C] See legend to Figure 28A. [Figure 28D] See legend to Figure 28A. [Figure 29A] Figures 29A-29D show the results of isoproterenol-induced arrhythmias in PKP2-cKO hearts treated with AAVrh.74-PKP2a. Figure 29A shows representative electrocardiogram (ECG) traces from PKP2-cKO mice treated with formulation buffer (FB). Figure 29B shows representative ECG traces from PKP2-cKO mice treated with AAVrh.74-PKP2a 6x10 vg / kg 14 days after tamoxifen injection. Figure 29C shows the percentage of mice that exhibited >100 premature ventricular contractions (PVCs) after isoproterenol (ISO). Figure 29D shows the number of ISO-induced ectopic beats in PKP2-cKO mice treated with FB or AAVrh.74-PKP2a. Data shown in Figures 29C and 29D were quantified over a 30-minute period after ISO injection, and ECGs were recorded 21 days after tamoxifen. Data are presented as mean ± SD. Left bar, PKP2-cKO mice treated with FB; middle bar, PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 10 vg / kg; and right bar, PKP2-cKO mice treated with AAVrh.74-PKP2a 2 × 10 vg / kg. Statistical analysis was performed using one-way ANOVA followed by Dunn's post-hoc analyses. PVC: premature ventricular contraction; FB: formulation buffer. [Figure 29B] See legend to Figure 29A. [Figure 29C] See legend to Figure 29A. [Figure 29D] See legend to Figure 29A. [Figure 30A]Figures 30A-30D show PKP2 transduction in non-human primate hearts. Figures 30A-B are photomicrographs after immunohistochemical labeling for PKP2 protein in sections of whole hearts from non-human primate animals, either control FB (Figure 30A) or "low dose" treated animals (Figure 30B). Figures 30C and 30D show quantification of PKP2 protein by Western blot and transgene mRNA (hPKP2a) in the ventricles of non-human primate hearts after intravenous injection of either "low" (8 x 10 vg / kg) or "high" (3 x 10 vg / kg) doses of AAVrh.74-PKP2a or FB (control). [Figure 30B] See legend to Figure 30A. [Figure 30C] See legend to Figure 30A. [Figure 30D] See legend to Figure 30A. [Figure 31-1]Figures 31A-31D show cardiac expression of ankyrin B in AAVrh.74-PKP2a-treated PKP2-cKO mice. Figure 31A shows the method used for data acquisition and analysis of immunofluorescence (IF) ankyrin B (AnkB) signals collected from paraffin-embedded tissue sections. The red line indicates the range of fluorescence intensity as plotted in the right panel. Peak depth was defined as the range between the peak and the valley. Figure 31B shows representative images of IF staining for AnkB (green) and nuclei (blue) in hearts from control mice treated with formulation buffer (FB), PKP2-cKO mice treated with FB, and PKP2-cKO mice treated with AAVrh.74-PKP2a 6 x 10 vg / kg 28 days before tamoxifen injection. Scale bar = 100 μm. Figure 31C shows quantification of AnkB peak depth (left panel) and mean intensity (right panel) in left ventricular sections from three different groups. Figure 31D shows quantification of AnkB peak depth (left panel) and mean intensity (right panel) in right ventricular sections from three different groups. Data are shown as mean ± SD. Left bar: control mice treated with FB; middle bar: PKP2-cKO mice treated with FB; right bar: PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 10 vg / kg. Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc analysis. [Figure 31-2] See description of Figure 31-1. [Figure 32A]Figures 32A-32C show cardiac expression of desmin in AAVrh.74-PKP2a-treated PKP2-cKO mice. Figure 32A shows representative images of immunofluorescence staining for desmin (green) and nuclei (blue) in hearts from control mice treated with formulation buffer (FB), PKP2-cKO mice treated with FB, and PKP2-cKO mice treated with AAVrh.74-PKP2a 6 x 10 vg / kg 28 days before tamoxifen injection. Scale bar = 100 μm. Figure 32B shows quantification of desmin peak depth (left panel) and mean intensity (right panel) in left ventricular sections from three different groups. Figure 32C shows quantification of desmin peak depth (left panel) and mean intensity (right panel) in right ventricular sections from three different groups. Data are presented as mean ± SD. Black bars, control mice treated with FB; red bars, PKP2-cKO mice treated with FB; purple bars, PKP2-cKO mice treated with AAVrh.74-PKP2a 6 x 10 vg / kg. Statistical significance was assessed by one-way ANOVA followed by Tukey's post-hoc test. [Figure 32B] See legend to Figure 32A. [Figure 32C] See legend to Figure 32A. DETAILED DESCRIPTION OF THE INVENTION

[0103] Detailed Description of the Disclosure The present disclosure provides gene therapy vectors for PKP2 and methods of use for delivering polynucleotides encoding PKP2 polypeptides or functional variants thereof, as well as other compositions and methods.

[0104] In certain aspects, the present disclosure relates to a gene therapy vector comprising a promoter sequence operably linked to a polynucleotide encoding a PKP2 polypeptide or a functional variant thereof. In some aspects, the promoter is a myosin heavy chain creatine kinase 7 (MHCK7) promoter. In some aspects, the AAV vector is an AAV9 vector. In some aspects, the promoter is an MHCK7 promoter, and the AAV vector is an AAV9 vector. In some aspects, the promoter is an hTNNT2 promoter. In some aspects, the promoter is an hTNNT2 promoter, and the AAV vector is an AAV9 vector. In some aspects, the PKP2 is human PKP2a. In some aspects, the PKP2 is human PKP2b. In some aspects, the AAV vector is an AAVrh.74 vector. In some aspects, the promoter is an MHCK7 promoter, and the AAV vector is an AAVrh.74 vector. In some aspects, the promoter is an hTNNT2 promoter. In some aspects, the promoter is an hTNNT2 promoter, and the AAV vector is an AAVrh.74 vector. In some embodiments, the PKP2 is human PKP2a. In some embodiments, the PKP2 is human PKP2b.

[0105] This disclosure further provides methods of treating a disorder or disorder in a subject by administering to the subject a gene therapy vector of the disclosure. In certain embodiments, the disorder or disorder is arrhythmogenic cardiomyopathy (ACM).

[0106] In certain embodiments, the subject being treated is an ACM patient with one or more mutations in the PKP2 gene. More than half of ACM patients have mutations in the desmosomal gene PKP2, which encodes the protein plakophilin-2 (PKP2). PKP2 is also associated with Brugada syndrome (BrS) and idiopathic ventricular fibrillation. It is a member of the armadillo repeat and plakophilin protein family. This protein contains nine central, conserved armadillo repeat domains flanked by N- and C-terminal domains. It functions to connect cadherins and intermediate filaments in the cytoskeleton.

[0107] Plakophilin 2 localizes to cellular desmosomes and the nucleus and binds to plakoglobin, desmoplakin, and desmosomal cadherins via its N-terminal head domain. PKP2 provides lateral stabilizing forces to desmosome-intermediate filament assemblies that facilitate cell-cell contact. PKP2 may also play a role in regulating intracellular signaling, regulating electrophysiology and transport, and controlling transcriptional processes.

[0108] Intravenous injection of an AAV9 vector encoding a C-terminal deletion mutant (R735X) of PKP2a into the hearts of wild-type mice accelerates the development of ACM when the treated mice are subjected to exercise training. Cruz et al. J Am Coll Cardiol. 65(14):1438-50 (2015). Mutant PKP2a causes a disease phenotype. A control AAV9 vector expressing non-mutant PKP2a does not cause phenotypic changes in wild-type mice. Heterologous expression of wild-type human PKP2a does not induce disease or functional changes. These studies demonstrate that mutant PKP2a can cause a disease phenotype. However, these studies do not demonstrate a therapeutic role for PKP2, because heterologous expression of non-mutant PKP2 in wild-type mice did not result in phenotypic changes.

[0109] According to the present disclosure, polynucleotides encoding PKP2 or functional variants thereof may be used in generating gene therapy vectors, where PKP2 or functional variants thereof comprise at least 800 or at least 830 amino acids (e.g., no C-terminal truncation at Arg-735). The resulting vectors may be used in treating diseases or disorders, such as PKP2-associated diseases or disorders, such as ACM, Brugada syndrome (BrS), idiopathic ventricular fibrillation, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and the like.

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although methods and materials similar to or equivalent to those described herein can be used in the practice of this disclosure, suitable methods and materials are described below. Furthermore, the materials, methods, and examples described herein are merely illustrative and not intended to be limiting.

[0111] All publications and patents mentioned herein, including but not limited to PCT Application Publication No. WO2022 / 032226 and U.S. Patent Application Nos. 17 / 670,389 and 17 / 670,390, are incorporated herein by reference in their entirety, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will prevail. However, any references, papers, publications, patents, patent publications, and patent applications cited herein are not intended to be an admission or suggestion that they constitute legally valid prior art or are part of the general knowledge in any country in the world, and should not be construed as an admission or suggestion that they constitute legally valid prior art or are part of the general knowledge in any country in the world.

[0112] In this description, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the recited range, and fractions thereof, as appropriate (e.g., 1 / 10 and 1 / 100 of an integer), unless otherwise specified. The term "about," when immediately preceding a number or numeral, means that the number or numeral varies by plus or minus 10%. As used herein, the terms "a" and "an" shall be understood to refer to "one or more" of the recited components, unless otherwise specified. The use of alternatives (e.g., "or") shall be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" shall be understood to mean either one or both of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably.

[0113] As used herein, the term "over time in a subject" refers to an effect in a subject that occurs over about a day, about a month, about a year, about a decade, and / or about several decades.

[0114] As used herein, the terms "identity" and "identical" with respect to polypeptide or polynucleotide sequences refer to the percentage of exact residue matches in an alignment between a "query" sequence and a "subject" sequence, e.g., an alignment generated by the BLAST algorithm. Identity is calculated over the full length of the subject sequence unless otherwise specified. Thus, if, when the query sequence is aligned with the subject sequence, at least x% (truncated) of the residues in the subject sequence align as exact matches with the corresponding residues in the query sequence, then the query sequence "shares at least x% identity with" the subject sequence. When the subject sequence has a variable position (e.g., a residue designated X), alignment with any residue in the query sequence counts as a match. Sequence alignment may be performed using the NCBI Blast service (BLAST+ version 2.12.0).

[0115] As used herein, the term "operably linked" refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, this term refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically contiguous with the transcribed sequence, i.e., cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous or located in close proximity to the coding sequence that enhances transcription.

[0116] As used herein, " AAV vector " or " rAAV vector " refers to a recombinant vector that contains one or more polynucleotides (or transgenes) of interest flanked by AAV inverted terminal repeats (ITRs).Such AAV vectors can replicate and be packaged into infectious viral particles when present in host cells that are transfected with a plasmid that encodes and expresses rep and cap gene products.Alternatively, AAV vectors can be packaged into infectious viral particles using host cells that are stably engineered to express rep and cap genes.

[0117] As used herein, "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a viral particle that is composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector.As used herein, when a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), it is typically referred to as an "AAV vector particle" or simply an "AAV vector."Therefore, the production of an AAV vector particle necessarily includes the production of an AAV vector, and therefore, the vector is contained within an AAV vector particle.

[0118] As used herein, "promoter" refers to a polynucleotide sequence capable of promoting the initiation of RNA transcription from a polynucleotide in a eukaryotic cell.

[0119] As used herein, "vector genome" refers to a polynucleotide sequence packaged by a vector (e.g., an rAAV virion), including flanking sequences (in AAV, inverted terminal repeat sequences). The terms "expression cassette" and "polynucleotide cassette" refer to the portion of a vector genome that is between the flanking ITR sequences. "Expression cassette" means that the vector genome contains at least one gene encoding a gene product operably linked to elements that drive expression (e.g., a promoter).

[0120] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk for or suffering from a disease, disorder, or condition that can be treated or ameliorated by the recombinant gene therapy vector or gene editing system disclosed herein. A patient or subject in need may be, for example, a patient or subject diagnosed with a heart-related disorder. The subject may have a PKP2 gene mutation that causes abnormal expression of the PKP2 protein, or may have a deletion of all or part of the PKP2 gene or gene regulatory sequence. The terms "subject" and "patient" are used interchangeably herein. The subject treated by the methods described herein may be an adult or a child. The subject may range in age.

[0121] As used herein, the term "variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein. As used herein, the term "functional variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein and retains one or more desirable activities of the parent protein.

[0122] As used herein, "treating" refers to alleviating one or more symptoms of a disease or disorder. The term "preventing" refers to delaying or halting the onset of one or more symptoms of a disease or disorder, or slowing the progression of a PKP2-related disease or disorder, such as arrhythmogenic cardiomyopathy (ACM).

[0123] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long, including two approximately 145-nucleotide inverted terminal repeats (ITRs). There are several known AAV variants, sometimes called serotypes, classified by antigenic epitopes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is shown in GenBank accession number NC_002077. The complete genome of AAV-2 is shown in GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983). The complete genome of AAV-3 is shown in GenBank accession number NC_1829. The complete genome of AAV-4 is shown in GenBank accession number NC_001829. The complete genome of AAV-5 is shown in GenBank accession number AF085716. The complete genome of AAV-6 is set forth in GenBank accession number NC_00 1862. At least portions of the AAV-7 and AAV-8 genomes are set forth in GenBank accession numbers AX753246 and AX753249, respectively. The AAV-9 genome is set forth in Gao et al., J. Virol., 78: 6381-6388 (2004). The AAV-10 genome is set forth in Mol. Ther., 13(1): 67-76 (2006). The AAV-11 genome is set forth in Virology, 330(2): 375-383 (2004). The sequence of the AAVrh.74 genome is set forth in U.S. Patent No. 9,434,928, which is incorporated herein by reference. Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 after their relative map locations) drive the expression of two AAV internal open reading frames encoding the rep and cap genes.Differential splicing of two rep promoters (p5 and p19) and one AAV intron (at nucleotides 2107 and 2227) produces four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins have multiple enzymatic properties that ultimately are responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0124] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cultured cells is noncytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cell types, enabling the potential for targeting many different tissues in vivo. Furthermore, AAV slowly transduces dividing and nondividing cells and can persist essentially for the lifetime of the cell as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted into a plasmid as cloned DNA, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb region of the genome (encoding the replication and structural capsid proteins rep-cap) can be exchanged with foreign DNA. The rep and cap proteins can be provided in trans to generate AAV vectors. Another important feature of AAV is that it is an extremely stable and robust virus. AAV readily survives the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV unnecessary. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0125] Gene delivery viral vectors useful in the practice of the present disclosure can be constructed using methodologies well known in the art of molecular biology.Typically, viral vectors carrying transgenes are assembled from a polynucleotide encoding the transgene, appropriate regulatory elements, and elements required for the production of viral proteins that mediate cell transduction.Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses.Representative examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with baculovirus helper vectors), 293 cells, etc.As described in US20170218395A1, AAV vectors can be produced using a herpesvirus-based system. Detailed protocols for generating such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and WO94 / 19478, the complete contents of each of which are incorporated herein by reference.

[0126] The present disclosure contemplates compositions and methods of use related to plakophilin-2 (PKP2) proteins or polypeptides. Various PKP2 mutations are known to be associated with cardiomyopathy and heart failure, including diseases such as those described in van Tintelen et al. Circulation 113:1650-58 (2006); Novelli Front. Cardiovasc. Med. (2008); and other sources. Therefore, viral vector-mediated PKP2 gene delivery may serve as a viable therapeutic agent for PKP2-related human diseases, such as cardiomyopathy and heart failure.

