Skp2 gene therapy using aav vectors

Abstract

Provided herein are gene therapies against PKP2 (spot phenanthrin protein-2), such as the use of adeno-associated virus (AAV) vectors. The promoter of the vector can be an MHCK7 promoter or a cardiac troponin T (cTnT2) promoter. The capsid may be AAV9 or AAVrh.74 or a capsid protein comprising AAV9 or AAVrh.74, or a functional variant thereof. Other promoters or capsids may be used. Also provided are methods of treatment, such as by intravenous, intracoronary, intracarotid, or intracardiac administration of the rAAV vector, as well as other compositions and methods.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 437,616 filed on January 6, 2023, U.S. Provisional Patent Application No. 63 / 460,557 filed on April 19, 2023, and U.S. Provisional Patent Application No. 63 / 525,426 filed on July 7, 2023, which are incorporated herein by reference in their entireties.

[0003] References to electronic sequence listings

[0004] 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 its entirety.

[0005] background

[0006] Arrhythmogenic cardiomyopathy (ACM) is an adult-onset form of heart disease that affects an estimated 1 in 1,000 to 1 in 1,250 people. Because this cardiomyopathy typically manifests in the free wall of the right ventricle, ACM is also known as arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic right ventricular dysplasia (ARVD). However, the observation of left-ventricle-dominant and biventricular forms has led to the recent use of the term "ACM" to encompass all forms of the disease. It manifests as a breakdown of the heart's muscle wall (myocardium) over time, which leads to an increased risk of abnormal heartbeats (arrhythmias) and sudden death when affected individuals exercise vigorously. Individuals may also experience a fluttering or pounding sensation in the chest (palpitations), dizziness, fainting (syncope), shortness of breath, and abnormal swelling of the legs or abdomen. Over time, ACM can lead to heart failure.

[0007] At least 13 genes have been implicated in ACM, many of which are involved in the biogenesis of desmosomes, which are intracellular junctions that provide strong adhesion between cells. When desmosomes fail to form normally, cardiomyocytes may separate from each other and die. The right ventricle, in particular, may develop weakness, and fatty deposits and scar tissue may replace the damaged myocardium, leading to right ventricular dilation. These changes ultimately prevent efficient heart pumping and disrupt the electrical signals that control the heartbeat, leading to arrhythmias. Autosomal dominant plakophilin-2 (PKP2) cardiomyopathy (also known as ARVD or ARVC) is an inherited form of ACM in which mutations affecting PKP2 have been detected.

[0008] Thus, there remains an unmet need in the art for treatments for PKP2-related diseases and disorders, including ACM. The compositions and methods disclosed herein address this need. Summary of the Invention

[0009] The present disclosure generally relates to gene therapy for a disease or condition, such as a heart disease or condition, using a vector that expresses PKP2 or a functional variant thereof.

[0010] In one aspect, the present disclosure provides a polynucleotide comprising an expression cassette and optionally flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein the polynucleotide comprises a polynucleotide sequence encoding plaquefhyrin-2 (PKP2) or a functional variant thereof, the polynucleotide sequence being operably linked to a promoter.

[0011] In some embodiments, the promoter is a cardiac-specific promoter.

[0012] In some embodiments, the promoter is a muscle-specific promoter.

[0013] In some embodiments, the promoter is a cardiomyocyte-specific promoter.

[0014] In some embodiments, the promoter is the myosin heavy chain creatine kinase 7 (MHCK7) promoter.

[0015] In some embodiments, the MHCK7 promoter is 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% identical to SEQ ID NO:31.

[0016] In some embodiments, the promoter is the cardiac troponin T (hTNNT2) promoter.

[0017] In some embodiments, the hTNNT2 promoter is 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% identical to SEQ ID NO:32.

[0018] In some embodiments, the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene, wherein optionally the hTNNT2 promoter and exon 1 together are 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% identical to SEQ ID NO: 32.

[0019] In some embodiments, the promoter is a pan-promoter, optionally a CMV promoter or a CAG promoter.

[0020] In some embodiments, the expression cassette comprises a polyA signal.

[0021] In some embodiments, the polyA signal is human growth hormone (hGH) polyA.

[0022] In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE), optionally a WPRE(x) containing a mutation that disables potential synthesis of the Woodchuck Hepatitis Virus X protein.

[0023] In some embodiments, the plaquefhyrin-2 (PKP2) or a functional variant thereof is PKP2.

[0024] In some embodiments, the PKP2 is functional PKP2.

[0025] In some embodiments, the PKP2 is human PKP2.

[0026] In some embodiments, the PKP2 is PKP2 isoform A.

[0027] In some embodiments, the PKP2 isoform A is 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% identical to SEQ ID NO: 1.

[0028] In some embodiments, the PKP2 is PKP2 isoform B.

[0029] In some embodiments, the PKP2 is 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% identical to SEQ ID NO:2.

[0030] In some embodiments, the polynucleotide sequence encoding PKP2 is a human PKP2 polynucleotide.

[0031] In some embodiments, the polynucleotide sequence encoding PKP2 is 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% identical to SEQ ID NO:3.

[0032] In some embodiments, the polynucleotide sequence encoding PKP2 is 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% identical to SEQ ID NO:4.

[0033] 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.

[0034] In some embodiments, the polynucleotide comprises 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.

[0035] In some embodiments, the polynucleotide comprises 4.0 kb to 4.6 kb, 4.0 kb to 4.5 kb, or 4.0 kb to 4.4 kb, or wherein the polynucleotide comprises 4.0 kb to 4.3 kb, 4.0 kb to 4.2 kb, or 4.0 kb to 4.1 kb.

[0036] In some embodiments, the PKP2 or a functional variant thereof comprises at least 800 or at least 830 amino acids.

[0037] In some embodiments, the polynucleotide is 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% identical to any one of SEQ ID NOs: 8-15, 89-96, and 97-102.

[0038] In some embodiments, the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs).

[0039] In some embodiments, the ITR is an AAV2 ITR and / or the ITR is 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% identical to any one of SEQ ID NOs: 20-26.

[0040] In another aspect, the present disclosure provides a gene therapy vector comprising the polynucleotide of any of the preceding embodiments.

[0041] In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.

[0042] In some embodiments, the rAAV vector is AAV9 or a functional variant thereof.

[0043] In some embodiments, the rAAV vector comprises a capsid protein that is 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% identical to SEQ ID NO:77.

[0044] In some embodiments, the rAAV vector is AAVrh.10 or a functional variant thereof.

[0045] In some embodiments, the rAAV vector comprises a capsid protein that is 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% identical to SEQ ID NO:79.

[0046] In some embodiments, the rAAV vector is AAV6 or a functional variant thereof.

[0047] In some embodiments, the rAAV vector comprises a capsid protein that is 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% identical to SEQ ID NO:78.

[0048] In some embodiments, the rAAV vector is AAVrh.74 or a functional variant thereof.

[0049] In some embodiments, the rAAV vector comprises a capsid protein that is 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% identical to SEQ ID NOs: 81-83.

[0050] 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 the vector of any one of the preceding embodiments.

[0051] In some embodiments, the disease or condition is cardiac disease.

[0052] In some embodiments, the disease or disorder is cardiomyopathy.

[0053] In some embodiments, the cardiomyopathy is arrhythmogenic cardiomyopathy (ACM).

[0054] In some embodiments, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC) or arrhythmogenic right ventricular dysplasia (ARVD).

[0055] In some embodiments, the cardiomyopathy is hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0056] In some embodiments, the disease or disorder is characterized by fibrofatty infiltration of the myocardium.

[0057] In some embodiments, the disease or condition is heart failure.