[0127] More than 230 PKP2 gene mutations have been identified in people with arrhythmogenic cardiomyopathy (ACM). (See "PKP2 Gene," MedlinePlus.) The condition most commonly affects the heart muscle surrounding the right ventricle, one of the heart's two lower chambers. ACM increases the risk of abnormal heartbeats (arrhythmias) and sudden death. Some PKP2 gene mutations lead to the production of abnormally short versions of plakophilin 2. Other mutations alter the structure of plakophilin 2 by adding, deleting, or modifying one or more of its protein building blocks (amino acids). Research suggests that the altered protein impairs desmosome formation and function.

[0128] Without normal desmosomes, heart muscle cells separate from one another and die, especially when the heart muscle is under stress (for example, during strenuous exercise). The damaged muscle is gradually replaced by fat and scar tissue. As this abnormal tissue grows, the wall of the right ventricle stretches, preventing the heart from pumping blood effectively. These changes also disrupt the electrical signals that control the heartbeat, which can lead to arrhythmias. Descriptions of PKP2-related diseases are described in the following references: Bonne et al. Genomics 51:452-454 (1998) [PubMed: 9721216]; Bonne et al. Cytogenet. Cell Genet. 88:286-287 (2000) [PubMed: 10828611]; Dalal et al., Circulation 113:1641-1649 (2006) [PubMed: 16549640]; Gerull et al. Nature Genet. 36:1162-1164 (2004) [PubMed: 15489853]; Grossmann et al. J. Cell Biol. 167:149-160 (2004) [PubMed: 15479741]; Marcus et al. Circulation 65:384-398 (1982) [PubMed: 7053899]; and Mertens et al. J. Cell Biol. 135:1009-1025 (1996) [PubMed: 8922383]. See also OMIM.org entry 602861 ("PLAKOPHILIN 2; PKP2").

[0129] The native sequences of human PKP2a and its isoform PKP2b are shown below, with Arg-735 underlined. TIFF2026502465000002.tif195111

[0130] In some embodiments, the PKP2 protein comprises a polypeptide sequence that is at least or about 75%, at least 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to any one of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the PKP2 protein is a wild-type or native PKP2 protein, e.g., human PKP2a or human PKP2b.

[0131] In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding PKP2 or a functional variant thereof, operably linked to a promoter. In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding PKP2, operably linked to a promoter.

[0132] In certain embodiments, a polynucleotide encoding PKP2a may comprise a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:3.

[0133] In certain embodiments, a polynucleotide encoding PKP2b may comprise a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% (9 at least or about 9%), or 100% identical to SEQ ID NO:4.

[0134] Optionally, the polynucleotide sequence encoding the vector genome may comprise a Kozak sequence, including, but not limited to, GCCACCATGG (SEQ ID NO:5). The Kozak sequence may overlap with the polynucleotide sequence encoding the PKP2a protein or a functional variant thereof. For example, the vector genome may comprise a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:6 (the first 10 nucleotides constitute the Kozak sequence).

[0135] In certain embodiments, the Kozak sequence may overlap with a polynucleotide sequence encoding a PKP2 (e.g., PKP2a or PKP2b) protein or functional variant thereof. For example, the vector genome may comprise a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:7 (the first 10 nucleotides constitute the Kozak sequence).

[0136] In some aspects, the Kozak sequence is TIFF2026502465000003.tif37128.

[0137] In some aspects, the vector genome does not include a Kozak sequence.

[0138] The polynucleotide sequence may be codon-optimized. For example, the vector genome may comprise a polynucleotide sequence encoding PKP2a that shares at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NO:87. The vector genome may comprise a polynucleotide sequence encoding PKP2b that shares at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NO:88.

[0139] The AAV virion of the present disclosure comprises a vector genome. The vector genome may include an expression cassette (or a polynucleotide cassette for gene editing applications that do not require expression of a polynucleotide sequence). Any suitable inverted terminal repeat (ITR) may be used. The ITR may be an AAV ITR from the same serotype as the capsid present in the AAV virion, or an AAV ITR from a different serotype than the capsid (e.g., AAV2 ITRs may be used with AAV virions having an AAV9 capsid or an AAVrh.74 capsid). In each case, the serotype of the capsid determines the name given to the virion. The ITRs are generally the 5'-most and 3'-most elements of the vector genome. The vector genome also generally contains, in 5'-to-3' order, a promoter, a transgene, a 3'-untranslated region (UTR) sequence (e.g., a WPRE element), and a polyadenylation sequence. In variations, the vector genome includes an enhancer element (generally 5' to the promoter) and / or an exon (generally 3' to the promoter). In variations, the vector genome of the present disclosure encodes a partial or complete transgene sequence that is used as a repair template in a gene editing system. In such variations, the vector genome may include an exogenous promoter, or the gene editing system may insert the transgene into a locus in the genome that has an endogenous promoter, such as a cardiac-specific promoter or a myocyte-specific promoter.

[0140] In some embodiments, the 5' ITR comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:20.

[0141] In some embodiments, the 5' ITR comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:21.

[0142] In some embodiments, the 5' ITR comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:22.

[0143] In some embodiments, the 5' ITR comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:23.

[0144] In some embodiments, the 3' ITR comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:24.

[0145] In some embodiments, the 3' ITR comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:25.

[0146] In some embodiments, the 3' ITR comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:26.

[0147] In some embodiments, the vector genome comprises one or more filler sequences that are at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to, e.g., SEQ ID NO:27; SEQ ID NO:28; or SEQ ID NO:29.

[0148] In some embodiments, the polynucleotide sequence encoding the PKP2 protein or its functional variant is operably linked to a promoter. In certain embodiments, the promoter is an MHCK7 promoter. In certain embodiments, the promoter is a TNNT2 promoter.

[0149] The present disclosure contemplates the use of various promoters. Promoters useful in embodiments of the present disclosure include, but are not limited to, the cytomegalovirus (CMV) promoter, the phosphoglycerate kinase (PGK) promoter, or a promoter sequence consisting of a CMV enhancer, the chicken β-actin promoter, and a portion of the rabbit β-globin gene (CAG). In some cases, the promoter may be a synthetic promoter. An exemplary synthetic promoter is provided by Schlabach et al. PNAS USA. 107(6):2538-43 (2010). In some embodiments, the promoter comprises a polynucleotide sequence at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:30.

[0150] In some embodiments, the polynucleotide sequence encoding the PKP2 protein or its functional variant is operably linked to an inducible promoter. The inducible promoter may be configured to transcriptionally express or not transcriptionally express the polynucleotide sequence in response to the addition or accumulation of a drug, or in response to the removal, degradation, or dilution of the drug. The drug may be a drug. The drug may be tetracycline or one of its derivatives, including but not limited to doxycycline. In some cases, the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically regulated promoter, or a physically regulated promoter (i.e., a promoter that responds to the presence or absence of light or low or high temperatures). Inducible promoters include heavy metal ion-inducible promoters (e.g., mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters) and promoters derived from T7 phage that are active in the presence of T7 RNA polymerase. This list of inducible promoters is not limiting.

[0151] In some cases, the promoter is a tissue-specific promoter, for example, a promoter that can drive expression in cardiac cells to a greater extent than in non-cardiac cells. In some embodiments, the tissue-specific promoter is selected from any of a variety of cardiac cell-specific promoters, including, but not limited to, desmin (Des), α-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (cTnC), cardiac troponin T (hTNNT2), muscle creatine kinase (CK), and their promoter / enhancer regions, such as MHCK7 combinations. In some cases, the promoter is a ubiquitous promoter. "Ubiquitous promoter" refers to a promoter that is not tissue-specific under either experimental or clinical conditions. In some cases, the ubiquitous promoter is any one of cytomegalovirus (CMV), cytomegalovirus early enhancer element, chicken β-actin gene intron with splice acceptor of rabbit β-globin gene (CAG), ubiquitin C (UBC), phosphoglycerate kinase (PGK), eukaryotic translation elongation factor 1 alpha 1 (EF1-α), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), simian virus 40 (SV40), hepatitis B virus (HBV), chicken β-actin, and human β-actin promoter.

[0152] In some embodiments, the promoter sequence is selected from Table 3. In some embodiments, the promoter comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to any one of SEQ ID NOs: 31-51. In some embodiments, the promoter comprises a fragment of the polynucleotide sequence of any one of SEQ ID NOs: 31-48, e.g., a fragment comprising at least 25%, at least 50%, at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of SEQ ID NOs: 31-48.

[0153] [Table 3] TIFF2026502465000005.tif226152TIFF2026502465000006.tif226152TIFF20265024650 00007.tif222152TIFF2026502465000008.tif222152TIFF2026502465000009.tif230152

[0154] In certain embodiments, the vector genome comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO: 31. In certain embodiments, the vector genome comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO: 32. In certain embodiments, the vector genome comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to SEQ ID NO:33.

[0155] Further illustrative examples of promoters are the SV40 late promoter from Simian Virus 40, the baculovirus polyhedron enhancer / promoter element, herpes simplex virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV), and various retroviral promoters containing LTR elements. A wide variety of other promoters are known and generally available in the art, and the sequences of many such promoters are available in sequence databases such as the GenBank database.

[0156] In some cases, the vectors of the present disclosure further comprise one or more regulatory elements selected from the group consisting of an enhancer, an intron, a poly-A signal, a sequence encoding a 2A peptide, a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), and a HPRE (hepatitis B post-transcriptional regulatory element).

[0157] In some embodiments, the vector comprises a CMV enhancer.

[0158] In certain embodiments, the vector comprises one or more enhancers. In certain embodiments, the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a β-actin enhancer sequence, or an EF1-α enhancer sequence. The above sequences are known in the art. For example, the sequence of the CMV immediate early (IE) enhancer is SEQ ID NO:50.

[0159] In certain embodiments, the vector comprises one or more introns, hi certain embodiments, the intron is a rabbit globin intron sequence, a chicken β-actin intron sequence, a synthetic intron sequence, an SV40 intron, or an EF1-α intron sequence.

[0160] In certain embodiments, the vector comprises a poly A sequence. In certain embodiments, the poly A sequence is a rabbit globin poly A sequence, a human growth hormone poly A sequence, a bovine growth hormone poly A sequence, a PGK poly A sequence, an SV40 poly A sequence, or a TK poly A sequence. In some embodiments, the poly A signal may be a bovine growth hormone polyadenylation signal (bGHpA).

[0161] In certain embodiments, the vector comprises one or more transcript stabilizing elements. In certain embodiments, the transcript stabilizing elements are a WPRE sequence, an HPRE sequence, a scaffold attachment region, a 3'UTR, or a 5'UTR. In certain embodiments, the vector comprises both a 5'UTR and a 3'UTR.

[0162] In some embodiments, the vector comprises a 5' untranslated region (UTR) selected from Table 4. In some embodiments, the vector genome comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to any one of SEQ ID NOs:51-61.

[0163] [Table 4] TIFF2026502465000011.tif224152TIFF2026502465000012.tif225152TIFF2026502465000013.tif180152

[0164] In some embodiments, the vector comprises a 3' untranslated region selected from Table 5. In some embodiments, the vector genome comprises a polynucleotide sequence that is at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identical to any one of SEQ ID NOs:62-70.

[0165] [Table 5] TIFF2026502465000015.tif224152TIFF2026502465000016.tif127152

[0166] In some embodiments, the vector comprises a polyadenylation (polyA) signal selected from Table 6. In some embodiments, the polyA signal comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:71-75.

[0167] [Table 6]

[0168] Exemplary vector genomes are illustrated in Figures 1-4 and are designated as SEQ ID NOs: 12-15, 89-92, and 97-99. The expression cassettes for each vector genome sequence are SEQ ID NOs: 8-11, 93-96, and 100-102, respectively. In some embodiments, the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to any one of SEQ ID NOs: 12-15, 89-92, or 97-99, optionally with or without ITR sequences. In some embodiments, the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to any one of SEQ ID NOs:8-11, 93-96, or 100-102. In certain embodiments, the vectors and expression cassettes disclosed herein demonstrate advantageous properties compared to alternatives, such as, for example, higher expression in mammalian cells (including target cells), more selective expression in target tissue (e.g., cardiac cells such as cardiomyocytes), higher infection rates, and / or increased manufacturability, e.g., higher production yields. In certain embodiments, the target tissue is heart tissue or cardiac tissue.

[0169] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a PKPa transgene; a WPRE(x) element; a pAGH-HS sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequences SEQ ID NO:31; any one of SEQ ID NOs:3, 6, and 87; SEQ ID NO:63; and SEQ ID NO:75; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0170] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a PKPa transgene; a WPRE(x) element; a pAGH-HS sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequences SEQ ID NO:32 or 33; any one of SEQ ID NOs:3, 6, and 87; SEQ ID NO:63; and SEQ ID NO:75; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0171] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a PKPb transgene; a WPRE(x) element; a pAGH-HS sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequences SEQ ID NO:31; any one of SEQ ID NOs:4, 7, and 88; SEQ ID NO:63; and SEQ ID NO:75; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0172] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a PKPb transgene; a WPRE(x) element; a pAGH-HS sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequences SEQ ID NO:32 or 33; any one of SEQ ID NOs:4, 7, and 88; SEQ ID NO:63; and SEQ ID NO:75; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0173] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a PKPa transgene; optionally, a WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequences SEQ ID NO:31; any one of SEQ ID NOs:3, 6, and 87; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0174] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a PKPa transgene; optionally, a WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO:32 or 33; any one of SEQ ID NOs:3, 6, and 87; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0175] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a PKPb transgene; optionally, a WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequences SEQ ID NO:31; any one of SEQ ID NOs:4, 7, and 88; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0176] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a PKPb transgene; optionally, a WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence SEQ ID NO:32 or 33; any one of SEQ ID NOs:4, 7, and 88; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0177] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; an SV40 intron; a PKPa transgene; optionally, a WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequences SEQ ID NO:31; SEQ ID NO:53 or 61; or any one of SEQ ID NOs:3, 6, and 87; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0178] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; an SV40 intron; a PKPa transgene; optionally, a WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequences SEQ ID NO:32 or 33; SEQ ID NO:53 or 61; or any one of SEQ ID NOs:3, 6, and 87; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0179] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; an SV40 intron; a PKPb transgene; optionally, a WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequences SEQ ID NO:31; SEQ ID NO:53 or 61; or any one of SEQ ID NOs:4, 7, and 88; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0180] In certain embodiments, the vector genome comprises, from 5' to 3', a 5' ITR; an hTnnT2 promoter; an SV40 intron; a PKPb transgene; optionally, a WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, from 5' to 3', the polynucleotide sequences SEQ ID NO:32 or 33; SEQ ID NO:53 or 61; or any one of SEQ ID NOs:4, 7, and 88; or a polynucleotide sequence sharing at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to each of the foregoing. In certain embodiments, the vector genome is packaged into an AAV9 or AAVrh.74 vector. The PKP2a transgene of this embodiment is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2a of at least 800, at least 837, or at least 830 amino acids. In particular embodiments, the PKP2a polypeptide comprises 837 amino acids. In a related embodiment, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding a PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In particular embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0181] In each case, optionally, a WPRE element may or may not be present.

[0182] In certain embodiments, the vector is AAVrh.74, and the vector genome encodes PKP2-A (shorter isoform, predominantly expressed in the heart), with or without codon optimization (e.g., optimized for human expression and CpG island reduction), operably linked to a cardiac-specific promoter. The AAVrh.74 vector is administered intravenously (IV) at a dose range of mid-E13vg / kg to mid-E14vg / kg, for example, for patient populations requiring a high vector load, e.g., adults presenting at an average age of 35 years.

[0183] Adeno-associated virus vector AAV vectors useful in practicing the present disclosure can be packaged into AAV virions (virus particles) using a variety of systems, including adenovirus-based systems and helper-free systems. Standard methods in AAV biology include, but are not limited to, those described in Kwon and Schaffer. Pharm Res. (2008) 25(3):489-99; Wu et al. Mol. Ther. (2006) 14(3):316-27; Burger et al. Mol. Ther. (2004) 10(2):302-17; Grimm et al. Curr Gene Ther. (2003) 3(4):281-304; Deyle DR, Russell DW. Curr Opin Mol Ther. (2009) 11(4):442-447; McCarty et al. Gene Ther. (2001) 8(16):1248-54; and Duan et al. Mol Ther. (2001) 4(4):383-91. Exemplary helper-free systems include, but are not limited to, those described in US Pat. No. 6,004,797; US Pat. No. 7,588,772; and US Pat. No. 7,094,604.

[0184] The AAV DNA in the rAAV genome can be derived from any AAV variant or serotype from which a recombinant virus can be obtained, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAVrh.10, including wild-type and variant versions of any of these serotypes. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0185] In some cases, the rAAV contains a self-complementary genome. As defined herein, a rAAV containing a "self-complementary" or "double-stranded" genome refers to a rAAV that has been engineered so that its coding region is configured to form an intramolecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors promoter efficient transduction independently of DNA synthesis. Gene Therapy. 8 (16): 1248-54 (2001). The present disclosure contemplates the use of a rAAV containing a self-complementary genome, in some cases. This is because, upon infection (such transduction), rather than waiting for cellular synthesis of the second strand of the rAAV genome, the two complementary halves of the scAAV join to form a single double-stranded DNA (dsDNA) unit that is ready to replicate and transcribe. Rather than the full coding capacity found in rAAV (4.7-6 kb), rAAV containing self-complementary genomes are understood to only possess about half that amount (about 2.4 kb).

[0186] In other cases, rAAV vectors contain a single-stranded genome. As defined herein, a "single, standard" genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAVs have a single-stranded DNA genome. Some have suggested that rAAVs must be scAAVs to achieve efficient cell transduction. However, with the understanding that other genetic modifications of rAAV vectors may be beneficial to achieve optimal gene transcription in target cells, the present disclosure contemplates rAAV vectors that may have a single-stranded genome rather than a self-complementary genome. In some cases, the present disclosure relates to single-stranded rAAV vectors that can achieve efficient gene transfer to the anterior segment of the mouse eye. See Wang et al. Single-stranded adeno-associated virus achieves efficient gene transfer to the anterior segment in the mouse eye. PLoS ONE 12(8): e0182473 (2017).

[0187] In some cases, the rAAV vector is an rAAV vector of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.10, or AAVrh.74. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). In some cases, the rAAV vector is an rAAV vector of serotype AAV9. In some embodiments, the rAAV vector is an rAAV vector of serotype AAV9 and comprises a single-stranded genome. In some embodiments, the rAAV vector is an AAV9 serotype rAAV vector and comprises a self-complementary genome. In some embodiments, the rAAV vector comprises an AAV2 inverted terminal repeat (ITR) sequence. In some embodiments, the rAAV vector comprises an AAV2 genome, such that the rAAV vector is an AAV-2 / 9 vector, an AAV-2 / 6 vector, or an AAV-2 / 8 vector.

[0188] The full-length sequence of the capsid gene of most known AAVs is set forth in US Pat. No. 8,524,446, which is incorporated herein in its entirety.

[0189] The AAV vector may comprise a wild-type AAV sequence or may comprise one or more modifications to the wild-type AAV sequence. In certain embodiments, the AAV vector comprises one or more amino acid modifications, optionally substitutions, deletions, or insertions, within the capsid protein, optionally VP1, VP2, and / or VP3. In certain embodiments, the modifications result in reduced immunogenicity when the AAV vector is provided to a subject.

[0190] The capsid protein of the rAAV may be modified so that the rAAV is targeted to a specific target tissue of interest, such as cardiomyocytes. In some embodiments, the rAAV is injected directly into the intraventricular space of a subject.

[0191] In some embodiments, the rAAV virion is an AAV2 rAAV virion. The capsid can be an AAV2 capsid or a functional variant thereof. In some embodiments, the AAV2 capsid comprises a capsid protein that shares at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identity with a reference AAV2 capsid protein, for example, SEQ ID NO: 76.

[0192] In some embodiments, the rAAV virion is an AAV9 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAV9 capsid comprises a capsid protein that shares at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identity with a reference AAV9 capsid protein, for example, SEQ ID NO: 77.

[0193] In some embodiments, the rAAV virion is an AAV6 rAAV virion. The capsid may be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAV6 capsid comprises a capsid protein that shares at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identity with a reference AAV6 capsid protein, for example, SEQ ID NO: 78.

[0194] In some embodiments, the rAAV virion is an AAVrh.10 rAAV virion. The capsid may be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAVrh.10 capsid comprises a capsid protein that shares at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identity with a reference AAVrh.10 capsid protein, e.g., SEQ ID NO:79.

[0195] In some embodiments, the rAAV virion is an AAVrh.74 rAAV virion. The capsid can be an AAVrh.74 capsid or a functional variant thereof. In some embodiments, the AAVrh.74 capsid comprises a capsid protein that shares at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identity with a reference AAVrh.74 capsid protein, e.g., SEQ ID NOs:81-83.

[0196] The polynucleotide sequence of wild-type AAVrh.74 cap is set forth as SEQ ID NO: 80. The present disclosure further provides the protein sequences of AAVrh.74 VP1, VP2, and VP3, including SEQ ID NOs: 81-83, and homologs or functional variants thereof. TIFF2026502465000018.tif175146TIFF2026502465000019.tif64145

[0197] In certain instances, the AAVrh.74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the rAAV vector comprises a polypeptide that comprises, consists essentially of, or even consists of a sequence identical to the amino acid sequence of AAVrh.74 VP1 set forth in SEQ ID NO: 81, e.g., a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74 VP1 set forth in SEQ ID NO: 81. In some embodiments, the rAAV vector comprises a polypeptide that comprises, consists essentially of, or even consists of a sequence identical to the amino acid sequence of AAVrh.74 VP2 set forth in SEQ ID NO:82, e.g., a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74 VP2 set forth in SEQ ID NO:82. In some embodiments, the rAAV vector comprises a polypeptide that comprises, consists essentially of, or even consists of a sequence identical to the amino acid sequence of AAVrh.74 VP3 set forth in SEQ ID NO:83, e.g., a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74 VP3 set forth in SEQ ID NO:83.

[0198] In some embodiments, the rAAV virion is an AAV-PHP.B rAAV virion or a neurotrophic variant thereof, such as, but not limited to, the rAAV virions disclosed in International Patent Publication Nos. WO2015 / 038958 A1 and WO2017 / 100671 A1. For example, the AAV capsid protein may include at least four consecutive amino acids from the sequence TLAVPFK (SEQ ID NO:85) or KFPVALT (SEQ ID NO:86), e.g., inserted between the sequence encoding amino acids 588 and 589 of the AAV9 capsid protein.

[0199] The capsid may be an AAV-PHP.B capsid or a functional variant thereof. In some embodiments, the AAV-PHP.B capsid comprises a capsid protein that shares at least 98%, 99%, or 100% identity to a reference AAV-PHP.B capsid protein, e.g., SEQ ID NO:84.

[0200] Additional AAV capsids for use in the rAAV virions of the present disclosure include the AAV capsids disclosed in Patent Publication Nos. WO2009 / 012176A2 and WO2015 / 168666A2.

[0201] Without being bound by theory, the present inventors have determined that an AAV9 vector, such as an AAVrh.74 or AAVrh.10 vector, confers desirable cardiac tropism to the vector. Without being bound by theory, the present inventors have further determined that an AAV9 vector, such as an AAVrh.74 or AAVrh.10 vector, confers desirable specificity to cardiac cells.

[0202] In an aspect, the present disclosure provides a pharmaceutical composition comprising an rAAV virion of the present disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0203] For administration purposes, various solutions can be used, including sterile aqueous solutions, optionally for administration by injection. Such aqueous solutions may be buffered, if desired, and the liquid diluent may first be made isotonic with saline or glucose. Solutions of rAAV as the free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water appropriately mixed with a surfactant, such as 0.001% or 0.01% poloxamer 188. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, all sterile aqueous media employed are readily available by standard techniques well known to those skilled in the art.

[0204] Pharmaceutical forms suitable for use as injectables include, but are not limited to, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. The form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The activity of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0205] Sterile injectable solution can be prepared by incorporating the required amount of rAAV into suitable solvent with various other components as mentioned above as necessary, and then sterilize by filtration.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle that contains the basic dispersion medium and other necessary components from above.For the preparation of sterile injectable solution, some specific preparation methods are vacuum drying and freeze-drying, which produce powder of active ingredient and any other desired components from the solution that has been previously sterilized and filtered.

[0206] In another aspect, the disclosure includes a kit comprising the rAAV virions of the disclosure and instructions for use.

[0207] In one aspect, the present disclosure provides a method for increasing PKP2 activity in a cell, comprising contacting the cell with the rAAV of the present disclosure. In another aspect, the present disclosure provides a method for increasing PKP2 activity in a subject, comprising administering the rAAV of the present disclosure to the subject. In some embodiments, the cell and / or subject has a defective expression level and / or activity of PKP2 messenger RNA or PKP2 protein, and / or contains a loss-of-function mutation in PKP2. The cell may be a cardiac cell, for example, a cardiomyocyte. In certain embodiments, the subject is a mammal, for example, a human.

[0208] In some embodiments, the methods promote survival of cardiac cells, e.g., cardiomyocytes, in cell culture and / or in vivo, hi some embodiments, the methods promote and / or restore cardiac function.

[0209] In another aspect, the present disclosure provides a method for treating a disease or disorder in a subject in need thereof, comprising administering an effective amount of an rAAV virion of the present disclosure to the subject. In some embodiments, the disease or disorder is a cardiac disease or disorder. Exemplary cardiac disorders include heart failure, arrhythmogenic right ventricular cardiomyopathy (ACM), Brugada syndrome (BrS), and idiopathic ventricular fibrillation. In certain embodiments, the subject suffers from or is at risk of arrhythmogenic right ventricular cardiomyopathy (ACM). In certain embodiments, the subject is a mammal, e.g., a human, with a loss-of-function mutation in the PKP2 gene. In certain methods, rAAV virion treatment results in expression of the PKP2 protein encoded by the rAAV virion in the subject, e.g., in the heart or cardiac tissue of the subject. In certain embodiments, rAAV virion treatment results in detectable PKP2 protein levels in the subject's heart that are at least 2-fold, at least 5-fold, at least 10-fold, or greater.

[0210] High-risk PKP2 patients include, for example, 1) carriers of protein-truncating PKP2 variants, including nonsense, frameshift, deletion, and stop-gain mutations, 2) patients with ICD implants, or 3) patients who meet HRS Class I or IIa criteria for ICD placement (potentially having an ICD in place). In certain embodiments, the methods of the present disclosure may be used to treat any of the disclosed patient populations.

[0211] A stop-gain variant is a type of substitution that results in a nonsense mutation, such as a stop codon, resulting in loss of function (or loss of function due to nonsense-mediated degradation, degradation of the truncated transcript, and a significant reduction in protein abundance). Non-limiting examples of stop-gain variant mutations include, for example, Arg79X, Tyr86X, Gln133X, Val406SerfsX3, Tyr616X, Trp676X, Tyr807X, Trp848X, Y86X, V406X, Y616X, W848X, and Y807X, compared to the human PKP2 gene encoding human PKP2 having the sequence of SEQ ID NO: 2, where X is a premature translation termination codon ( ) caused by a nonsense mutation or frameshift and premature termination. * (also called Ter) indicates high-end PKP2 prevalence (163kJ) * PKP2 Stop Gain Frequency (0.28) * PKP2 ICD frequency (0.41) = 18.7 kpts. High-end PKP2 prevalence (153 kpts) * PKP2 Stop Gain Frequency (0.28) * PKP2 ICD frequency (0.41) = 17.5 kpts.

[0212] In certain embodiments, the subjects include patients with PKP2-ACM who have a pathogenic PKP2 variant and a clinical diagnosis. The prevalence of ACM is 1:1000 to 1:5000 (Peters S, Trummel M, Meyners W. Prevalence of right ventricular dysplasia-cardiomyopathy in a non-referral hospital. Int J Cardiol. 2004;97(3):499-501; and McKenna WJ, Judge DP. Epidemiology of the inherited cardiomyopathies. Nat Rev Cardiol. 2021;18(1):22-36). Among 2,572 ACM patients evaluated from 13 publications, an aggregated mean of 32.9% had PKP2 mutations. TIFF2026502465000020.tif103160

[0213] Taking advantage of the modest ACM prevalence (1:5000) and the PKP2 mutation frequency in ACM of 32.9%, the prevalence of PKP2-ACM patients across the US and EU is approximately 50,000 patients.

[0214] In certain embodiments, the subject comprises a PKP2 stop-gain variant mutation, including but not limited to, any of the PKP2 stop-gain variant mutations specifically disclosed herein, while in other embodiments, the subject may comprise a different type of PKP2 mutation.

[0215] In certain embodiments, the subject may include an implantable cardioverter defibrillator (ICD), while in other embodiments, the subject may not include an ICD.

[0216] In certain embodiments, the subject comprises a PKP2 stop-gain variant mutation, including but not limited to, any of the PKP2 stop-gain variant mutations disclosed herein, while in other embodiments, the subject may comprise a different type of PKP2 mutation and an implantable cardioverter-defibrillator (ICD).

[0217] In certain embodiments, subjects treated with the disclosed vectors or treated according to the disclosed methods contain a PKP2 stop-gain variant mutation and / or have an implantable cardioverter defibrillator (ICD). https: / / www.mayoclinic.org / tests-procedures / implantable-cardioverter-defibrillators / about / pac-20384692

[0218] AAV-mediated PKP2 protein delivery to the heart may extend lifespan and prevent or attenuate cardiac cell degeneration, heart failure, scarring, reduced ejection fraction, arrhythmias, angina, low exercise intolerance, angina (chest pain), sudden cardiac death, exertional muscle pain, and seizures. AAV-mediated PKP2 protein delivery to the heart may show improvement from or prevent the usual disease course as detected by pathological electrocardiograms, cardiac MRI, cardiac biopsies, reduced paroxysmal ventricular arrhythmias, reduced sudden cardiac death, and / or reduced or absent further development of fibrofatty deposits in the right ventricular myocardium. The disclosed methods may prevent a decrease in right ventricular ejection fraction (RVEF), restore right ventricular ejection fraction (RVEF), and / or increase right ventricular ejection fraction (RVEF).

[0219] The methods disclosed herein may provide efficient biodistribution in the heart. The methods disclosed herein may result in sustained expression in all or a significant proportion of cardiac cells, such as cardiomyocytes. In particular, the methods disclosed herein may provide long-term PKP2 protein expression throughout a subject's life after AAV vector administration. In some embodiments, PKP2 protein expression in response to treatment lasts for at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 25 years, 30 years, 35 years, or 40 years.

[0220] Combination therapy is also contemplated by the present disclosure. Combination of the methods of the present disclosure with standard medical treatments (e.g., corticosteroids or local pressure-reducing drugs) is particularly contemplated, as is combination with novel therapies. In some cases, subjects may be treated with a combination of steroids and / or immunosuppressants to prevent or reduce immune responses to the administration of the rAAV described herein.