[0058] In some embodiments, the subject is a mammal.

[0059] In some embodiments, the subject is a primate.

[0060] In some embodiments, the subject is a human.

[0061] In some embodiments, the subject has a mutation in the PKP2 gene.

[0062] In some embodiments, the mutation is a "stop-gain" variant mutation of the PKP2 gene.

[0063] In some embodiments, the subject has an implantable cardioverter-defibrillator (ICD).

[0064] In some embodiments, the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheterization.

[0065] In some embodiments, administration increases PKP2 expression by at least about 5%.

[0066] In some embodiments, administration increases PKP2 expression by at least about 30%.

[0067] In some embodiments, administration increases PKP2 expression by at least about 70%.

[0068] In some embodiments, administration increases PKP2 expression by about 5% to about 10%.

[0069] In some embodiments, administration increases PKP2 expression by about 30% to about 50%.

[0070] In some embodiments, administration increases PKP2 expression by about 50% to about 70%.

[0071] In some embodiments, administration increases PKP2 expression by about 70% to about 100%.

[0072] In a specific embodiment, the increased PKP2 expression is PKP2 expression in the heart or cardiac tissue of a subject to which the vector is administered. In a specific embodiment, the increased PKP2 expression is PKP2 expression in the left ventricle. In a specific embodiment, the increased PKP2 expression is PKP2 expression in the right ventricle.

[0073] In some embodiments, the methods treat and / or prevent a disease or disorder.

[0074] In some embodiments, the method comprises administering an effective amount of a vector.

[0075] In some embodiments, the disease or disorder is associated with or caused by loss of function of PKP2 in the subject.

[0076] In some embodiments, the disease or disorder is associated with or caused by a gain of function of PKP2 in the subject.

[0077] In some embodiments, the subject has a mutation resulting in an amino acid substitution selected from the group consisting of Arg490Trp, Asp26Asn, Thr50_Val51SerfsX60, Arg79X, Tyr86X, Gln133X, Val406SerfsX3, Tyr616X, Trp676X, Cys796Arg, Cys796E, Tyr807X, Glu62Lys, S688P, Trp848X, Y86X, V406X, Y616X, W848X, and Y807X relative to a human PKP2 gene encoding a human PKP2 having a sequence of SEQ ID NO: 2.

[0078] In some embodiments, the method comprises administering a pharmaceutical composition comprising an effective amount of a carrier.

[0079] In some embodiments, the method comprises administering to the subject about 1×10 11 Vector genomes / kg is about 1×10 13 Vector genomes / kg of vector, approximately 1×10 12 Vector genomes / kg is about 1×10 14 vector genomes / kg of vector, or administering approximately 1×10 13 Vector genomes / kg is about 1×10 15In some embodiments, the vector is AAV9, optionally comprising a sequence at least 90% or at least 95% identical to any one of SEQ ID NOs: 12, 8, 13, 9, 97, 100, 89, 93, 90, or 94, and the method comprises administering to the subject approximately 1×10 13 to about 1×10 14 vector genomes / kg, for example, about 1×10 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 about 1×10 14 In some embodiments, the vector is AAV.rh.74, optionally comprising a sequence at least 90% or at least 95% identical to any one of SEQ ID NOs: 12, 8, 13, 9, 97, 100, 89, 93, 90, or 94, and the method comprises administering to the subject approximately 5×10 13 to about 5×10 14 vector genomes / kg, for example, about 5×10 13 , about 6×10 13 , about 7×10 13 , about 8×10 13 , about 9×10 13 , about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg or about 5×10 14 In some embodiments, the vector is administered intravenously or systemically or locally to the heart.

[0080] In another aspect, the present disclosure provides a pharmaceutical composition comprising the carrier of any one of the preceding embodiments.

[0081] 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.

[0082] In another aspect, the present disclosure provides use of a vector according to any of the preceding embodiments for treating a disease or disorder, optionally according to a method according to any of the preceding embodiments.

[0083] In another aspect, the present disclosure provides a vector according to any of the preceding embodiments for use in treating a disease or disorder, optionally according to a method according to any of the preceding embodiments.

[0084] In another aspect, the present disclosure provides a polynucleotide comprising a polynucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical 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.

[0085] In some embodiments, the promoter is the MHCK7 promoter.

[0086] In some embodiments, the MHCK7 promoter is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31.

[0087] In some embodiments, PKP2 is human PKP2.

[0088] In some embodiments, PKP2 is PKP2 isoform A.

[0089] In some embodiments, PKP2 isoform A is at least 80%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:1.

[0090] In another aspect, the present disclosure provides a gene therapy vector comprising the polynucleotide of any of the preceding embodiments.

[0091] In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.

[0092] In some embodiments, the rAAV vector is an AAV9 vector.

[0093] In some embodiments, the rAAV vector is an AAVrh.74 vector.

[0094] In another aspect, the present disclosure provides a method of treating and / or preventing a cardiac disorder in a subject identified as having a mutation in the PKP2 gene, comprising administering to the subject the vector of any one of the preceding embodiments.

[0095] In some embodiments, the cardiac disorder is a cardiomyopathy, optionally arrhythmogenic cardiomyopathy (ACM), hypertrophic cardiomyopathy, or dilated cardiomyopathy.

[0096] In some embodiments, the cardiac disorder is heart failure.

[0097] In some embodiments, the subject is a mammal.

[0098] In some embodiments, the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and / or cardiac catheterization.

[0099] In some embodiments, the method prevents or reduces a decrease in left ventricular ejection fraction percentage (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 an untreated subject identified as having a mutation in the PKP2 gene.

[0100] In some embodiments, the method prevents or reduces a decrease in left ventricular fractional shortening percentage (FS%), 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 mutation in the PKP2 gene.

[0101] In some embodiments, the method prevents or reduces right ventricular area (RV area) in square millimeters (mm 2 ), optionally preventing or reducing the increase 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 increase observed in untreated subjects identified as having a mutation in the PKP2 gene.

[0102] In some embodiments, the method prevents or reduces a decrease in the right ventricular velocity time integral (RVVTI) (mm / sec) in millimeters / second, 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 mutation in the PKP2 gene.

[0103] In some embodiments, the method prevents or reduces an increase in left or right ventricular fibrosis, optionally by preventing or reducing an increase of 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 increase observed in an untreated subject identified as having a mutation in the PKP2 gene.

[0104] Various other aspects and embodiments are disclosed in the detailed description that follows.The present disclosure is limited only by the following claims.

[0105] BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figures 1A-1B Shown are diagrams illustrating non-limiting examples of vector genomes. Figure 1A A version without the SV40 intron is shown.The full-length polynucleotide sequence of one embodiment of the vector genome is SEQ ID NO: 12, and the associated expression cassette is SEQ ID NO:8. Figure 1B A version with the SV40 intron is shown. The full-length 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 as described herein is labeled "Enhancer / MHCK7" in the figure.

[0107] Figure 2 Figures illustrating non-limiting examples of vector genomes are shown. Although not shown, the hTNNT2 element also includes exon 1. The full-length 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 full-length polynucleotide sequence of another embodiment of the vector genome is SEQ ID NO: 97, and the associated expression cassette is SEQ ID NO: 100.

[0108] Figure 3 Figures illustrating non-limiting examples of vector genomes are shown. The full-length 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 full-length 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 as described herein is labeled "Enhancer / MHCK7" in the figure.

[0109] Figure 4 Figures illustrating non-limiting examples of vector genomes are shown. The full-length 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 full-length polynucleotide sequence of another embodiment of the vector genome is SEQ ID NO: 99, and the associated expression cassette is SEQ ID NO: 102.