[0221] In some embodiments, the AAV vector is administered at a dose of about 1 x 10 per kilogram (kg) of total body mass of the subject.12 ~5×10 14 Vector genome (vg) or approximately 1 × 10 12 ~6×10 14 In some embodiments, the AAV vector is administered at a dose of about 1 x 10 vg of AAV vector (vg / kg). 13 ~5×10 14 In some embodiments, the AAV vector is administered at a dose of about 1 x 10 vg / kg. 13 ~1×10 14 In some embodiments, the AAV vector is administered at a dose of about 3 x 10 vg / kg. 13 ~3×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 vg / kg. 13 ~3×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 vg / kg. 13 ~5×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 vg / kg. 13 ~1×10 14 In some embodiments, the AAV vector is administered at a dose of about 1 x 10 vg / kg. 12 Less than 3 × 10 vg / kg 12 Less than 5 × 10 vg / kg 12 Less than 7 × 10 vg / kg 12 Less than 1 × 10 vg / kg 13 Less than 3 × 10 vg / kg 13 Less than 5 × 10 vg / kg 13 Less than 7 × 10 vg / kg 13 Less than 1 × 10 vg / kg 14 Less than 3 × 10 vg / kg 14 Less than 5 × 10 vg / kg 14 Less than 7 × 10 vg / kg 14 Less than 1 × 10 vg / kg 15 Less than 3 × 10 vg / kg 15 Less than 5 × 10 vg / kg 15 vg / kg or less, or approximately 7 × 10 15In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh.74 vector, e.g., a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In some cases, it may be advantageous to use a higher dose for the AAVrh.74 vector than for the AAV9 vector. In certain embodiments, the AAV9 vector is administered at a dose of about 1 x 10 13 ~Approx. 1×10 14 vg / kg, e.g., about 1 × 10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , or about 5 × 10 13 In certain embodiments, the AAVrh.74 vector is administered at a dose of about 5 x 10 vg / kg. 13 ~Approx. 5×10 14 vg / kg, e.g., about 5 × 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , or about 5 × 10 14 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered at a dose of at least about 5 x 10 vg / kg. 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least approximately 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14vg / kg, or at least about 7 × 10 14 It is administered at a dose of 1000 mg / kg.

[0222] In some embodiments, the AAV vector is about 1 x 10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 2×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 4×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 2×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 6×10 14 vg / kg, approximately 7×10 14 vg / kg, approx. 8×10 14 vg / kg, approx. 9×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg, or approximately 7 × 10 15 vg / kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh.74 vector, e.g., a vector comprising a sequence disclosed in any of SEQ ID NOs:8-15 or 89-96. In certain embodiments, the AAV9 vector is administered at a dose of about 1 x 10 13 ~Approx. 1×10 14 vg / kg, e.g., about 1 × 10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13, or about 5 × 10 13 In certain embodiments, the AAVrh.74 vector is administered at a dose of about 5 x 10 vg / kg. 13 ~Approx. 5×10 14 vg / kg, e.g., about 5 × 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , or about 5 × 10 14 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered at a dose of at least about 5 x 10 vg / kg. 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least approximately 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 It is administered at a dose of 1000 mg / kg.

[0223] In some embodiments, the AAV vector is 1 x 10 12 vg / kg, 3 × 10 12 vg / kg, 5 × 10 12 vg / kg, 7 × 10 12 vg / kg, 1 × 10 13 vg / kg, 2 × 10 13 vg / kg, 3 × 10 13 vg / kg, 4 × 10 13 vg / kg, 5 × 10 13 vg / kg, 6 × 10 13 vg / kg, 7 × 1013 vg / kg, 8 × 10 13 vg / kg, 9 × 10 13 vg / kg, 1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 3 × 10 14 vg / kg, 4 × 10 14 vg / kg, 5 × 10 14 vg / kg, 6 × 10 14 vg / kg, 7 × 10 14 vg / kg, 8 × 10 14 vg / kg, 9 × 10 14 vg / kg, 1 × 10 15 vg / kg, 3 × 10 15 vg / kg, 5 × 10 15 vg / kg, or 7 × 10 15 vg / kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh.74 vector, e.g., a vector comprising a sequence disclosed in any of SEQ ID NOs:8-15 or 89-96. In certain embodiments, the AAV9 vector is administered at a dose of about 1 x 10 13 ~Approx. 1×10 14 vg / kg, e.g., about 1 × 10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , or about 5 × 10 13 In certain embodiments, the AAVrh.74 vector is administered at a dose of about 5 x 10 vg / kg. 13 ~Approx. 5×10 14 vg / kg, e.g., about 5 × 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , or about 5 × 10 14 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered at a dose of at least about 5 x 10 vg / kg. 13vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least approximately 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 It is administered at a dose of 1000 mg / kg.

[0224] In some embodiments, the AAV vector is administered at a dose of about 1 x 10 per kilogram (kg) of total body mass of the subject. 12 ~5×10 14 The AAV vector is administered systemically at a dose of vector genome (vg) (vg / kg). In some embodiments, the AAV vector is administered at a dose of about 1 x 10 13 ~5×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 vg / kg. 13 ~3×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 vg / kg. 13 ~1×10 14 In some embodiments, the AAV vector is administered systemically at a dose of about 1 x 10 vg / kg. 12 Less than 3 × 10 vg / kg 12 Less than 5 × 10 vg / kg 12 Less than 7 × 10 vg / kg 12 Less than 1 × 10 vg / kg 13 Less than 3 × 10 vg / kg 13 Less than 5 × 10 vg / kg 13 Less than 7 × 10 vg / kg 13 Less than 1 × 10 vg / kg 14 Less than 3 × 10 vg / kg 14 Less than 5 × 10 vg / kg 14Less than 7 × 10 vg / kg 14 Less than 1 × 10 vg / kg 15 Less than 3 × 10 vg / kg 15 Less than 5 × 10 vg / kg 15 vg / kg or less, or approximately 7 × 10 15 In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh.74 vector, e.g., a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In certain embodiments, the AAV9 vector is administered systemically at a dose of about 1 x 10 13 ~Approx. 1×10 14 vg / kg, e.g., about 1 × 10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , or about 5 × 10 13 In certain embodiments, the AAVrh.74 vector is administered at a dose of about 5 x 10 vg / kg. 13 ~Approx. 5×10 14 vg / kg, e.g., about 5 × 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , or about 5 × 10 14 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered at a dose of at least about 5 x 10 vg / kg. 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least approximately 3 × 10 14 vg / kg, at least about 4 × 1014 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 It is administered at a dose of 1000 mg / kg.

[0225] In some embodiments, the AAV vector is about 1 x 10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 2×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 4×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 2×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 6×10 14 vg / kg, approximately 7×10 14 vg / kg, approx. 8×10 14 vg / kg, approx. 9×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg, or approximately 7 × 10 15 vg / kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh.74 vector, e.g., a vector comprising a sequence disclosed in any of SEQ ID NOs:8-15 or 89-96. In certain embodiments, the AAV9 vector is administered systemically at a dose of about 1 x 10 13 ~Approx. 1×10 14 vg / kg, e.g., about 1 × 1013 , about 2×10 13 , about 3×10 13 , about 4×10 13 , or about 5 × 10 13 In certain embodiments, the AAVrh.74 vector is administered at a dose of about 5 x 10 vg / kg. 13 ~Approx. 5×10 14 vg / kg, e.g., about 5 × 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , or about 5 × 10 14 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered at a dose of at least about 5 x 10 vg / kg. 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least approximately 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 It is administered at a dose of 1000 mg / kg.

[0226] In some embodiments, the AAV vector is 1 x 10 12 vg / kg, 3 × 10 12 vg / kg, 5 × 10 12 vg / kg, 7 × 10 12 vg / kg, 1 × 10 13 vg / kg, 2 × 10 13 vg / kg, 3 × 10 13 vg / kg, 4 × 1013 vg / kg, 5 × 10 13 vg / kg, 6 × 10 13 vg / kg, 7 × 10 13 vg / kg, 8 × 10 13 vg / kg, 9 × 10 13 vg / kg, 1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 3 × 10 14 vg / kg, 4 × 10 14 vg / kg, 5 × 10 14 vg / kg, 6 × 10 14 vg / kg, 7 × 10 14 vg / kg, 8 × 10 14 vg / kg, 9 × 10 14 vg / kg, 1 × 10 15 vg / kg, 3 × 10 15 vg / kg, 5 × 10 15 vg / kg, or 7 × 10 15 vg / kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh.74 vector, e.g., a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In certain embodiments, the AAV9 vector is administered systemically at a dose of about 1 x 10 13 ~Approx. 1×10 14 vg / kg, e.g., about 1 × 10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , or about 5 × 10 13 In certain embodiments, the AAVrh.74 vector is administered at a dose of about 5 x 10 13 ~Approx. 5×10 14 vg / kg, e.g., about 5 × 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , or about 5 × 10 14In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered at a dose of at least about 5 x 10 vg / kg. 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least approximately 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 It is administered at a dose of 1000 mg / kg.

[0227] In some embodiments, the AAV vector is administered at a dose of about 1 x 10 per kilogram (kg) of total body mass of the subject. 12 ~5×10 14 The AAV vector is administered intravenously at a dose of about 1 x 10 vector genomes (vg) (vg / kg). In some embodiments, the AAV vector is administered intravenously at a dose of about 1 x 10 13 ~5×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 vg / kg. 13 ~3×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 vg / kg. 13 ~1×10 14 In some embodiments, the AAV vector is administered intravenously at a dose of about 1 x 10 vg / kg. 12 Less than 3 × 10 vg / kg 12 Less than 5 × 10 vg / kg 12 Less than 7 × 10 vg / kg 12 Less than 1 × 10 vg / kg 13 Less than 3 × 10 vg / kg 13 Less than 5 × 10 vg / kg 13 Less than 7 × 10 vg / kg 13 Less than 1 × 10 vg / kg14 Less than 3 × 10 vg / kg 14 Less than 5 × 10 vg / kg 14 Less than 7 × 10 vg / kg 14 Less than 1 × 10 vg / kg 15 Less than 3 × 10 vg / kg 15 Less than 5 × 10 vg / kg 15 vg / kg or less, or approximately 7 × 10 15 In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh.74 vector, e.g., a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In certain embodiments, the AAV9 vector is administered intravenously at a dose of about 1 x 10 13 ~Approx. 1×10 14 vg / kg, e.g., about 1 × 10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , or about 5 × 10 13 In certain embodiments, the AAVrh.74 vector is administered at a dose of about 5 x 10 13 ~Approx. 5×10 14 vg / kg, e.g., about 5 × 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , or about 5 × 10 14 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered at a dose of at least about 5 x 10 vg / kg. 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 1014 vg / kg, at least approximately 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 It is administered at a dose of 1000 mg / kg.

[0228] In some embodiments, the AAV vector is about 1 x 10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 7×10 12 vg / kg, approximately 1×10 13 vg / kg, approx. 2×10 13 vg / kg, approx. 3×10 13 vg / kg, approx. 4×10 13 vg / kg, approx. 5×10 13 vg / kg, approximately 6×10 13 vg / kg, approximately 7×10 13 vg / kg, approx. 8×10 13 vg / kg, approx. 9×10 13 vg / kg, approximately 1×10 14 vg / kg, approx. 2×10 14 vg / kg, approx. 3×10 14 vg / kg, approx. 4×10 14 vg / kg, approx. 5×10 14 vg / kg, approximately 6×10 14 vg / kg, approximately 7×10 14 vg / kg, approx. 8×10 14 vg / kg, approx. 9×10 14 vg / kg, approximately 1×10 15 vg / kg, approx. 3×10 15 vg / kg, approx. 5×10 15 vg / kg, or approximately 7 × 10 15 In certain embodiments, the AAV9 vector is administered intravenously at a dose of about 1 x 10 vg / kg. 13 ~Approx. 1×10 14 vg / kg, e.g., about 1 × 10 13 , about 2×10 13 , about 3×10 13 , about 4×1013 , or about 5 × 10 13 In certain embodiments, the AAVrh.74 vector is administered at a dose of about 5 x 10 vg / kg. 13 ~Approx. 5×10 14 vg / kg, e.g., about 5 × 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , or about 5 × 10 14 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered at a dose of at least about 5 x 10 vg / kg. 13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least approximately 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 It is administered at a dose of 1000 mg / kg.

[0229] In some embodiments, the AAV vector is 1 x 10 12 vg / kg, 3 × 10 12 vg / kg, 5 × 10 12 vg / kg, 7 × 10 12 vg / kg, 1 × 10 13 vg / kg, 2 × 10 13 vg / kg, 3 × 10 13 vg / kg, 4 × 10 13 vg / kg, 5 × 10 13 vg / kg, 6 × 10 13vg / kg, 7 × 10 13 vg / kg, 8 × 10 13 vg / kg, 9 × 10 13 vg / kg, 1 × 10 14 vg / kg, 2 × 10 14 vg / kg, 3 × 10 14 vg / kg, 4 × 10 14 vg / kg, 5 × 10 14 vg / kg, 6 × 10 14 vg / kg, 7 × 10 14 vg / kg, 8 × 10 14 vg / kg, 9 × 10 14 vg / kg, 1 × 10 15 vg / kg, 3 × 10 15 vg / kg, 5 × 10 15 vg / kg, or 7 × 10 15 In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh.74 vector, e.g., a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In certain embodiments, the AAV9 vector is administered intravenously at a dose of about 1 x 10 13 ~Approx. 1×10 14 vg / kg, e.g., about 1 × 10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 , or about 5 × 10 13 In certain embodiments, the AAVrh.74 vector is administered at a dose of about 5 x 10 vg / kg. 13 ~Approx. 5×10 14 vg / kg, e.g., about 5 × 10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , or about 5 × 10 14 In some embodiments, the AAV vector, e.g., the AVrh.74 vector, is administered at a dose of at least about 5 x 10 vg / kg.13 vg / kg, at least about 6 × 10 13 vg / kg, at least about 7 × 10 13 vg / kg, at least about 8 × 10 13 vg / kg, at least about 9 × 10 13 vg / kg, at least about 1 × 10 14 vg / kg, at least about 2 × 10 14 vg / kg, at least approximately 3 × 10 14 vg / kg, at least about 4 × 10 14 vg / kg, at least about 5 × 10 14 vg / kg, at least about 6 × 10 14 vg / kg, or at least about 7 × 10 14 It is administered at a dose of 1000 mg / kg.

[0230] Evidence of functional improvement, clinical benefit, or efficacy in patients may be evidenced by changes in New York Heart Association functional classification (NYHA Class), pathological electrocardiograms, cardiac MRI, cardiac biopsies, a reduction in paroxysmal ventricular arrhythmias, a reduction in sudden cardiac death, and / or a reduction or absence of further development of fibrofatty deposits in the right ventricular myocardium. Benefit may be observed in electrocardiographic features commonly associated with arrhythmogenic right ventricular cardiomyopathy, such as T wave inversion, prolonged S-wave upstroke, localized QRS widening, and / or paroxysmal episodes of ventricular tachycardia.

[0231] In some embodiments, the method prevents or reduces a decrease in left ventricular ejection fraction percent (LVEF%) by, optionally, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject suffering from or at risk of a disease or disorder associated with or caused by loss of function of PKP2.

[0232] In some embodiments, the method prevents or reduces a decrease in left ventricular fractional shortening percent (FS%) by, optionally, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject suffering from or at risk of a disease or disorder associated with or caused by loss of function of PKP2.

[0233] In some embodiments, the method prevents or reduces the increase in right ventricular area in square millimeters of RV area (mm2) by, optionally, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject suffering from or at risk for a disease or disorder associated with or caused by loss of function of PKP2.

[0234] In some embodiments, the method prevents or reduces a decrease in right ventricular velocity time integral in millimeters per second (RV VTI) (mm / sec) by, optionally, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject suffering from or at risk for a disease or disorder associated with or caused by loss of function of PKP2.

[0235] In some embodiments, the method prevents or reduces an increase in left ventricular fibrosis or right ventricular fibrosis by, optionally, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject suffering from or at risk of a disease or disorder associated with or caused by loss of function of PKP2.

[0236] Administration of an effective dose of the composition may be by any route standard in the art, including, but not limited to, systemic, local, direct injection, intravenous, or intracardiac administration. In some cases, administration includes systemic, local, direct injection, intravenous, or intracardiac injection. Administration may also be by cardiac catheter.

[0237] In some embodiments, the present disclosure provides for local and systemic administration of an effective dose of the rAAV and compositions of the present disclosure. For example, systemic administration can be administration into the circulatory system to affect the entire body. Systemic administration includes parenteral administration by injection, infusion, or implantation. Routes of administration of the compositions disclosed herein include intravenous ("IV"), intraperitoneal ("IP"), intramuscular ("IM"), intralesional, or subcutaneous ("SC") administration, or implantation of a sustained-release device, such as a mini-osmotic pump, a depot formulation, or the like. In some embodiments, the method of the present disclosure comprises administering an AAV vector or pharmaceutical composition of the present disclosure by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intravenous, iontophoretic, or intracranial administration.

[0238] In particular, administration of the rAAV of the present disclosure can be accomplished using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal, including, but not limited to, injection into the heart.

[0239] In some embodiments, the methods of the present disclosure include intracardiac delivery. Infusion may be performed using a syringe / needle with a dedicated cannula, catheter, or infusion pump. Administration may include delivery of an effective amount of rAAV virions or a pharmaceutical composition containing rAAV virions to the heart. This may be accomplished, for example, via intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoretic, or intracranial administration. The compositions of the present disclosure may also be administered intravenously.

[0240] The treatment methods disclosed herein may reduce and / or prevent one or more symptoms, including, but not limited to, ventricular hypertrophy, ventricular tachycardia, hypoexercise capacity, angina, and reduced RVEF. The benefits of AAV-mediated PKP2 overexpression may be demonstrated by increased survival and attenuation of the normal progression of cardiomyopathy observed on echocardiograms from the left and / or right ventricles (e.g., greater left ventricular ejection fraction, greater left ventricular fractional shortening, and longer right ventricle velocity time intervals compared to PKP cKO formulation buffer control animals).

[0241] Electrophysiological evidence for the functional benefits of AAV-mediated PKP2 protein delivery can be demonstrated by the reduction of disease-associated disrupted calcium dynamics in affected cardiomyocytes, most notably measures of L-type calcium current, sarcoplasmic reticulum calcium leak, and diastolic calcium leak, as well as standard measures of calcium transients, such as time to peak amplitude and relaxation time constant, in affected (e.g., PKP2-deficient) cardiomyocytes. Histological analysis may reveal the benefits of AAV-mediated PKP2 overexpression by a reduced appearance of disease-associated collagen deposits in various cardiac regions, including the ventricles (e.g., via Trichrome staining). Further benefits may also be revealed by assessing cardiomyocyte ventricular proteins involved in calcium signaling pathways, as measured by increased (i.e., normalized) relative levels of Casq2, Trdn, Cav1.2, and / or AnkB and / or RyR2.

[0242] Effect of rAAV administration In some aspects, administration of an rAAV of the present disclosure can have a beneficial effect on a subject.

[0243] lifespan In some embodiments, administration of an rAAV of the present disclosure may extend the lifespan of a subject compared to a subject not administered an rAAV of the present disclosure or compared to a baseline.

[0244] In some embodiments, administration of an rAAV of the present disclosure extends lifespan by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to a subject not administered an rAAV of the present disclosure or to a baseline.

[0245] In some embodiments, administration of an rAAV of the present disclosure increases lifespan by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, or about 35% compared to a subject not administered an rAAV of the present disclosure or to baseline. %, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, or about 400% to about 500%.

[0246] ejection fraction In some embodiments, administration of an rAAV of the present disclosure limits the decline in ejection fraction, restores the ejection fraction, and / or increases the ejection fraction in a subject compared to a subject not administered an rAAV of the present disclosure or to a baseline. In some embodiments, administration of an rAAV of the present disclosure limits the decline in ejection fraction, restores the ejection fraction, and / or increases the ejection fraction in a subject over time.

[0247] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases ejection fraction by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0248] In some embodiments, administration of an rAAV of the present disclosure limits the decrease in ejection fraction to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0249] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases ejection fraction by at least about 11%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0250] In some embodiments, administration of an rAAV of the present disclosure limits the decrease in ejection fraction over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0251] In some embodiments, administration of an rAAV of the present disclosure increases or decreases ejection fraction by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, or about 10% to about 15% compared to a subject not administered an rAAV of the present disclosure or to baseline. , about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0252] In some embodiments, administration of an rAAV of the present disclosure improves or decreases ejection fraction over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, or %, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0253] In some embodiments, administration of an rAAV of the present disclosure reduces ejection fraction by about 1% to less than about 90%, about 20% to less than about 80%, about 30% to less than about 80%, about 40% to less than about 80%, about 50% to less than about 80%, about 1% to less than about 2%, about 2% to less than about 3%, about 3% to less than about 4%, about 4% to less than about 5%, about 5% to less than about 6%, about 6% to less than about 7%, about 7% to less than about 8%, about 8% to less than about 9%, about 9% to 10%, or about 10% to about 15% compared to a subject not administered an rAAV of the present disclosure or to baseline. less than about 15% to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0254] In some embodiments, administration of an rAAV of the present disclosure results in a decrease in ejection fraction over time in a subject, the decrease being between about 1% and about 90%, between about 20% and about 80%, between about 30% and about 80%, between about 40% and about 80%, between about 50% and about 80%, between about 1% and about 2%, between about 2% and about 3%, between about 3% and about 4%, between about 4% and about 5%, between about 5% and about 6%, between about 6% and about 7%, between about 7% and about 8%, between about 8% and about 9%, between about 9% and 10%, between about 10% and about 15%, or between about 15% and about 20%. The range is limited to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0255] Left ventricular ejection fraction (LVEF) In some embodiments, administration of an rAAV of the present disclosure prevents a decrease in LVEF, restores LVEF, and / or increases LVEF in a subject compared to a subject not administered an rAAV of the present disclosure or compared to baseline. In some embodiments, administration of an rAAV of the present disclosure limits a decrease in LVEF, restores LVEF, and / or increases LVEF in a subject over time.

[0256] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases LVEF by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0257] In some embodiments, administration of an rAAV of the present disclosure limits the decrease in LVEF to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0258] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases LVEF by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0259] In some embodiments, administration of an rAAV of the present disclosure limits the decrease in LVEF over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0260] In some embodiments, administration of an rAAV of the present disclosure improves LVEF by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, or about 15% compared to a subject not administered an rAAV of the present disclosure or to baseline. %, about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0261] In some embodiments, administration of an rAAV of the present disclosure improves LVEF over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, or Recovery and / or increase by 0%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0262] In some embodiments, administration of an rAAV of the present disclosure reduces LVEF by about 1% to less than about 90%, about 20% to less than about 80%, about 30% to less than about 80%, about 40% to less than about 80%, about 50% to less than about 80%, about 1% to less than about 2%, about 2% to less than about 3%, about 3% to less than about 4%, about 4% to less than about 5%, about 5% to less than about 6%, about 6% to less than about 7%, about 7% to less than about 8%, about 8% to less than about 9%, about 9% to 10%, or about 10% to less than about 15% compared to a subject not administered an rAAV of the present disclosure or to baseline. %, less than about 15% to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0263] In some embodiments, administration of an rAAV of the present disclosure reduces LVEF over time in a subject by about less than 1% to less than about 90%, about 20% to less than about 80%, about 30% to less than about 80%, about 40% to less than about 80%, about 50% to less than about 80%, about 1% to less than about 2%, about 2% to less than about 3%, about 3% to less than about 4%, about 4% to less than about 5%, about 5% to less than about 6%, about 6% to less than about 7%, about 7% to less than about 8%, about 8% to less than about 9%, about 9% to 10%, about 10% to less than about 15%, or less than about 15%. The range is limited to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0264] Right ventricular ejection fraction (RVEF) In some embodiments, administration of an rAAV of the present disclosure prevents a decrease in RVEF, restores RVEF, and / or increases RVEF in a subject compared to a subject not administered an rAAV of the present disclosure or compared to baseline. In some embodiments, administration of an rAAV of the present disclosure limits a decrease in LVEF, restores LVEF, and / or increases LVEF in a subject over time.

[0265] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases RVEF by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0266] In some embodiments, administration of an rAAV of the present disclosure limits the decrease in RVEF to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0267] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases RVEF by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0268] In some embodiments, administration of an rAAV of the present disclosure limits the decrease in RVEF over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0269] In some embodiments, administration of an rAAV of the present disclosure increases RVEF by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, or about 15% compared to a subject not administered an rAAV of the present disclosure or to baseline. %, about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0270] In some embodiments, administration of an rAAV of the present disclosure increases RVEF over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 3 ... Recovery and / or increase by 0%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0271] In some embodiments, administration of an rAAV of the present disclosure results in a decrease in RVEF of less than about 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, less than about 9% to 10%, or less than about 10% to about 15% compared to a subject not administered an rAAV of the present disclosure or to baseline. %, less than about 15% to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0272] In some embodiments, administration of an rAAV of the present disclosure results in a decrease in RVEF over time in a subject of between about 1% and about 90%, between about 20% and about 80%, between about 30% and about 80%, between about 40% and about 80%, between about 50% and about 80%, between about 1% and about 2%, between about 2% and about 3%, between about 3% and about 4%, between about 4% and about 5%, between about 5% and about 6%, between about 6% and about 7%, between about 7% and about 8%, between about 8% and about 9%, between about 9% and 10%, between about 10% and about 15%, or between about 15% and about 20%. The range is limited to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0273] Right ventricular (RV) area In some embodiments, administration of an rAAV of the present disclosure prevents an increase in RV area, restores RV area, and / or reduces RV area in a subject compared to a subject not administered an rAAV of the present disclosure or to a baseline. In some embodiments, administration of an rAAV of the present disclosure prevents an increase in RV area, restores RV area, and / or reduces RV area in a subject over time.

[0274] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in RV area, restores RV area, and / or reduces RV area by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to a baseline.

[0275] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in serotonin levels by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 3% or more compared to a subject not administered an rAAV of the present disclosure or to a baseline. Prevents RV area increase, restores RV area, and / or reduces RV area by 5%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0276] In some embodiments, administration of an rAAV of the disclosure prevents an increase in RV area, restores RV area, and / or reduces RV area by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0277] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in HIV-1 response over time in a subject of about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about 40%, about 3 ... Prevents RV area increase, restores RV area, and / or reduces RV area by about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0278] Left ventricular (LV) area In some embodiments, administration of an rAAV of the present disclosure prevents an increase in LV area, restores LV area, and / or reduces LV area in a subject compared to a subject not administered an rAAV of the present disclosure or to a baseline. In some embodiments, administration of an rAAV of the present disclosure prevents an increase in RV area, restores RV area, and / or reduces RV area in a subject over time.

[0279] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in LV area, restores LV area, and / or reduces LV area by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0280] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in serotonin levels by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 3% or more compared to a subject not administered an rAAV of the present disclosure or to a baseline. Prevents the increase in LV area, restores LV area, and / or reduces LV area by 5%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0281] In some embodiments, administration of an rAAV of the disclosure prevents an increase in LV area, restores LV area, and / or reduces LV area by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% over time in a subject.

[0282] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in HIV-1 response over time in a subject of about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about 40%, about 3 ... Prevents the increase in LV area, restores LV area, and / or reduces LV area by about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0283] Right ventricular (RV) velocity time integral (VTI) In some embodiments, administration of an rAAV of the present disclosure limits the decrease in RV VTI, restores RV VTI, and / or increases RV VTI in a subject compared to a subject not administered an rAAV of the present disclosure or compared to baseline. In some embodiments, administration of an rAAV of the present disclosure limits the decrease in RV VTI, restores RV VTI, and / or increases RV VTI in a subject over time.

[0284] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases RV VTI by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0285] In some embodiments, administration of an rAAV of the present disclosure limits the reduction in RV VTI to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0286] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases RV VTI by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0287] In some embodiments, administration of an rAAV of the present disclosure limits the decrease in RV VTI over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0288] In some embodiments, administration of an rAAV of the present disclosure improves RV VTI by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, or about 15% to about 20% compared to a subject not administered an rAAV of the present disclosure or to baseline. , about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0289] In some embodiments, administration of an rAAV of the present disclosure improves RV VTI over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about Restore and / or increase by 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0290] In some embodiments, administration of an rAAV of the present disclosure results in a reduction in RV VTI of less than about 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, less than about 9% to 10%, less than about 10% to less than about 15%, less than about 15% to less than about 20%. The range is limited to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0291] In some embodiments, administration of an rAAV of the present disclosure results in a reduction in RV VTI over time in a subject by about less than 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, less than about 9% to 10%, less than about 10% to less than about 15%, less than about 15% to less than about 20%, less than about 20% to less than about 35%. %, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0292] Left ventricular (LV) velocity time integral (VTI) In some embodiments, administration of an rAAV of the present disclosure limits the decrease in LV VTI, restores LV VTI, and / or increases LV VTI in a subject compared to a subject not administered an rAAV of the present disclosure or compared to baseline. In some embodiments, administration of an rAAV of the present disclosure limits the decrease in LV VTI, restores LV VTI, and / or increases LV VTI over time in a subject. Thus, the methods disclosed herein can be used to limit the decrease in LV VTI, restore LV VTI, and / or increase LV VTI in a subject in need thereof, e.g., a subject with ACM, e.g., ARVC or ARVD.

[0293] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases LV VTI by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0294] In some embodiments, administration of an rAAV of the present disclosure limits the decrease in LV VTI to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0295] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases LV VTI by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0296] In some embodiments, administration of an rAAV of the disclosure limits the decrease in LV VTI over time in a subject to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0297] In some embodiments, administration of an rAAV of the present disclosure improves LV VTI by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, or about 15% to about 20% compared to a subject not administered an rAAV of the present disclosure or to baseline. , about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0298] In some embodiments, administration of an rAAV of the present disclosure improves LV VTI over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 25% to about Restore and / or increase by 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0299] In some embodiments, administration of an rAAV of the present disclosure results in a reduction in LV VTI of less than about 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, less than about 9% to 10%, less than about 10% to less than about 15%, less than about 15% to less than about 20%. The range is limited to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0300] In some embodiments, administration of an rAAV of the present disclosure may result in a decrease in LV VTI over time in a subject by about less than 1% to less than about 90%, about 20% to less than about 80%, about 30% to less than about 80%, about 40% to less than about 80%, about 50% to less than about 80%, about 1% to less than about 2%, about 2% to less than about 3%, about 3% to less than about 4%, about 4% to less than about 5%, about 5% to less than about 6%, about 6% to less than about 7%, about 7% to less than about 8%, about 8% to less than about 9%, about 9% to 10%, about 10% to less than about 15%, about 15% to less than about 20%, or about 20% to less than about 35%. %, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0301] Left ventricular (LV) fibrosis In some embodiments, administration of an rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis in a subject compared to a subject not administered an rAAV of the present disclosure or compared to baseline. In some embodiments, administration of an rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis in a subject over time. Thus, the methods disclosed herein can be used to prevent an increase in LV fibrosis and / or reduce LV fibrosis in a subject in need thereof, for example, a subject in need thereof, for example, a subject with ACM, e.g., ARVC or ARVD.

[0302] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to a baseline.