[0110] Figures 5A-5B PKP2 protein expression in CHO-Lec2 cells is shown. Figure 5AWestern blots (WB) of PKP2 (upper panel) or loading control GAPDH (lower panel) are shown. Figure 5B Bar graphs of Western blots are shown. AAV vector serotype (AAV9 or AAVrh.74) and promoter (MHCK7 or hTnT) are indicated (AA9 represents AAV9 and rh.74 represents AAVrh.74). Controls include GFP vector (CON-GFP) and no transduction (no Tdxn).

[0111] Figure 6 Figure 20: Left ventricular ejection fraction percentage (LVEF%) of normal mice (control) or PKP2 knockout mice (cKOPKP2) receiving an intravenous formulation control (FB) or mice receiving intravenous delivery of the indicated AAV vectors of human PKP2a. All AAV doses were 3E13 vg / kg. Echocardiography was used to determine the LVEF of 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 comparison relative to the PKP2-cKO-FB control group (****p<0.0001). Data are plotted as mean ± SEM and the number of people per group (N).

[0112] Figure 7 The left ventricular fractional shortening percentage (FS%) of normal mice (control) or untreated PKP2 knockout mice (cKO PKP2) receiving an intravenous formulation control (FB) or mice receiving the indicated intravenous AAV vectors delivering human PKP2a is shown. All AAV doses were 3E13 vg / kg. FS in the left ventricle was determined by echocardiography 28 days after tamoxifen induction (which was 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 injection 56 days after injection). Statistical analysis was performed for the PKP2-cKO-FB control group using one-way analysis of variance and Dunnett's post hoc comparison (*p≤0.05, ****p<0.0001). Data are plotted as mean ± SEM and number of each group (N).

[0113] Figure 8 Shown are the right ventricular area (RV area (mm2)) of normal mice (control) or untreated PKP2 knockout mice (cKO PKP2) that received an intravenous formulation control (FB) or mice that received the indicated intravenous AAV vectors delivering human PKP2a. 2 ) (right bars). All AAV doses were 3E13 vg / kg. RV area was calculated by echocardiography 28 days after tamoxifen treatment (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) or FB control injection. Statistical analysis (one-way ANOVA followed by Dunnett's post hoc comparison for PKP2-cKO-FB control group) was performed (*p≤0.05, **p≤0.005, ***p≤0.0005, ****p<0.0001). Data are expressed as mean ± SEM and the number of each group (N).

[0114] Figure 9 Shown are right ventricular velocity time integral (RV VTI (mm / sec)) in mm / sec for normal mice (control) or untreated PKP2 knockout mice (cKO PKP2) receiving an intravenous formulation control (FB) or mice receiving the indicated intravenous AAV vectors delivering human PKP2a. All AAV doses were 3E13 vg / kg. Calculated by echocardiography. RV-VTI was measured 28 days after tamoxifen treatment and 56 days after injection with either the 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 relative to the PKP2-cKO-FB control using one-way ANOVA followed by Tukey's post hoc multiple comparison test (*p≤0.05, **p≤0.01). Data are expressed as mean ± SEM and number (N) per group.

[0115] Figures 10A-10BThe extent of fibrosis in the left and right ventricles was illustrated by quantification of the percentage of collagen following trichrome histological staining of hearts from normal mice (control) or untreated PKP2 knockout mice (cKO PKP2) that received intravenous formulation control (FB) or mice that received the indicated intravenous AAV vectors delivering human PKP2a. All AAV doses were 3E13 vg / kg. Control animals without conditional PKP2 knockout (Cre Neg group) were found to have very little collagen, while control PKP2 knockout animals that received formulation buffer (CKO FB group) were found to have a significantly greater proportion of collagen in both the left and right ventricles. AAV-mediated overexpression of PKP2 resulted in robust attenuation of collagen in all AAV-injected groups to varying degrees [n=4 for all groups; p values ​​reflect results from one-way ANOVA and Bonferroni post hoc analysis]. Figure 10A is a bar graph of the percentage of fibrosis in the left ventricle. Figure 10B Figure 2 is a bar graph showing the percentage of fibrosis in the right ventricle. The percentage of fibrosis in the left and right ventricles of the heart was assessed by trichrome staining 28 days after tamoxifen treatment (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) in control and PKP2-cKO mice.

[0116] Figures 11A-11B PKP2 protein expression in mouse hearts is shown. All AAV doses were 3E13 vg / kg. Figure 11A Western blots (WB) of PKP2 (upper panel) or loading control GAPDH (lower panel) are shown. Figure 11B Bar graphs of Western blots are shown. AAV vector serotypes (AAV9 or rh.74) and promoters (MHCK7 or hTnT) are indicated. Gel images of PKP2 detected in the heart 28 days after tamoxifen injection (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)) Figure 11A ) and quantification normalized to GAPDH ( Figure 11B , obtained at the same time Figure 11A Data are presented as mean ± SEM and number of cells per group (N).

[0117] Figures 12A-12BLVEF and FS in mice are shown. Mice were administered with AAV vector or formulation control (FB) 4 weeks after treatment with tamoxifen. Figure 12A Shown are the percentages of left ventricular ejection fraction (LVEF%) at baseline (left bar) and 28 days after tamoxifen treatment. The AAV9 dose was aE13 vg / kg and the AAVrh.74 dose was 6E13 vg / kg. LVEF was measured by echocardiography of PKP2-cKO mice taken 28 days after tamoxifen injection and 56 days after injection with FB, AAV9-hTnT-PKP2a (1E13 vg / kg) or AAVrh.74-hTnT-PKP2a (6E13 vg / kg). Statistical analysis was performed using repeated measures two-way ANOVA (***p≤0.001, ****p<0.0001). Data are plotted as mean ± SEM and the number of groups (N); dpi TAM = days after tamoxifen injection. Figure 12B Shown are the percentage of left ventricular fractional shortening (FS%) at baseline (left bar) and 28 days after tamoxifen treatment. The AAV9 dose was aE13 vg / kg and the AAVrh.74 dose was 6E13 vg / kg. FS was measured from echocardiograms of PKP2-cKO mice taken 28 days after tamoxifen injection and 56 days after injection with FB, AAV9-hTnT-PKP2a (1E13 vg / kg) or AAVrh.74-hTnT-PKP2a (6E13 vg / kg). Statistical analysis was performed using repeated measures two-way ANOVA (***p≤0.001, ****p<0.0001). Data are plotted as mean ± SEM and the number of groups (N); dpi TAM = days after tamoxifen injection.

[0118] Figures 13A-13B RV area and RV VTI in mice are shown. Mice were dosed with AAV vector or formulation control (FB) 4 weeks after treatment with tamoxifen. Figure 13A Shown are right ventricular area (RV area (mm2) at baseline (left bar) and 28 days after tamoxifen treatment. 2 RV area was measured from echocardiograms obtained 28 days after tamoxifen and 56 days after intravenous injection with 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 and number of participants per group (N); dpi TAM = days after tamoxifen injection. Figure 13BShown are right ventricular velocity time integral (RV VTI (mm / sec)) in mm / sec at baseline (left bar) and 28 days after tamoxifen treatment. RV-VTI was measured from echocardiograms taken 28 days after tamoxifen treatment and 56 days after injection with FB (control), AAV9-hTnT-PKP2a (1E13 vg / kg), or AAVrh.74-hTnT-PKP2a (6E13 vg / kg). Statistical analysis was performed using a two-way mixed-effects ANOVA followed by Tukey's post hoc multiple comparison test (*p≤0.05). Data are plotted as mean ± SEM and the number of people per group (N); dpi TAM = days after tamoxifen injection.

[0119] Figures 14A-14B The extent of fibrosis in the left and right ventricles was assessed by quantification of the percentage of collagen after trichrome histological staining of the hearts. The extent of fibrosis was assessed by trichrome staining of the left and right ventricles of FB-injected controls 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. Figure 14A ) and right ventricle ( Figure 14B ) Quantification of the percentage of fibrotic cardiac tissue in the left and right ventricles. Statistical analysis was performed using one-way ANOVA. Tukey's post hoc comparisons were performed when appropriate. These figures illustrate the extent of fibrosis in the left and right ventricles based on quantification of the percentage of collagen following trichrome histological staining of the hearts. AAV-mediated overexpression of PKP2 resulted in robust attenuation of collagen in all AAV-injected groups to varying degrees [n=4 for all groups; p values ​​reflect results from one-way ANOVA and Bonferroni post hoc analysis]. Figure 14A is a bar graph of the percentage of collagen in the left ventricle. Figure 14B is a bar graph of the percentage of collagen in the right ventricle.

[0120] Figures 15A-15B PKP2 protein expression in mouse heart is shown. Figure 15A Western blots (WB) of PKP2 (upper panel) or loading control GAPDH (lower panel) are shown. PKP2 protein was assessed by Western blot in the hearts of 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 15BBar graphs of Western blots are shown. 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 and the number of groups (N). AAV vector serotype (AAV9 or rh.74) and dose (1E13 vg / kg or 6E13 vg / kg) are indicated.

[0121] Figure 16A -C shows a diagram illustrating the experimental timeline for experiments at days -28, +7, and +14. PKP2-cKO mice were administered AAV ( Figure 16C Echocardiography was performed at baseline (for AAV-treated animals) and 28 days after tamoxifen (for all animals), and at study endpoint at 5 months after tamoxifen ( Figure 16A and Figure 16B ).

[0122] Figure 17 Kaplan Meier survival curves are shown demonstrating the prevention of disease-related mortality following intravenous AAVrh.74-hTnT-PKP2a in the PKP2-cKO model of ACM. Kaplan-Meier survival curves depict the long-term survival of PKP2-cKO mice following intravenous infusion of AAVrh.74-hTnT-PKP2a when administered at +7 (6E13 vg / kg) or +14 (2E14 vg / kg) days after tamoxifen. Survival curves are also depicted for animals that received intravenous AAV9-hTnT-PKP2a (3E13 vg / kg) at +7 days after tamoxifen and for PKP2-cKO (PKP2-cKO-FB) and control (control-FB) mice injected with FB. 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 the two groups relative to the control (****p<0.0001). The line from top to bottom at 50 days corresponds to AAVrh.74-hTnt-6E13+7D, AAVrh.74-hTnt-2E14+14D, and PKP2-cKO-FB. The line from top to bottom at 150 days corresponds to AAVrh.74-hTnt-6E13+7D, AAVrh.74-hTnt-2E14+14D, and AAV9-hTnt-3E13+7D.

[0123] Figures 18A-18BShown are the left ventricular ejection fraction percentage (LVEF%) and left ventricular fractional shortening percentage (FS%) for the AAV delayed injection paradigm (days +7 and +14). Figure 18A Figure 20 shows the left ventricular ejection fraction percentage (LVEF%) after 28 days and 5 months of tamoxifen treatment. LVEF was calculated from echocardiograms performed on PKP2-cKO mice injected with FB (control-FB) or FB (PKP2-cKO-FB), AAVrh.74-hTnT-PKP2a (6E13 vg / kg, 7 days after tamoxifen and 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 relative to PKP2-cKO-FB 28 days after tamoxifen (***p≤0.001, ****p≤0.0001). Data are expressed as mean ± SEM and number (N) per group. Figure 18B Shown is the percentage of left ventricular fractional shortening (FS%) after 28 days and 5 months after tamoxifen treatment. FS was calculated from the echocardiogram performed 28 days and 5 months after tamoxifen injection. Statistical analysis (one-way analysis of variance followed by Dunnett's post hoc comparison) was performed. The p value reflects the significance (*p≤0.05, **p≤0.01, ****p≤0.0001) of the PKP2-cKO (PKP2-cKO-FB) injected with FB relative to 28 days after tamoxifen. Data are expressed as mean ± SEM and the number (N) of each group. The bar of the figure from left to right corresponds to the legend from top to bottom.

[0124] Figures 19A-19B : Shown are the right ventricular areas (mm) in the delayed AAV injection paradigm (days +7 and +14). 2 ) and right ventricular velocity time integral (RV VTI (mm / sec)) in mm / sec. Figure 19A The right ventricular area (RV area (mm2) is shown after 28 days and 5 months of tamoxifen treatment. 2)). RV area was calculated from echocardiograms performed on control mice injected with FB (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) 28 days and 5 months after tamoxifen injection. Statistical analysis was performed using one-way ANOVA followed by Dunnett's post hoc comparisons. P values ​​reflect significance relative to PKP2-cKO-FB mice 28 days after tamoxifen (****p≤0.0001). Data are expressed as mean ± SEM and number of groups (N). Figure 19B Shown are the right ventricular velocity time integral (RV VTI (mm / sec)) in millimeters per second after 28 days and 5 months of tamoxifen treatment. RV-VTI was calculated from echocardiograms performed 28 days and 5 months after tamoxifen injection on 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 for PKP2-cKO-FB 28 days after tamoxifen using one-way ANOVA followed by Dunnett's post hoc comparison (*p≤0.05, **p≤0.01, ***p≤0.001). Data are presented as mean ± SEM and the number of groups (N). The bars in the graphs from left to right correspond to the legends from top to bottom.

[0125] Figures 20A-20C Figure 5: AAVrh.74-mediated PKP2a overexpression in PKP2-cKO mice alleviates arrhythmias using an AAV delayed injection paradigm (+14 days). AAVrh.74-hTnT-PKP2a was injected 14 days after tamoxifen-induced PKP2 knockout. Electrocardiogram (ECG) data were collected 21 and 28 days after tamoxifen injection (7 and 14 days after AAV, respectively). Figure 20A Shown are the proportions of cases with >100 premature ventricular contractions (PVCs) in animals treated with AAVrh.74-hTnT-PKP2a compared to FB-treated PKP2-cKO controls. Figure 20BEctopic beat levels are shown, which reveal a reduction in arrhythmia burden. (*p≤0.05, **p≤0.01). Data are expressed as mean ± SEM and the number (N) of each group. For each treatment, the left bar is 21 days after tamoxifen injection, and the right bar is 28 days after tamoxifen injection. Figure 20C Shown are electrocardiograms of control mice (FB) and mice treated with AAVrh.74-PKP2a 21 days after tamoxifen treatment.

[0126] Figures 21A-21B Quantification of collagen percentage based on trichrome histological staining of the heart showed that the left ventricle ( Figure 21A ) and right ventricle ( Figure 21B ) in the left and right ventricles. AAV-mediated overexpression of PKP2 resulted in a robust attenuation of collagen in all AAV-injected groups to varying degrees [n = 4 or 8 per group; p values ​​reflect results from one-way ANOVA with Bonferroni post hoc analysis]. The extent of fibrosis 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) was assessed 5 months after tamoxifen induction. The left ventricle ( Figure 21A ) and right ventricle ( Figure 21B ) in the fibrotic cardiac tissue percentage. Statistical analysis was performed using one-way ANOVA followed by Dunnett's post hoc comparison for control mice treated with FB 28 days after tamoxifen (*p≤0.05, **p≤0.01). Data are expressed as mean ± SEM and the number (N) of each group.