[0303] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in serotonin levels by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 2% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 2% to about 30%, about 30% to about 35%, about 35% to about 40%, about 35% to about 45%, about 35% to about 50%, about 35% to about 50%, about 35% to about 50%, about 35% to about 6 ...70%, about 35% to about 70%, about 35% to about 80%, about 35% to about 80%, about 35% to Prevents an increase in LV fibrosis and / or reduces LV fibrosis by 0% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0304] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0305] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in a subject's response over time of about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about Prevents an increase in LV fibrosis and / or reduces LV fibrosis by 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0306] Right ventricular (RV) fibrosis In some embodiments, administration of the rAAV of the present disclosure prevents an increase in RV fibrosis and / or reduces RV fibrosis in a subject compared to a subject not administered the rAAV of the present disclosure or compared to baseline. In some embodiments, administration of the rAAV of the present disclosure prevents an increase in RV fibrosis and / or reduces RV fibrosis in a subject over time. Thus, the methods disclosed herein can be used to prevent an increase in RV fibrosis and / or reduce RV fibrosis in a subject in need thereof, for example, a subject in need thereof, for example, a subject with ACM, for example, ARVC or ARVD.

[0307] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in RV fibrosis and / or reduces RV fibrosis by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to subjects not administered an rAAV of the present disclosure or to a baseline.

[0308] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in serotonin levels by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 2% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 2% to about 30%, about 30% to about 35%, about 35% to about 40%, about 35% to about 45%, about 35% to about 50%, about 35% to about 50%, about 35% to about 50%, about 35% to about 6 ...70%, about 35% to about 70%, about 35% to about 80%, about 35% to about 80%, about 35% to Prevents an increase in RV fibrosis and / or reduces RV fibrosis by 0% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0309] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in RV fibrosis and / or reduces RV fibrosis by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0310] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in a subject's response over time of about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about Prevents an increase in RV fibrosis and / or reduces RV fibrosis by 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0311] Premature ventricular contractions (PVCs) In some embodiments, administration of the rAAV of the present disclosure prevents an increase in PVC and / or reduces PVC in a subject compared to a subject not administered the rAAV of the present disclosure or at baseline.In some embodiments, administration of the rAAV of the present disclosure prevents an increase in PVC and / or reduces PVC in a subject over time.Thus, the methods disclosed herein can be used to prevent an increase in PVC and / or reduce PVC in a subject in need thereof, for example, a subject in need thereof, for example, a subject with ACM, for example, ARVC or ARVD.

[0312] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in PVCs and / or reduces PVCs by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to a baseline.

[0313] In some embodiments, administration of an rAAV of the present disclosure improves or reduces inflammatory bowel disease by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, or about 1% to about 2% compared to a subject not administered an rAAV of the present disclosure or to baseline. The increase in PVC is prevented and / or the PVC is reduced by about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0314] In some embodiments, administration of an rAAV of the present disclosure prevents a rise in PVC and / or reduces PVC by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% over time in a subject.

[0315] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in HIV-1 response over time in a subject of about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, or about 20% to about 35%. , about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0316] Non-sustained ventricular tachycardia (NSVT) In some embodiments, administration of the rAAV of the present disclosure prevents an increase in NSVT and / or reduces NSVT in a subject compared to a subject not administered the rAAV of the present disclosure or compared to baseline.In some embodiments, administration of the rAAV of the present disclosure prevents an increase in NSVT and / or reduces NSVT in a subject over time.Therefore, the methods disclosed herein can be used to prevent an increase in NSVT and / or reduce NSVT in a subject in need thereof, for example, a subject in need thereof, for example, a subject with ACM, for example, ARVC or ARVD.

[0317] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in NSVT and / or reduces NSVT by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to a baseline.

[0318] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in serotonin levels by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about Prevents an increase in NSVT and / or reduces NSVT by 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0319] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in NSVT and / or reduces NSVT by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0320] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in HIV-1 response over time in a subject of about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, Prevents an increase in NSVT and / or reduces NSVT by about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0321] Ventricular tachycardia (VT) In some embodiments, administration of the rAAV of the present disclosure prevents an increase in VT and / or reduces VT in a subject compared to a subject not administered the rAAV of the present disclosure or to a baseline. In some embodiments, administration of the rAAV of the present disclosure prevents an increase in NSVT and / or reduces NSVT in a subject over time.

[0322] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in VT and / or reduces VT by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to a baseline. Thus, the methods disclosed herein may be used to prevent an increase in VT and / or reduce VT in a subject in need thereof, e.g., a subject with ACM, e.g., ARVC or ARVD.

[0323] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in serotonin levels by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, or about 15% to about 20% compared to a subject not administered an rAAV of the present disclosure or to baseline. , about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0324] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in VT and / or reduces VT by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% over time in a subject.

[0325] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in HIV-1 response over time in a subject of about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, or %, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0326] Ectopic beats In some embodiments, administration of rAAV of the present disclosure prevents the increase in ectopic beats and / or reduces ectopic beats in a subject compared to a subject that is not administered with rAAV of the present disclosure or baseline.In some embodiments, administration of rAAV of the present disclosure prevents the increase in ectopic beats and / or reduces ectopic beats in a subject over time.Therefore, the method disclosed herein can be used to prevent the increase in ectopic beats and / or reduce ectopic beats in a subject in need thereof, for example, a subject in need thereof, for example, a subject with ACM, for example, ARVC or ARVD.

[0327] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to a baseline.

[0328] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in serotonin levels by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 2% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 2% to about 30%, about 30% to about 35%, about 35% to about 40%, about 35% to about 45%, about 35% to about 50%, about 35% to about 50%, about 35% to about 50%, about 35% to about 6 ...70%, about 35% to about 70%, about 35% to about 80%, about 35% to about 80%, about 35% to Prevents an increase in ectopic beats and / or reduces ectopic beats by 0% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0329] In some embodiments, administration of an rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in a subject over time.

[0330] In some embodiments, administration of an rAAV of the present disclosure results in an improvement in a subject's response over time of about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about Prevents an increase in ectopic beats and / or reduces ectopic beats by 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%.

[0331] Intervening plate In some embodiments, administration of an rAAV of the present disclosure limits the loss of intercalated discs, restores intercalated discs, and / or increases intercalated discs in a subject compared to a subject not administered an rAAV of the present disclosure or to a baseline. In cardiac muscle, connections between adjacent cells are formed by intercalated discs. To enable the heart to beat, intercalated discs are highly specialized, allowing the coordinated function of cardiac cells. In some embodiments, administration of an rAAV of the present disclosure limits the loss of intercalated discs, restores intercalated discs, and / or increases intercalated discs in a subject over time. Thus, the methods disclosed herein can be used to limit the loss of intercalated discs, restore intercalated discs, and / or increase intercalated discs in a subject in need thereof, for example, a subject in need thereof, for example, a subject with ACM, e.g., ARVC or ARVD.

[0332] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases intercalated discs by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject not administered an rAAV of the present disclosure or to a baseline.

[0333] In some embodiments, administration of an rAAV of the present disclosure limits the reduction of intercalated discs to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% compared to a subject not administered an rAAV of the present disclosure or to baseline.

[0334] In some embodiments, administration of an rAAV of the present disclosure restores and / or increases intercalated discs by at least about 11%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% over time in a subject.

[0335] In some embodiments, administration of an rAAV of the present disclosure limits the loss of intercalated discs in a subject over time to less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, less than about 70%, at least about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100%.

[0336] In some embodiments, administration of an rAAV of the present disclosure improves interstitial disc function by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, or about 10% to about 15% compared to a subject not administered an rAAV of the present disclosure or to baseline. , about 15% to about 20%, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0337] In some embodiments, administration of an rAAV of the present disclosure reduces intercalated disc size over time in a subject by about 1% to about 90%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%, about 7% to about 8%, about 8% to about 9%, about 9% to 10%, about 10% to about 15%, about 15% to about 20%, or %, about 20% to about 35%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100% recovery and / or increase.

[0338] In some embodiments, administration of an rAAV of the present disclosure results in a reduction in intercalated discs of less than about 1% to less than about 90%, less than about 20% to less than about 80%, less than about 30% to less than about 80%, less than about 40% to less than about 80%, less than about 50% to less than about 80%, less than about 1% to less than about 2%, less than about 2% to less than about 3%, less than about 3% to less than about 4%, less than about 4% to less than about 5%, less than about 5% to less than about 6%, less than about 6% to less than about 7%, less than about 7% to less than about 8%, less than about 8% to less than about 9%, less than about 9% to 10%, or less than about 10% to about 15% compared to a subject not administered an rAAV of the present disclosure or to baseline. less than about 15% to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%.

[0339] In some embodiments, administration of an rAAV of the present disclosure results in a reduction of intercalated discs over time in a subject by between about 1% and about 90%, between about 20% and about 80%, between about 30% and about 80%, between about 40% and about 80%, between about 50% and about 80%, between about 1% and about 2%, between about 2% and about 3%, between about 3% and about 4%, between about 4% and about 5%, between about 5% and about 6%, between about 6% and about 7%, between about 7% and about 8%, between about 8% and about 9%, between about 9% and 10%, between about 10% and about 15%, or between about 15% and about 20%. The range is limited to less than about 20%, less than about 20% to less than about 35%, less than about 25% to less than about 30%, less than about 30% to less than about 35%, less than about 35% to less than about 40%, less than about 40% to less than about 45%, less than about 45% to less than about 50%, less than about 50% to less than about 55%, less than about 55% to less than about 60%, less than about 60% to less than about 65%, less than about 65% to less than about 70%, less than about 70% to less than about 75%, less than about 75% to less than about 80%, less than about 80% to less than about 85%, less than about 85% to less than about 90%, less than about 90% to less than about 95%, or less than about 95% to about 100%. [Example]

[0340] Example 1: In vitro testing of adeno-associated viral vectors AAV vectors described herein (having the structures shown in Figure 1A (e.g., SEQ ID NO:12), Figure 2 (e.g., SEQ ID NO:13 or 97), Figure 3 (e.g., SEQ ID NO:14 or 98), and Figure 4 (e.g., SEQ ID NO:15 or 99)) were prepared and used to transduce CHO-Lec2 cells, and PKP2 expression levels were assessed by Western blot (Figures 5A-5B; individual lanes in the SDS-PAGE gel shown in Figure 5A correspond to the respective PKP2 levels depicted in Figure 5B). Surprisingly, the MHCK7 promoter drove robust PKP2 expression in cardiomyocytes (lanes 1 and 3 in Figure 5A), whereas the hTnnT2 promoter ("hTnT") produced negligible PKP2 levels above background under these test conditions (lanes 2 and 4 in Figure 5A). The AAVrh.74 serotype induced higher PKP2 expression than the AAV9 serotype vector (Fig. 5A , lanes 3 and 4).

[0341] These results demonstrate that AAV9 and AAVrh.74 vectors can be used effectively to express PKP2 in cardiomyocytes and that the MHCK7 promoter is superior to the hTnnT2 promoter when assessing only the relative levels of PKP2 expression using this in vitro assay.

[0342] Example 2: In vivo efficacy of adeno-associated viral vectors Evidence for the benefits of AAV-mediated PKP2 overexpression was confirmed using a cardiomyocyte-specific, tamoxifen-activated PKP2 knockout mouse line (αMHC-Cre-ER(T2) / Pkp2 fl / fl; referred to as "PKP2-cKO"), as described in Cerrone et al., Nat Comm., 2017. This mouse model allows for controlled onset of PKP2 loss, restricting it to adult myocytes. The progression of molecular and functional events leading to arrhythmogenic cardiomyopathy (ACM) begins with an early right ventricular predominance, although both the left and right ventricles are involved in these mice. The time course of molecular, structural, and functional events as a result of PKP2-cKO has been well characterized (Cerrone et al., Nat Comm., 2017). PKP2 deficiency in adult ventricular myocytes is sufficient to cause an RV-predominant arrhythmogenic cardiomyopathy that includes functional, molecular, and structural hallmarks consistent with the disease phenotype of ACM.

[0343] PKP2-cKO mice were injected with tamoxifen to induce myocyte-specific PKP2 knockout. 3 × 10 cells were administered intravenously (tail vein) via tamoxifen injection (as described below). 13 Mice were injected with 1000 ng / kg of AAV vector. After 4 weeks, mice were treated with tamoxifen to induce myocyte-specific PKP2 knockout. The vector genomes tested were: 5'ITR; MHCK7 promoter (and its enhancer element); SV40 intron; Kozak sequence; PKP2a transgene; WPRE(x); hGH polyadenylation sequence; 3'ITR (structure illustrated in Figure 1B; e.g., SEQ ID NO:89); and 5'ITR; hTnnT2 promoter (and exon 1); Kozak sequence; PKP2a transgene; WPRE(x); hGH polyadenylation sequence; 3'ITR (structure illustrated in Figure 2; e.g., SEQ ID NO: 13 or 97) Each vector genome was tested in an AAV9 or AAVrh.74 serotype vector.

[0344] After 28 days of tamoxifen treatment and 56 days of AAV treatment, mice were evaluated for various physiological parameters essentially as described in Cerrone et al., Nat Comm., 2017, or using standard methodologies known in the art. Efficacy in treating disease was assessed using left ventricular ejection fraction percent (LVEF%) (FIG. 6), left ventricular fractional shortening percent (FS%) (FIG. 7), RV area in square millimeters (mm 2 RV area in millimeters per second (Figure 8), RV velocity time integral in millimeters per second (RV VTI) (mm / sec) (Figure 9), and the degree of fibrosis (Figures 10A-10B). These measures are among the key parameters indicative of ACM in human disease and are therefore appropriate functional and structural indices for assessing the potential efficacy of AAV-mediated PKP2 overexpression in cardiomyocytes. Generally, the amount of fibrosis in the right ventricle is usually slightly greater (regardless of disease), and this was further exacerbated with PKP2 deficiency in the cKO model. Progressive deterioration of these parameters was observed within 28 days of tamoxifen injection, because tamoxifen injection causes myocyte-specific PKP gene knockout. In vivo expression of the transgene was demonstrated using Western blots detecting PKP2 levels (Figures 11A-11B). 28 days after tamoxifen injection, AAV (3 × 10 13 Gel image (Figure 11A) and quantification of PKP2 normalized to GAPDH (Figure 11B), detected in the heart 56 days after injection of AAV9-MHCK7-PKP2a, AAV9-hTnT-PKP2a, AAVrh.74-MHCK7-PKP2a, or AAVrh.74-hTnT-PKP2a at a dose of 1000 mg / kg (quantified from the simultaneously acquired top gel in panel A).

[0345] Evidence of alleviation of the disease phenotype was observed to varying degrees following AAV9-mediated and AAVrh.74-mediated PKP expression. 13A pretreatment paradigm (AAV injection 4 weeks prior to tamoxifen-induced PKP cKO) was used, along with a dose of 1000 mg / kg (vg / kg). Given the cardiotropy of AAVrh.74 and the biological effects (e.g., LVEF%, FS%, and right ventricular area) observed with AAVrh.74-mediated PKP2 overexpression in this model, we also tested the optimization of the dose of AAVrh.74 in combination with the appropriate promoter (i.e., either MHCK7 or hTnnT2).

[0346] In the next set of experiments, 6 × 10 13 A dose of 1 × 10 vg / kg was tested, and 1 × 10 for AAV9-hTnT-PKP2a. 13 Doses of 1000 mg / kg were tested. After 28 days of treatment with tamoxifen and 56 days of treatment with AAV, cardiac parameters were evaluated as in the previous examples. Efficacy in treating disease was assessed by measuring the percent left ventricular ejection fraction (LVEF%) (FIG. 12A), percent left ventricular fractional shortening (FS%) (FIG. 12B), and RV area in square millimeters (mm 2 RV area in millimeters per second (RV VTI (mm / sec)) (Figure 13A), RV velocity time integral in millimeters per second (RV VTI (mm / sec)) (Figure 13B), and the extent of fibrosis (Figures 14A-14B). Western blots detecting PKP2 were used to demonstrate in vivo expressed protein levels (Figures 15A-15B). 28 days after tamoxifen injection, AAV (1 x 10 13 vg / kg dose of AAV9-hTnT-PKP2a or 6 × 10 13 Gel image (Figure 15A) and quantification of PKP2 normalized to GAPDH (Figure 15B, quantified from the simultaneously acquired top gel of panel A) detected in the heart 56 days after injection of AAVrh.74-hTnT-PKP2a at a dose of 1000 mg / kg.