[0127] Figure 22Shows PKP2 protein expression in the mouse heart 5 months after AAV injection (tamoxifen +7 days or +14 days paradigm). The bar graph shows the quantification of the Western blot. The AAV vector serotype was AAVrh.74, at a dose of 6E13 vg / kg (AAV 7 days after tamoxifen) or 2E14 vg / kg (AAV injection 14 days after tamoxifen). Five months after AAV injection (tamoxifen +7 days), PKP2 protein was evaluated by Western blot in the hearts of control mice administered FB (CON-FB) and mice administered AAVrh.74-hTnT-PKP2a (6E13 vg / kg and 2E14 vg / kg). Quantification of the PKP2 protein band was presented relative to GAPDH. Statistical analysis did not find significant differences between the groups. Data are represented as mean ± SEM and the number (N) of each group.

[0128] Figures 23A-23C Shows dose-dependent detection in the heart tissue of animals injected with AAVrh.74-PKP2a relative to control Figure 23A ) vector genome (ddPCR), Figure 23B ) transgenic PKP2a mRNA (RT-ddPCR) and Figure 23C ) PKP2 protein expression (Western blot). Figure 23C The red dotted line in indicates the endogenous expression level of PKP2 protein normalized to GAPDH in control-FB mice. Data are represented 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 3×10 13 vg / kg; right bar, PKP2-cKO mice treated with AAVrh.74-PKP2a 6×10 13 vg / kg. The number of mice (N) in the study was noted in the corresponding bar. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc analysis. <LoQ: less than the limit of quantification; FB: formulation buffer.

[0129] Figures 24A-24C Shows 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 6×10 13Representative images of immunofluorescence staining for PKP2 (red) and nuclei (DAPI; blue) in hearts from vg / kg PKP2-cKO mice. Mice were treated with FB or AAVrh.74-PKP2a 28 days before tamoxifen injection. White arrows in panels A and C highlight the localization of PKP2 at the intercalated disc. FB: formulation buffer. Scale bar = 150 μm for the left panel and 50 μm for the right panel.

[0130] Figures 25A-25C Representative images of left ventricular (LV) and right ventricular (RV) echocardiograms are shown to evaluate control and PKP2-cKO mice treated with formulation buffer (FB) and with AAVrh.74-PKP2a 6 × 10 28 days before tamoxifen injection. 13 The contractile force of PKP2-cKO mice treated with vg / kg Figure 25A ). Measurements were collected 28 days after tamoxifen injection. Figure 25B Quantification of left ventricular ejection fraction (LVEF) measured by long-axis B-mode echocardiography is shown. Figure 25C Figure 3 shows right ventricular (RV) area measured by modified long-axis B-mode echocardiography. The red dashed line indicates the mean recorded from PKP2-cKO mice injected with FB. Data are expressed 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, mice treated with AAVrh.74-PKP2a3 × 10 13 vg / kg treated PKP2-cKO mice; right bar, treated with AAVrh.74-PKP2a 6×10 13 vg / kg treated PKP2-cKO mice. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc analysis. LVID: left ventricular internal diameter, LVEF: left ventricular ejection fraction, RV: right ventricle, FB: formulation buffer.

[0131] Figures 26A-26C Cardiac fibrosis in AAVrh.74-PKP2a-treated PKP2-cKO mice is shown. Figure 26A The left panel shows representative images of Masson's trichrome staining of longitudinal sections of hearts 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. Figure 26A The right panel shows a high-contrast mask of the same section, emphasizing the blue collagen deposition. For all images, scale bar = 1 mm. Hearts were removed 28 days after tamoxifen injection. Figure 26BQuantification of the percentage of left ventricular fibrosis is shown. Figure 26C Figure 4 shows right ventricular fibrosis in hearts from four different groups. Data are expressed 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, mice treated with AAVrh.74-PKP2a3 × 10 13 vg / kg treated PKP2-cKO mice; right bar, treated with AAVrh.74-PKP2a6 × 10 13 vg / kg treated PKP2-cKO mice. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc analysis. FB: formulation buffer.

[0132] Figure 27 Figure 2 is a graph showing survival after AAVrh.74-PKP2a injection in PKP2-cKO mice 7 or 14 days after tamoxifen. Kaplan Meier curves depict the survival after AAVrh.74-PKP2a (6×10 13 vg / kg, 7 days after tamoxifen or 2×10 14 Long-term survival of PKP2-cKO mice after administration of (vg / kg, 14 days after tamoxifen).

[0133] Figures 28A-28D Shown are disease progression following 7 or 14 days of AAVrh.74-PKP2a treatment after tamoxifen in PKP2-cKO mice. Figure 28A The upper panel shows control mice treated with formulation buffer (FB) and tamoxifen 14 days after treatment with 2 × 10 14 Representative images of Masson's trichrome staining of longitudinal cardiac sections from PKP2-cKO mice treated with vg / kg of AAVrh.74-PKP2a. Hearts were removed 5 months after tamoxifen injection. Figure 28A The lower panel shows a high-contrast mask of the same section, emphasizing the collagen deposition in blue. For all images, scale bar = 1 mm. Figure 28B The results of the experiments of control mice treated with FB and tamoxifen 7 days later (6×10 13 vg / kg) or 14 days (2×10 14 Quantification of left ventricular fibrosis percentage (left panel) and right ventricular fibrosis percentage (right panel) by Masson's trichrome staining of longitudinal cardiac sections from PKP2-cKO mice treated with AAVrh.74-PKP2a (vg / kg). Hearts were removed 5 months after tamoxifen. The red dashed line indicates the level of fibrosis in PKP2-cKO mice injected with formulation buffer (FB). Figure 28CShown is the quantification of left ventricular ejection fraction (LVEF) over time in PKP2-cKO mice treated with AAVrh.74-PKP2a at the doses indicated at the bottom of the bars, 7 or 14 days after tamoxifen. Figure 28D The corresponding quantification of right ventricular area (RV area) is shown. Echocardiography was performed 28 days and 5 months after tamoxifen. Echocardiography was performed only on control and PKP2-cKO mice treated with FB 28 days after tamoxifen. Data are expressed as mean ± SD. The left bar is a control mouse treated with FB; the second bar from the left is a PKP2-cKO mouse treated with FB; the third and fourth bars from the left are mice treated with AAVrh.74-PKP2a 6×10 13 vg / kg treated PKP2-cKO mice; the two right bars are AAVrh.74-PKP2a 2×10 14 vg / kg treated PKP2-cKO mice. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc analysis. FB: formulation buffer.

[0134] Figures 29A-29D Shown are the results of isoproterenol-induced arrhythmias in PKP2-cKO hearts treated with AAVrh.74-PKP2a. Figure 29A Shown are representative electrocardiogram (ECG) traces from PKP2-cKO mice treated with formulation buffer (FB). Figure 29B Figure 2 shows the results from AAVrh.74-PKP2a 6×10 cells 14 days after tamoxifen injection. 13 Representative ECG traces of PKP2-cKO mice treated with vg / kg. Figure 29C Shown are the percentages of mice that experienced >100 premature ventricular contractions (PVCs) following isoproterenol (ISO). Figure 29D Shown are the numbers of ISO-induced ectopic beats in PKP2-cKO mice treated with FB or AAVrh.74-PKP2a. Figure 29C and Figure 29D The data shown were quantified over a 30-minute period after ISO injection, and ECGs were recorded 21 days after tamoxifen. Data are expressed as mean ± SD. Left bar, PKP2-cKO mice treated with FB; middle bar, mice treated with AAVrh.74-PKP2a 6 × 10 13 vg / kg treated PKP2-cKO mice; and right bars, treated with AAVrh.74-PKP2a 2×10 14vg / kg treated PKP2-cKO mice. Statistical analysis was performed using one-way ANOVA followed by Dunn's post hoc analysis. PVC: premature ventricular contraction, FB: formulation buffer.

[0135] Figures 30A-30D Transduction of PKP2 in nonhuman primate hearts is shown. Figure 30A -B is in contrast to FB( Figure 30A ) or “low-dose” treated animals ( Figure 30B ) non-human primate heart sections after immunohistochemical labeling for PKP2 protein. Figure 30C and Figure 30D showed that the intravenous infusion of "low" (8 × 10 13 vg / kg) or "high" (3×10 14 Western blot quantification of PKP2 protein and transgenic mRNA (hPKP2a) in the ventricles of nonhuman primate hearts following administration of 5 vg / kg) doses of AAVrh.74-PKP2a or FB (control).

[0136] Figures 31A-31D Cardiac expression of ankyrin B in AAVrh.74-PKP2a-treated PKP2-cKO mice is shown. Figure 31A Shown is a method for data acquisition and analysis of immunofluorescence (IF) ankyrin B (AnkB) signals collected from paraffin-embedded tissue sections. The red line represents the fluorescence intensity range as plotted in the right figure. Peak depth is defined as the range between the peak and the valley. Figure 31B Shown are 6×10 PKP2a from control mice treated with formulation buffer (FB), PKP2-cKO mice treated with FB, and mice treated with AAVrh.74-PKP2a 28 days before tamoxifen injection. 13 Representative images of IF staining of AnkB (green) and nuclei (blue) in hearts of vg / kg-treated PKP2-cKO mice. Scale bar = 100 μm. Figure 31C Shown are quantifications of AnkB peak depth (left panel) and mean intensity (right panel) in left ventricular sections from three different groups. Figure 31D Quantification of AnkB peak depth (left panel) and mean intensity (right panel) in right ventricular sections from three different groups is shown. Data are expressed as mean ± SD. Left bar, control mice treated with FB; middle bar, PKP2-cKO mice treated with FB; right bar, mice treated with AAVrh.74-PKP2a 6×10 13 vg / kg treated PKP2-cKO mice. Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc analysis.

[0137] Figures 32A-32C Cardiac expression of desmin in AAVrh.74-PKP2a-treated PKP2-cKO mice is shown. Figure 32A Shown are 6×10 PKP2a from control mice treated with formulation buffer (FB), PKP2-cKO mice treated with FB, and mice treated with AAVrh.74-PKP2a 28 days before tamoxifen injection. 13 Representative images of immunofluorescence staining of desmin (green) and nuclei (blue) in hearts of vg / kg treated PKP2-cKO mice. Scale bar = 100 μm. Figure 32B Shown are quantifications of desmin peak depth (left panel) and mean intensity (right panel) in left ventricular sections from three different groups. Figure 32C Quantification of desmin peak depth (left panel) and mean intensity (right panel) in right ventricular sections from three different groups is shown. Data are expressed as mean ± SD. Black bars, control mice treated with FB; red bars, PKP2-cKO mice treated with FB; purple bars, mice treated with AAVrh.74-PKP2a 6×10 13 vg / kg treated PKP2-cKO mice. Statistical significance was assessed by one-way ANOVA followed by Tukey's post hoc test.

[0138] Detailed Description of the Disclosure

[0139] The present disclosure provides gene therapy vectors directed against PKP2 that deliver polynucleotides encoding PKP2 polypeptides or functional variants thereof, as well as methods of use and other compositions and methods.

[0140] In a specific embodiment, 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 embodiments, the promoter is the myosin heavy chain creatine kinase 7 (MHCK7) promoter. In some embodiments, the AAV vector is an AAV9 vector. In some embodiments, the promoter is the MHCK7 promoter and the AAV vector is an AAV9 vector. In some embodiments, the promoter is the hTNNT2 promoter. In some embodiments, the promoter is the hTNNT2 promoter and the AAV vector is an AAV9 vector. In some embodiments, PKP2 is human PKP2a. In some embodiments, PKP2 is human PKP2b. In some embodiments, the AAV vector is an AAVrh.74 vector. In some embodiments, the promoter is the MHCK7 promoter and the AAV vector is an AAVrh.74 vector. In some embodiments, the promoter is the hTNNT2 promoter. In some embodiments, the promoter is the hTNNT2 promoter and the AAV vector is an AAVrh.74 vector. In some embodiments, PKP2 is human PKP2a. In some embodiments, PKP2 is human PKP2b.

[0141] The present disclosure also provides methods of treating a disease or condition in a subject by administering to the subject a gene therapy vector of the present disclosure.In certain embodiments, the disease or condition is arrhythmogenic cardiomyopathy (ACM).

[0142] 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 carry mutations in the desmosome gene PKP2 encoding the protein plaqueflanone-2 (PKP2). PKP2 is also associated with Brugada syndrome (BrS) and idiopathic ventricular fibrillation. It is a member of the armadillo repeat and plaqueflanone protein families. The protein contains nine central conserved armadillo repeat domains flanked by N-terminal and C-terminal domains. It functions to connect cadherins to intermediate filaments in the cytoskeleton.

[0143] PKP2 localizes to desmosomes and the nucleus, binding plakoglobin, desmoplakin, and desmosomal cadherin via its N-terminal head domain. PKP2 provides lateral stabilization and, together with desmosome-intermediate filament assembly, promotes intercellular contacts. It also plays a role in regulating intracellular signaling, electrophysiology, trafficking, and transcriptional processes.

[0144] Intravenous injection of an AAV9 vector encoding a C-terminal deletion mutant (R735X) of PKP2a into the hearts of wild-type mice accelerated the development of ACM when the treated mice underwent exercise training. Cruz et al. J Am Coll Cardiol. 65(14):1438-50(2015). Mutant PKP2a caused a disease phenotype; and a control AAV9 vector expressing non-mutant PKP2a did not cause phenotypic changes in wild-type mice. Heterologous expression of wild-type human PKP2a did not induce disease or alter function. These studies indicate that mutant PKP2a can cause a disease phenotype. They failed to demonstrate a curative effect of PKP2 because heterologous expression of non-mutant PKP2 resulted in no phenotypic changes in wild-type mice.

[0145] According to the present disclosure, polynucleotides encoding PKP2 or a functional variant thereof, wherein the PKP2 or a functional variant thereof comprises at least 800 or at least 830 amino acids (e.g., no C-terminal truncation at Arg-735), can be used to produce gene therapy vectors. The resulting vectors can be used to treat diseases or conditions, such as PKP2-related diseases or conditions, such as ACM, Brugada syndrome (BrS), idiopathic ventricular fibrillation, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and other diseases or conditions.

[0146] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used to practice this disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0147] All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated as being incorporated by reference herein, including but not limited to PCT Application Publication No. WO 2022 / 032226 and U.S. Application Serial Nos. 17 / 670,389 and 17 / 670,390. In the event of a conflict, the present application, including any definitions herein, will control. However, reference to any reference, article, publication, patent, patent disclosure, or patent application cited herein is not and should not be taken as an admission, or any form of suggestion, that it constitutes valid prior art or forms part of the common general knowledge in any country in the world.

[0148] Unless otherwise stated, in this specification, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the stated range, and where appropriate, include its fraction (e.g., one tenth and one hundredth of an integer). The term "about", when immediately preceding a number or numerical range, refers to that number or numerical range plus or minus 10%. It should be understood that, unless otherwise stated, the terms "a" and "an" used herein refer to "one or more" listed components. The use of alternatives (e.g., "or") should be understood to mean any one, two, or any combination thereof in the alternatives. The term "and / or" should be understood to mean any one or two in the alternatives. As used herein, the terms "include" and "comprising" are used synonymously.