[0347] For further analysis, samples were collected from either control (Cre-negative) mice injected with formulation buffer (control-FB; n = 4 mice), PKP2cKO mice injected with formulation buffer (PKP2cKO-FB; n = 4 mice), or PKP2-cKO mice injected with AAVrh.74-PKP2a (PKP2cKO-AAVrh.74-PKP2a; n = 4 mice). Mice were treated with FB or AAVrh.74-PKP2a 28 days before tamoxifen injection. Immunofluorescence images were obtained from paraffin-embedded tissue slices. Fluorescence intensity maps were collected from a spread of five to eight sarcomeres that appeared to be correctly aligned for analysis to obtain the fluorescence intensity map. Figure 31A shows an example of adult ventricular tissue stained with an antibody against AnkB (Cerrone et al., 2017). The red line indicates the range of fluorescence intensity as plotted in the right panel. Two parameters were collected: mean intensity and peak depth (defined as the range between the peak and valley; see Figure 31A, right panel). Representative images obtained under the three test conditions are shown in Figure 31B. The values ​​of each intensity map were averaged and considered as one data point. Multiple regions were mapped per tissue section, and data were collected separately from right ventricular (RV) or left ventricular (LV) samples. Results collected from the LV and RV are shown in Figure 31C and Figure 31D, respectively. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc analysis. Statistical significance (p-value) is indicated above the bars. The results demonstrate that re-expression of PKP2 in PKP2cKO cardiac myocytes reversed a previously reported molecular phenotype, i.e., reduced AnkB expression.

[0348] Similarly, tissue sections were immunostained for desmin (Figures 32A–32C; the immunostaining method was the same as described for PKP2; the desmin antibody was the same as that used in Perez-Hernandez et al. 2022; PMID: 35959657). Representative examples and results are shown in Figures 32A–32C. In this case, increased intensity from PKP2cKO cardiomyocytes (see Supplementary Figure 8 in Perez-Hernandez et al. 2022; PMID: 35959657) and a statistically significant recovery of signal intensity in AAV-treated mice were confirmed, along with a trend toward recovery of peak depth in both RV and LV samples, although the wide data distribution affected statistical power. The data show that two molecular hallmarks of PKP2cKO hearts—reduced AnkB and increased desmin—were reversed in hearts treated with the AAV-delivered PKP2 transgene.

[0349] These results demonstrate that AAV9 and AAVrh.74 can provide benefits in relevant animal models of PKP2-associated ACM (also known as arrhythmogenic right ventricular dysplasia (ARVD) or arrhythmogenic cardiomyopathy (ACM)). Furthermore, AAV vectors carrying either the MHCK7 promoter or the hTnnT2 promoter have proven effective in treating PKP2-associated diseases.

[0350] Example 3: In vivo efficacy of adeno-associated viral vectors After evaluating the in vivo efficacy of various AAV-PKP2 vector constructs at different doses in the PKP-cKO model of ACM, we selected the AAVrh.74-hTnT-PKP2a vector construct (Figure 2; e.g., SEQ ID NO: 13 or 97) for further R&D and clinical development based on its robust functional and anatomical benefits, the safety profile observed to date, and the potential evasion of immune responses to the AAV9 capsid that have been observed in clinical trials using high-dose intravenous delivery in adults.

[0351] To further evaluate and characterize the potential benefits of the lead vector, AAVrh.74-hTnT-PKP2, additional experiments were conducted, specifically to assess the extent to which benefits could be observed in this model of ACM after disease onset using a PKP2-cKO mouse model. An example experimental model and timeline is illustrated in Figure 16A-C. Animals were initially injected with tamoxifen, followed by intravenous injections of different doses of AAVrh.74-hTnT-PKP2 or control formulation buffer (FB) on days -28, +7, or +14. Treatment groups across the various studies included control animals, AAV9-hTnT-PKP2, and various doses of AAVrh.74-hTnT-PKP2 as follows (see figures and figure legends for specific doses used in separate experiments): WT (Cre-): formulation buffer (control); PKP2-cKO: formulation buffer (negative control); PKP2-cKO:AAV9-hTnT-PKP2:3E13vg / kg; PKP2-cKO:AAVrh.74-hTnT-PKP2:6E13vg / kg; and PKP2-cKO:AAVrh.74-hTnT-PKP2:2E14vg / kg. The expression cassette of the vector was as depicted in Figure 2. Echocardiography was performed after 21 days, 28 days, and 5 months of tamoxifen, depending on the experiment.

[0352] Findings from experiments using a delayed AAVrh.74-hTnT-PKP2a injection paradigm in the PKP2-cKO model are shown in Figures 17-20 and 27-29. It was found that the previously defined "effective dose" of AAVrh.74-hTnT-PKP2, 6E13 vg / kg, resulted in 100% survival at the predefined time point of 5 months after tamoxifen, compared with 100% mortality by day 50 in PKP2-cKO control animals (Figure 17). A "high dose" of AAVrh.74-hTnT-PKP2a (2E14 vg / kg) resulted in 90% survival, with one animal death found to be unrelated to AAV-hTnT-PKP2a. All AAVrh.74-hTnT-PKP2-injected animals were found to have preserved ejection fraction (Figure 18A), fractional shortening (Figure 18B), and right ventricular area at 28 days, which persisted for up to 5 months (Figure 19A). Importantly, AAVrh.74-hTnT-PKP2a administered after disease onset (14 days after tamoxifen) significantly attenuated isoproterenol-induced premature ventricular contractions, which are typically observed as a result of PKP2 loss in this mouse model as well as in ACM patients (Figures 20A-20C). Figures 21A-B show the extent of fibrosis after 5 months of tamoxifen in the left ventricle (Figure 21A) and right ventricle (Figure 21B), based on quantification of percent collagen after cardiac trichrome histological staining.

[0353] The presence and abundance of AAV vector DNA, transgene mRNA transcripts, and PKP2 protein in the heart were confirmed by ddPCR, RT-ddPCR, and Western blot of heart lysates, respectively. Cumulative results are shown in Figures 22 and 23A-C. The results demonstrate efficient transduction and subsequent expression of human PKP2a (hPKP2a) transgene mRNA and PKP2 protein in the heart in all AAVrh.74-PKP2a-injected mice.

[0354] Immunofluorescence images acquired from fixed sections of cardiac tissue are shown in Figures 24A-C. Figure 24A shows the immunolocalization of native PKP2 in the heart of a control (Cre-negative, TAM-injected) mouse. The image shows clear immunoreactive PKP2-positive signals visualized as distinct plaques aligned perpendicular to the fiber direction, as expected for intercalated disc proteins (see arrows in Figure 24A). Figures 24B and 24C show cardiac sections from PKP2-cKO mice euthanized 28 days after TAM and previously (28 days before TAM) injected with either FB (Figure 24B) or FB + AAVrh.74-PKP2a (Figure 24C). Notably, immunolabeled PKP2 signals were absent in hearts from PKP2-cKO mice injected with FB alone (Figure 24B). However, in hearts receiving AAVrh.74-PKP2a treatment, there was a high density of PKP2 signals oriented perpendicular to the fiber direction (Figure 24C; arrows). These results were confirmed in four mice per group. The data demonstrate that the exogenous PKP2 gene was transcribed and translated, and the expressed protein correctly localized to the subcellular domains predicted for the native PKP2 protein.

[0355] The ability of exogenous proteins to prevent the cardiomyopathy phenotype was also examined. 28 days after TAM injection, cardiac function was assessed in treated mice by echocardiography. Figures 25A-C show the cardiac function of mice treated with FB alone (control, first bar from the left; PKP2-cKO, second bar from the left), or AAVrh.74-PKP2a (PKP2-cKO + 3x10 13 vg / kg AAVrh.74-PKP2a, third bar from the left; PKP2-cKO+6 × 10 13Echocardiographic images (Figure 25A) and cumulative data (Figures 25B and 25C) obtained from hearts of mice injected with 1000 mg / kg AAVrh.74-PKP2a (4th bar from the left) are shown. As shown in Figures 25A-C, AAVrh.74-PKP2a or FB were injected 56 days prior to recording and 28 days prior to TAM injection. As expected, loss of PKP2 expression significantly reduced left ventricular ejection fraction (LVEF; dotted line and second bar in Figure 25B) and increased right ventricular (RV) area (dotted line and second bar from the left in Figure 25C). In contrast, AAVrh.74-mediated hPKP2a expression dose-dependently attenuated or prevented the loss of LV contractile function and the increase in RV area.

[0356] Cardiac fibrosis is a common feature in PKP2-deficient hearts. As shown in Figure 26A-C, visualization and quantification of the extent of collagen abundance in the ventricular free wall revealed the presence of extensive fibrosis in PKP2-cKO animals (second bar from the left), which is particularly evident in the high (6 × 10 13 Fibrosis was significantly reduced by administration of AAVrh.74-PKP2a at a dose of 1000 mg / kg (4th bar from the left) (third and fourth bars from the left). Notably, a dose-related reduction in cardiac fibrosis was observed in the left and right ventricles of AAVrh.74-PKP2a-injected animals.

[0357] The data presented in the previous figure demonstrate that treatment with AAVrh.74-PKP2a prior to TAM-mediated PKP2 knockout can attenuate the development of a cardiomyopathy phenotype in PKP2-cKO mice. To assess whether AAVrh.74-PKP2a delivery after tamoxifen-mediated disease induction can halt the progression of the ARVC phenotype, PKP2-cKO mice were injected with AAVrh.74-PKP2a 7 or 14 days after TAM injection (see Figure 16B-C). In addition to echocardiographic analysis, we also tracked the long-term survival of mice. As illustrated by the Kaplan-Meier curves in Figure 27, only FB-injected PKP2-cKO animals died 30–50 days after TAM injection, consistent with previous reports. In contrast, all but one of the AAVrh.74-PKP2a-injected mice survived for 5 months (155 days after TAM). At this time, the animals were euthanized and protein expression and cardiac structure were examined.

[0358] Furthermore, as shown by the representative images and cumulative data in Figures 28A-B, Trichrome staining analysis demonstrated that a high dose of AAVrh.74-PKP2a (2 x 10 14 The percentage of LV free wall and percentage of RV free wall occupied by collagen in animals injected with low doses of AAVrh.74-PKP2a (6 × 10 vg / kg, third bar from the left) were found to be similar (albeit with a trend towards higher values) when compared to those observed in control animals injected with FB (first bar from the left). 13Collagen abundance in hearts from mice fed TAM (vg / kg, second bar from the left) was higher than in control animals. Importantly, comparison with collagen abundance in PKP2-cKO animals at the same time point was not possible due to early lethality in this group. However, collagen abundance in treated animals was lower than that documented by historical data from PKP2-cKO mice 42 days after TAM injection (close to the time of death according to the Kaplan-Meier curve in Figure 27). Finally, cumulative data obtained from echocardiographic analysis of hearts from mice injected with AAVrh.74-PKP2a 7 or 14 days after TAM are shown in Figures 28C-D. Hearts from FB-injected PKP2-cKO mice exhibited a dramatic decrease in LVEF (Figure 28C) and an increase in RV area (Figure 28D) 28 days after TAM injection (compare the second and first bars from the left in Figures 28C-D), consistent with previous findings. These changes were mitigated by AAVrh.74-PKP2a even when injected 14 days after TAM, and the beneficial effects persisted up to 5 months after TAM injection, the longest time point evaluated.

[0359] Previous studies have demonstrated that a bolus of 3 mg / kg isoproterenol (ISO) 21 days after TAM injection in anesthetized PKP2-cKO mice induces premature ventricular contractions. Therefore, we used an ISO exposure protocol to determine whether AAVrh.74-PKP2a delivered 14 days after TAM could reduce arrhythmia burden in PKP2-cKO animals. Representative ECG traces are shown in Figures 29A-B. FB-injected PKP2-cKO animals exhibited multiple PVCs, with 6 of 10 animals showing more than 100 PVCs within 30 minutes of recording after ISO injection (Figure 29C), and the total number of PVCs averaged approximately 300 (Figure 29D). In contrast, both parameters of arrhythmia burden were significantly reduced by AAVrh.74-PKP2a administration at both doses tested (6 × 10 13 vg / kg and 2 × 10 14 vg / kg, purple and orange bars in Figures 29C-D, respectively) were also dramatically reduced.

[0360] Safety and toxicology studies in mice consistently demonstrated adequate vector DNA biodistribution. Transgene mRNA was 10-100-fold enriched in the heart compared with all other organs, including the liver. Clinical and histopathological analyses demonstrated that AAVrh.74-PKP2a delivered 3 × 10 transgenes to mice across multiple studies. 14 Doses up to (and including) 2000mg / kg were found to be well tolerated and safe.

[0361] Example 4: Phase 1 Clinical Trial A multicenter dose-escalation study is conducted for PKP2-related ACM.Subjects may include those with mutations that lead to truncated PKP2 expression, such as "right-stop-gain" variant mutations.Subjects may have ICD.

[0362] Important inclusion / exclusion criteria include age ≥18 years; ACM diagnosis; PKP2 gene mutation; clinically significant arrhythmia burden; and preserved LV function.

[0363] Treatment with the PKP2 gene therapy vectors disclosed herein, such as AAV9 or AAVrh.74, is expected to improve one or more endpoints. Expected endpoints include, but are not strictly limited to: Primary: Safety (frequency and severity of AE / SAE) Secondary: Key Exploratory / Preliminary efficacy Myocardial PKP2 expression and vector copy number PVC decline during continuous Holter monitoring; other parameters affecting LTVA risk according to the ARVC Risk Calculator; or any parameter affecting LTVA risk Exploratory Endpoints VT / VF monitoring (ECG / Holter / AICD) Reduced incidence of ICD shocks RV function improvement (MRI / echo) LV ejection fraction (MRI / echo) Blood biomarkers: BNP, NT-proBNP, troponin I Kansas City Cardiomyopathy Questionnaire Stable LGE on MRI Left or right atrial volume Additional arrhythmia-related endpoints Physiological cardiac assessment and quality of life assessment Additional biomarkers

[0364] Example 5: In vivo expression of PKP2 in non-human primates (NHPs) To demonstrate that the vectors disclosed herein lead to expression in the heart of large mammals, studies were performed in non-human primates (NHPs).

[0365] NHPs were intravenously injected with a "low" or "high" dose of AAVrh.74-PKP2a (see Figure 2, e.g., SEQ ID NO: 13 or 97) or formulation buffer (FB; vehicle control). The low dose was 8 x 10 13 vg / kg, and the high dose is 3 × 10 14 The AAVrh.74-PKP2a vector delivered an expression cassette and flanking AAV inverted terminal repeats (ITRs) to cardiomyocytes. Polynucleotide sequence encoding PKP2, isoform a, of SEQ ID NO:1; the hTnnT2 promoter sequence (and exon 1) of SEQ ID NO:32 operably linked to a polynucleotide sequence encoding PKP2a; and Poly(A) sequence The polynucleotide comprises:

[0366] Three months after a single intravenous injection of the AAVrh.74-PKP2a vector, animals were humanely sacrificed and cardiac tissue was collected. Expression of human PKP2a transgene mRNA and PKP2a protein in cardiac tissue was confirmed by RT-PCR, immunostaining, and Western blot.