[0149] As used herein, the term "over time in a subject" refers to an effect occurring in a subject for about a day, about a month, about a year, about ten years, and / or about several decades.

[0150] As used herein, the terms "identity" and "identical" refer to the percentage of residues that are exactly matched in an alignment of a "query" sequence with a "subject" sequence, such as an alignment generated by the BLAST algorithm, with respect to a polypeptide or polynucleotide sequence. Unless otherwise indicated, identity is calculated across the full length of the subject sequence. Thus, a query sequence "has at least x% identity" to a subject sequence if, when the query sequence is aligned with the subject sequence, at least x% (rounded down) of the residues in the subject sequence exactly match the corresponding residues in the query sequence. In the case where the subject sequence has variable positions (e.g., residues represented by X), alignment with any residue in the query sequence is counted as a match. Sequence alignments can be performed using the NCBI Blast service (BLAST+ version 2.12.0).

[0151] As used herein, the term "operably linked" refers to the functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, if a promoter sequence stimulates or regulates the transcription of a coding sequence in an appropriate host cell or other expression system, the promoter sequence is operably linked to the coding sequence. Typically, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not necessarily have to be physically adjacent to or located in close proximity to the coding sequence whose transcription they enhance.

[0152] As used herein, "AAV vector" or "rAAV vector" refers to a recombinant vector comprising one or more polynucleotides of interest (or transgenes) flanked by AAV inverted terminal repeats (ITRs). Such AAV vectors can replicate and be packaged into infectious viral particles when present in host cells transfected with plasmids encoding and expressing the rep and cap gene products. Alternatively, host cells that have been stably engineered to express the rep and cap genes can be used to package AAV vectors into infectious particles.

[0153] As used herein, "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsulated polynucleotide AAV vector. As used herein, if the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is generally referred to as an "AAV vector particle" or simply "AAV vector". Therefore, the production of AAV vector particles necessarily includes the production of AAV vectors, because such vectors are contained within AAV vector particles.

[0154] As used herein, "promoter" refers to a polynucleotide sequence capable of promoting the initiation of RNA transcription from a polynucleotide in a eukaryotic cell.

[0155] As used herein, "vector genome" refers to the polynucleotide sequence packaged by a vector (e.g., rAAV virion), including flanking sequences (in AAV, inverted terminal repeats). The terms "expression cassette" and "polynucleotide cassette" refer to the portion of the vector genome between the flanking ITR sequences. "Expression cassette" means that the vector genome comprises at least one gene encoding a gene product that is operably linked to an element driving expression (e.g., a promoter).

[0156] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk of or suffering from a disease, disorder or condition that is suitable for treatment or improvement by the recombinant gene therapy vector or gene editing system disclosed herein. For example, a patient or subject in need can be a patient or subject diagnosed with a heart-related disorder. The subject may have a mutation in the PKP2 gene, or a deletion of all or part of the PKP2 gene, or a deletion of the gene regulatory sequence, which results in abnormal expression of the PKP2 protein. "Subject" and "patient" are used interchangeably herein. The subject treated by the methods described herein can be an adult or a child. The age range of the subject can vary.

[0157] As used herein, the term "variant" refers to a protein having 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 having one or more amino acid substitutions, insertions, or deletions compared to a parent protein and retaining one or more desired activities of the parent protein.

[0158] As used herein, "treating" refers to alleviating one or more symptoms of a disease or condition. The term "preventing" refers to delaying or interrupting the onset of one or more symptoms of a disease or condition or slowing the progression of a PKP2-related disease or condition (e.g., arrhythmogenic cardiomyopathy (ACM)).

[0159] Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome of approximately 4.7 kb in length, including two inverted terminal repeats (ITRs) of approximately 145 nucleotides. There are multiple known AAV variants, sometimes referred to as serotypes based on antigenic epitope classification. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and in Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least a portion of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 45:555-564 (1983). The AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The sequence of the AAVrh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. The cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained in the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) combined with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. Rep proteins possess diverse enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and a non-consensus translation start site are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome.The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0160] AAV has unique characteristics that make it attractive as a vector for delivering exogenous DNA to cells, for example in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cells, allowing for the potential to target many different tissues in vivo. Furthermore, AAV slowly transduces both dividing and non-dividing cells and can persist as a transcriptionally active nuclear episome (extrachromosomal element) for essentially the entire lifespan of these cells. The AAV proviral genome is inserted into a plasmid as cloned DNA, enabling the construction of recombinant genomes. Furthermore, because the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with exogenous DNA. To produce AAV vectors, the rep and cap proteins can be provided in trans. Another notable feature of AAV is that it is an extremely stable and robust virus. It readily tolerates the conditions used to inactivate adenoviruses (56 to 65°C for several hours), making cryopreservation of AAV less critical. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to superinfection.

[0161] Gene delivery viral vectors that can be used to implement the present disclosure can be constructed using methods well-known in the field of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding transgenes, suitable regulatory elements, and elements required for producing viral proteins that mediate cell transduction. Such recombinant viruses can be produced by techniques known in the art (e.g., by transfecting packaging cells or transiently transfecting with helper plasmids or viruses). Typical 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, a herpes virus-based system can be used to produce AAV vectors. Detailed protocols for generating such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and WO94 / 19478, the entire contents of which are incorporated herein by reference.

[0162] The present disclosure relates to compositions and methods of use related to plaquenil protein-2 (PKP2) proteins or polypeptides. Various mutations in PKP2 are known to be associated with cardiomyopathy and heart failure, including those described in van Tintelen et al. Circulation 113:1650-58 (2006); Novelli Front. Cardiovasc. Med. (2008); and other sources. Therefore, viral vector-mediated delivery of the PKP2 gene may be used as a viable therapy for PKP2-related human diseases, such as cardiomyopathy and heart failure.

[0163] More than 230 mutations in the PKP2 gene have been identified in people with arrhythmogenic cardiomyopathy (ACM). (See "PKP2 gene" on MedlinePlus.) This condition most commonly affects the heart muscle that surrounds 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 result in the production of an abnormally short form of the plakflytin 2 protein. Other mutations alter the structure of plakflytin 2 by adding, deleting, or changing one or more of its protein building blocks (amino acids). Studies have shown that the altered protein impairs the formation and function of desmosomes.

[0164] Without normal desmosomes, cardiomyocytes separate from each other and die, especially when the heart muscle is under stress, such as during strenuous exercise. The damaged heart muscle is gradually replaced by fat and scar tissue. As this abnormal tissue accumulates, the walls of the right ventricle become stretched, preventing the heart from pumping blood efficiently. These changes also disrupt the electrical signals that control the heartbeat, which can lead to abnormal heart rhythms. Descriptions of PKP2-related diseases can be found 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 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. 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").

[0165] The native sequences of human PKP2a and its isoform PKP2b are shown below, with Arg-735 underlined:

[0166] PKP2a (SEQ ID NO: 1)—837 amino acids

[0167]

[0168]

[0169] PKP2b (SEQ ID NO: 2)—881 amino acids

[0170]

[0171] 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, such as human PKP2a or human PKP2b.

[0172] In some embodiments, 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 embodiments, 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.

[0173] In certain embodiments, the polynucleotide encoding PKP2a can 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.

[0174] In certain embodiments, the 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%, or 100% identical to SEQ ID NO:4.

[0175] 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 a polynucleotide sequence encoding a 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 ten nucleotides constitute the Kozak sequence).

[0176] In certain embodiments, the Kozak sequence can overlap with a polynucleotide sequence encoding a PKP2 (e.g., PKP2a or PKP2b) protein or a functional variant thereof. For example, the vector genome can 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 ten nucleotides constitute the Kozak sequence).