[0367] As shown in Figures 30A-D, NHPs administered FB exhibited endogenous PKP2a expression in the heart, whereas NHPs administered AAVrh.74-PKP2a exhibited elevated levels of PKP2a mRNA and protein compared to controls. The data demonstrate that transgene PKP2a was detected in non-human primate hearts across multiple levels of analysis. Figures 30A-B show photomicrographs after immunohistochemical labeling of PKP2 protein in whole heart sections from non-human primate animals treated with either control FB (Figure 30A) or "low-dose" (Figure 30B). Moderate levels of endogenous PKP2 immunolabeling were observed in the intercalated discs, the interjunctional spaces between cardiomyocytes, in FB control-treated animals (see, e.g., open triangles in Figure 30A). This contrasts sharply with the intense and frequently detected PKP2-positive immunolabeling observed in the intercalated discs of "low-dose"-treated animals (see, e.g., open triangles in Figure 30B). Figures 30C and 30D show quantification of PKP2 protein and transgene mRNA (hPKP2a) by Western blot in the ventricles of non-human primate hearts after intravenous injection of "low" or "high" doses of AAVrh.74-PKP2a or FB (control).

[0368] These data demonstrate that the AAVrh.74-PKP2a vector transduced and expressed a potentially therapeutic transgene in cardiac tissue of a large mammal after intravenous administration.

[0369] Safety and toxicology studies in non-human primates (NHPs) demonstrated adequate vector DNA biodistribution. Clinical and histopathological analyses demonstrated that AAVrh.74-PKP2a delivered 3 × 10 in NHPs across multiple studies. 14Doses up to (and including) 2000 mg / kg were found to be well tolerated and safe.

[0370] array TIFF2026502465000021.tif127152TIFF2026502465000022.tif224152TIFF2026502465000023.tif224152TIF F2026502465000024.tif224152TIFF2026502465000025.tif224152TIFF2026502465000026.tif224152TIFF202 6502465000027.tif224152TIFF2026502465000028.tif224152TIFF2026502465000029.tif224152TIFF2026502 465000030.tif224152TIFF2026502465000031.tif224152TIFF2026502465000032.tif224152TIFF20265024650 00033.tif224152TIFF2026502465000034.tif224152TIFF2026502465000035.tif224152TIFF20265024650000 36.tif224152TIFF2026502465000037.tif224152TIFF2026502465000038.tif224152TIFF2026502465000039.t if224152TIFF2026502465000040.tif224152TIFF2026502465000041.tif224152TIFF2026502465000042.tif22 4152TIFF2026502465000043.tif224152TIFF2026502465000044.tif224152TIFF2026502465000045.tif120152

Claims

1. 1. A polynucleotide comprising an expression cassette and, optionally, flanking adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein the polynucleotide comprises a polynucleotide sequence encoding plakophilin-2 (PKP2) or a functional variant thereof operably linked to a promoter, and optionally, the polynucleotide shares 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%, or 100% identity with any one of SEQ ID NOs:8-15, 89-96, or 97-102.

2. 2. The polynucleotide of claim 1, wherein the promoter is a cardiac-specific promoter.

3. 3. The polynucleotide of claim 1 or claim 2, wherein the promoter is a muscle-specific promoter.

4. The polynucleotide of any one of claims 1 to 3, wherein the promoter is a cardiomyocyte-specific promoter.

5. 5. The polynucleotide of any one of claims 1 to 4, wherein the promoter is the myosin heavy chain creatine kinase 7 (MHCK7) promoter.

6. 6. The polynucleotide of claim 5, wherein the MHCK7 promoter shares 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% or 100% identity with SEQ ID NO:

31.

7. 5. The polynucleotide of any one of claims 1 to 4, wherein the promoter is a cardiac troponin T (hTNNT2) promoter.

8. The polynucleotide of claim 7, wherein the hTNNT2 promoter shares 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% or 100% identity with SEQ ID NO:

32.

9. 9. The polynucleotide of any one of claims 1 to 8, wherein the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene, and optionally the hTNNT2 promoter and exon 1 together share 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%, or 100% identity with SEQ ID NO:

32.

10. 4. The polynucleotide of any one of claims 1 to 3, wherein the promoter is a ubiquitous promoter, optionally a CMV promoter or a CAG promoter.

11. The polynucleotide of any one of claims 1 to 10, wherein the expression cassette comprises a polyA signal.

12. 12. The polynucleotide of claim 11, wherein the polyA signal is human growth hormone (hGH) polyA.

13. 13. The polynucleotide of any one of claims 1 to 12, wherein the expression cassette comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), optionally a WPRE(x).

14. 14. The polynucleotide of any one of claims 1 to 13, wherein the plakophilin-2 (PKP2) or functional variant thereof is PKP2.

15. 15. The polynucleotide of claim 14, wherein the PKP2 is functional PKP2.

16. 16. The polynucleotide of claim 14 or claim 15, wherein the PKP2 is human PKP2.

17. 17. The polynucleotide of claim 16, wherein the PKP2 is PKP2 isoform A.

18. 18. The polynucleotide of claim 17, wherein PKP2 isoform A shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:

1.

19. 17. The polynucleotide of claim 16, wherein the PKP2 is PKP2 isoform B.

20. 20. The polynucleotide of claim 19, wherein PKP2 shares 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% or 100% identity with SEQ ID NO:

2.

21. 21. The polynucleotide of any one of claims 1 to 20, wherein the polynucleotide sequence encoding PKP2 is a human PKP2 polynucleotide.

22. 22. The polynucleotide of any one of claims 1 to 21, wherein the polynucleotide sequence encoding PKP2 shares 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% or 100% identity with SEQ ID NO:

3.

23. 22. The polynucleotide of any one of claims 1 to 21, wherein the polynucleotide sequence encoding PKP2 shares 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% or 100% identity with SEQ ID NO:

4.

24. 24. The polynucleotide of any one of claims 1 to 23, comprising at least about 4.0 kb, at least about 4.1 kb, at least about 4.2 kb, at least about 4.3 kb, at least about 4.4 kb, or at least about 4.5 kb.

25. 25. The polynucleotide of any one of claims 1 to 24, comprising at most about 4.1 kb, at most about 4.2 kb, at most about 4.3 kb, at most about 4.4 kb, at most about 4.5 kb, or at most about 4.6 kb.

26. 26. The polynucleotide of any one of claims 1 to 25, comprising between 4.0kb and 4.6kb, between 4.0kb and 4.5kb, or between 4.0kb and 4.4kb, or consisting of between 4.0kb and 4.3kb, between 4.0kb and 4.2kb, or between 4.0kb and 4.1kb.

27. 27. The polynucleotide of any one of claims 1 to 26, wherein PKP2 or a functional variant thereof consists of at least 800 or at least 830 amino acids.

28. 28. The polynucleotide of any one of claims 1-27, sharing 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%, or 100% identity with any one of SEQ ID NOs:8-15, 89-96, or 97-102.

29. 29. The polynucleotide of any one of claims 1 to 28, wherein the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs).

30. 30. The polynucleotide of claim 29, wherein the ITR is an AAV2 ITR and / or the ITR shares 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%, or 100% identity with any one of SEQ ID NOs:20-26.

31. A gene therapy vector comprising the polynucleotide of any one of claims 1 to 30, and optionally comprising the sequence of any one of SEQ ID NOs: 8-15 89-96, or 97-102.

32. 32. The vector of claim 31, wherein the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.

33. The vector of claim 32, wherein the rAAV vector is AAV9 or a functional variant thereof.

34. The vector of claim 33, wherein the rAAV vector comprises a capsid protein that shares at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NO:

77.

35. The vector of claim 32, wherein the rAAV vector is AAVrh.10 or a functional variant thereof.

36. The vector of claim 35, wherein the rAAV vector comprises a capsid protein that shares at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NO:

79.

37. The vector of claim 32, wherein the rAAV vector is AAV6 or a functional variant thereof.

38. The vector of claim 37, wherein the rAAV vector comprises a capsid protein that shares at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NO:

78.

39. The vector of claim 38, wherein the rAAV vector is AAVrh.74 or a functional variant thereof.

40. 40. The vector of claim 39, wherein the rAAV vector comprises a capsid protein that shares at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or 100% identity to SEQ ID NOs:81-83.

41. 41. A method for treating and / or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject the vector of any one of claims 31 to 40.

42. 42. The method of claim 41, wherein the disease or disorder is a cardiac disorder.

43. 43. The method of claim 41 or 42, wherein the disease or disorder is cardiomyopathy.

44. 44. The method of claim 43, wherein the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ACM).

45. 44. The method of claim 43, wherein the cardiomyopathy is hypertrophic cardiomyopathy or dilated cardiomyopathy.

46. 42. The method of claim 41, wherein the disease or disorder is characterized by fibrofatty infiltration of the myocardium.

47. 43. The method of claim 41 or 42, wherein the disease or disorder is heart failure.

48. The method of any one of claims 41 to 47, wherein the subject is a mammal.

49. 49. The method of claim 48, wherein the subject is a primate.

50. 50. The method of claim 49, wherein the subject is a human.

51. 51. The method of any one of claims 41 to 50, wherein the subject has a mutation in the PKP2 gene, optionally a stop-gain variant mutation, and / or optionally the subject has an implantable cardioverter defibrillator (ICD).

52. 52. The method of any one of claims 41-51, wherein the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheter.

53. The method of any one of claims 41-52, wherein said administering increases PKP2 expression by at least about 5%.

54. The method of any one of claims 41-52, wherein said administering increases PKP2 expression by at least about 30%.

55. The method of any one of claims 41-52, wherein said administering increases PKP2 expression by at least about 70%.

56. The method of any one of claims 41-52, wherein said administering increases PKP2 expression by about 5% to about 10%.

57. The method of any one of claims 41 to 52, wherein said administering increases PKP2 expression by about 30% to about 50%.

58. The method of any one of claims 41-52, wherein said administering increases PKP2 expression by about 50% to about 70%.

59. The method of any one of claims 41-52, wherein said administering increases PKP2 expression by about 70% to about 100%.

60. 60. The method of any one of claims 41 to 59, for treating and / or preventing a disease or disorder.

61. 61. The method of any one of claims 41 to 60, comprising administering an effective amount of said vector.

62. 62. The method of any one of claims 41 to 61, wherein the disease or disorder is associated with or caused by loss of function of PKP2 in the subject.

63. 62. The method of any one of claims 41 to 61, wherein the disease or disorder is associated with or caused by a gain of function of PKP2 in the subject.

64. 64. The method of any one of claims 41 to 63, wherein the subject has a mutation causing an amino acid substitution selected from Arg490Trp, Asp26Asn, Thr50_Val51SerfsX60, Arg79X, Tyr86X, Gln133X, Val406SerfsX3, Tyr616X, Trp676X, Cys796Arg, Cys796E, Tyr807X, Glu62Lys, S688P, Trp848X, Y86X, V406X, Y616X, W848X, and Y807X compared to a human PKP2 gene encoding human PKP2 having the sequence of SEQ ID NO:

2.

65. 65. The method of any one of claims 41 to 64, comprising administering an effective amount of a pharmaceutical composition comprising said vector.

66. Approximately 1×10 11 Vector genome ~ approx. 1 x 10 13 administering to the subject a vector of the vector genome, 12 Vector genome ~ approx. 1 x 10 14 administering to a subject a vector of the vector genome, or about 1 x 10 13 Vector genome ~ approx. 1 x 10 15 administering a vector of the vector genome to a subject; Optionally, the vector is AAV9, and optionally comprises a sequence having at least 80%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 8-15, 89-96, or 97-102, and optionally comprises a sequence having at least 80%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 12, 8, 13, 9, 97, 100, 89, 93, 90, or 94, and the method comprises administering to about 1 x 10 13 ~Approx. 1×10 14 administering a genomic vector to a subject, Optionally, the vector is AAV.rh.74, and optionally comprises a sequence having at least 80%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 8-15, 89-96, or 97-102, and optionally comprises a sequence having at least 80%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 12, 8, 13, 9, 97, 100, 89, 93, 90, or 94, and the method comprises transfecting at least about 5×10 13 ~Approx. 5×10 14 The method of any one of claims 41 to 65, comprising administering a genomic vector to a subject.

67. A pharmaceutical composition comprising the vector of any one of claims 31 to 40.

68. A kit comprising the vector of any one of claims 31 to 40 or the pharmaceutical composition of claim 6, and optionally instructions for use.

69. 67. Use of a vector according to any one of claims 31 to 40 in the treatment of a disease or disorder, optionally according to a method according to any one of claims 41 to 66.

70. 67. A vector according to any one of claims 31 to 40 for use in the treatment of a disease or disorder, optionally according to a method according to any one of claims 41 to 66.

71. A polynucleotide comprising a polynucleotide sequence sharing at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 12-15, 89-92, or 97-99, or to any one of SEQ ID NOs: 8-11, 93-96, or 100-102.

72. 72. The polynucleotide of claim 71, wherein the promoter is the MHCK7 promoter.

73. 73. The polynucleotide of claim 72, wherein the MHCK7 promoter shares 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% or 100% identity with SEQ ID NO:

31.

74. 72. The polynucleotide of claim 71, wherein the PKP2 is human PKP2.

75. 75. The polynucleotide of claim 74, wherein the PKP2 is PKP2 isoform A.

76. 76. The polynucleotide of claim 75, wherein PKP2 isoform A shares at least 80%, at least 90%, at least 95%, at least 99% or 100% identity with SEQ ID NO:

1.

77. A gene therapy vector comprising the polynucleotide of any one of claims 71 to 76.

78. 78. The vector of claim 77, wherein the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.

79. The vector of claim 78, wherein the rAAV vector is an AAV9 vector.

80. The vector of claim 78, wherein the rAAV vector is an AAVrh.74 vector.

81. 81. A method of treating and / or preventing cardiac damage in a subject identified as having a mutation in the PKP2 gene, said method comprising administering to said subject a vector of any one of claims 77 to 80, optionally having a stop-gain variant mutation, and / or optionally wherein said subject has an implantable cardioverter-defibrillator (ICD).

82. 82. The method of claim 81, wherein the cardiac disorder is a cardiomyopathy, optionally arrhythmogenic cardiomyopathy (ACM), arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic right ventricular dysplasia (ARVD), hypertrophic cardiomyopathy, or dilated cardiomyopathy.

83. 82. The method of claim 81, wherein the cardiac disorder is heart failure.

84. 84. The method of any one of claims 81 to 83, wherein the subject is a mammal.

85. 85. The method of any one of claims 81-84, wherein said vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheter.

86. 86. The method of any one of claims 81-85, wherein the decrease in left ventricular ejection fraction percent (LVEF%) is prevented or reduced, optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as compared to the decrease observed in an untreated subject identified as having a mutation in the PKP2 gene.

87. The method of any one of claims 81-86, wherein the decrease in left ventricular fractional shortening percent (FS%) is prevented or reduced by, optionally, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as compared to the decrease observed in an untreated subject identified as having a mutation in the PKP2 gene.

88. The method of any one of claims 81-87, wherein the increase in right ventricular area in millimeters squared RV area (mm2) is prevented or reduced by, optionally, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% as compared to the increase observed in an untreated subject identified as having a mutation in the PKP2 gene.

89. 89. The method of any one of claims 81-88, wherein the decrease in right ventricular velocity time integral in millimeters / second RV VTI (mm / sec) is prevented or reduced by, optionally, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% as compared to the decrease observed in an untreated subject identified as having a mutation in the PKP2 gene.

90. The method of any one of claims 81-89, wherein the increase in left ventricular fibrosis or right ventricular fibrosis is prevented or reduced, optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, as compared to the increase observed in an untreated subject identified as having a mutation in the PKP2 gene.

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