[0177] In some embodiments, the Kozak sequence is a surrogate Kozak sequence comprising or consisting of any of the following:

[0178] (gcc)gccRccAUGG(SEQ ID NO:16);

[0179] AGNNAUGN;

[0180] ANNAUGG;

[0181] ANNAUGC;

[0182] ACCAUGG; and

[0183] GACACCAUGG (SEQ ID NO: 18).

[0184] In some embodiments, the vector genome does not contain a Kozak sequence.

[0185] The polynucleotide sequence can be codon-optimized. For example, the vector genome can comprise a polynucleotide sequence encoding PKP2a 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: 87. The vector genome may comprise a polynucleotide sequence encoding PKP2b 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:88.

[0186] The AAV virion of the present disclosure comprises a vector genome. The vector genome may comprise 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) can be used. ITR can be an AAV ITR of the same serotype as the capsid present in the AAV virion or a serotype different from the capsid (e.g., AAV2 ITR can be used with an AAV virion having an AAV9 capsid or an AAVrh.74 capsid). In each case, the serotype of the capsid determines the name applied to the virion. ITR is typically the 5' and 3' most elements of the vector genome. The vector genome will generally also contain a promoter, a transgene, a 3' untranslated region (UTR) sequence (e.g., a WPRE element) and a polyadenylation sequence in a 5' to 3' order. In a variation, the vector genome includes an enhancer element (generally located at the 5' of the promoter) and / or an exon (generally located at the 3' of the promoter). In a variation, the vector genome encoding of the present disclosure is used as a partial or complete transgenic sequence of a repair template in a gene editing system. In such variations, the vector genome can contain an exogenous promoter, or the gene editing system can insert the transgene into a locus in the genome that has an endogenous promoter, such as a cardiac-specific or myocyte-specific promoter.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] In some embodiments, the vector genome comprises one or more stuffer sequences, e.g., 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: 27; SEQ ID NO: 28; or SEQ ID NO: 29.

[0195] 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 the MHCK7 promoter. In certain embodiments, the promoter is the TNNT2 promoter.

[0196] The present disclosure contemplates the use of various promoters. Promoters that can be used in the embodiments of the present disclosure include, but are not limited to, cytomegalovirus (CMV) promoters, phosphoglycerol kinase (PGK) promoters, or promoter sequences (CAG) consisting of a CMV enhancer and a chicken beta-actin promoter and a portion of a rabbit beta-globin gene. In some cases, the promoter can be a synthetic promoter. Schlabach et al. PNAS USA. 107 (6): 2538-43 (2010) provides exemplary synthetic promoters. 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 SEQ ID NO: 30.

[0197] In some embodiments, the polynucleotide sequence encoding PKP2 protein or its functional variant is operably connected to an inducible promoter. Inducible promoter can be configured to make the polynucleotide sequence transcriptional expression or non-transcriptional expression in response to the addition or accumulation of agent or in response to the removal, degradation or dilution of agent. The agent can be a medicine. The agent can be one of tetracycline or its derivatives, including but not limited to doxycycline. In some cases, the inducible promoter is tet-on promoter, tet-off promoter, chemical regulation promoter, physical regulation promoter (that is, in response to the presence or absence of light or in response to a promoter at low or high temperature). Inducible promoter includes heavy metal ion inducible promoter (such as mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and in the presence of T7 RNA polymerase, there is an active promoter from T7 phage. This inducible promoter list is non-restrictive.

[0198] In some cases, the promoter is a tissue-specific promoter, such as a promoter that can drive expression to a greater extent in cardiac cells than in non-cardiac cells. In some embodiments, the tissue-specific promoter is selected from any various 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 a combination of promoter / enhancer regions thereof, such as MHCK7. In some cases, the promoter is a pan-promoter. The term "pan-promoter" refers to a promoter that is not tissue-specific under experimental or clinical conditions. In some cases, the pan-promoter is any one of cytomegalovirus (CMV), cytomegalovirus early enhancer element chicken β-actin gene intron and rabbit β-globin gene splice acceptor (CAG), ubiquitin C (UBC), phosphoglycerol kinase (PGK), eukaryotic translation elongation factor 1 alpha 1 (EF1-alpha), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), simian virus 40 (SV40), hepatitis B virus (HBV), chicken β-actin and human β-actin promoters.

[0199] 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, such as 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.

[0200] Table 3

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209] 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.

[0210] 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 including LTR elements. Many other promoters are known and commonly available in the art, and the sequences of many such promoters are available in sequence databases (e.g., the GenBank database).

[0211] 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 Posttranscriptional Regulatory Element), and a HPRE (Hepatitis B Posttranscriptional Regulatory Element).

[0212] In some embodiments, the vector comprises a CMV enhancer.

[0213] In certain embodiments, the vector comprises one or more enhancers. In specific embodiments, the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a β-actin enhancer sequence, or an EF1-α enhancer sequence. Such sequences are known in the art. For example, the sequence of the CMV immediate early (IE) enhancer is SEQ ID NO: 50.

[0214] In certain embodiments, the vector comprises one or more introns. In specific 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.

[0215] In certain embodiments, the vector comprises a polyA sequence. In specific embodiments, the polyA sequence is a rabbit globin polyA sequence, a human growth hormone polyA sequence, a bovine growth hormone polyA sequence, a PGK polyA sequence, an SV40 polyA sequence, or a TK polyA sequence. In some embodiments, the poly-A signal may be a bovine growth hormone polyadenylation signal (bGHpA).

[0216] In certain embodiments, the vector comprises one or more transcription stabilizing elements. In specific embodiments, the transcription stabilizing element is a WPRE sequence, an HPRE sequence, a scaffold attachment region, a 3'UTR, or a 5'UTR. In specific embodiments, the vector comprises both a 5'UTR and a 3'UTR.

[0217] 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.

[0218] Table 4

[0219]

[0220]

[0221]

[0222]

[0223] 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.

[0224] Table 5

[0225]

[0226]

[0227]

[0228]

[0229] In some embodiments, the vector comprises a polyadenylation (polyA) signal selected from Table 6. In some embodiments, the polyA signal comprises a polynucleotide sequence that is 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.

[0230] Table 6

[0231]

[0232] Exemplary vector genomes are shown in Figures 1-4 and are provided as SEQ ID NOs: 12-15, 89-92, and 97-99. The expression cassette for each vector genome sequence is 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 that is 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% identical 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 that is 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% identical to any one of SEQ ID NOs: 8-11, 93-96, or 100-102. In certain embodiments, the vectors and expression cassettes disclosed herein exhibit advantageous properties compared to alternatives, such as higher expression in mammalian cells (including target cells), more selective expression in target tissues (e.g., cardiac cells, such as cardiomyocytes), higher infection rates, and / or increased manufacturability, such as higher manufacturing yields. In specific embodiments, the target tissue is cardiac tissue or myocardial tissue.

[0233] 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 sequence of 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 having 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 in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In specific embodiments, the PKP2a polypeptide comprises 837 amino acids. In related embodiments, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding at least 800, at least 830, at least 837, or at least 881 amino acids of PKP2b. In specific embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0234] 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 sequence of 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 having 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 in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In specific embodiments, the PKP2a polypeptide comprises 837 amino acids. In related embodiments, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding at least 800, at least 830, at least 837, or at least 881 amino acids of PKP2b. In specific embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0235] 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 sequence of 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 having 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 in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In specific embodiments, the PKP2a polypeptide comprises 837 amino acids. In related embodiments, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding at least 800, at least 830, at least 837, or at least 881 amino acids of PKP2b. In specific embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0236] 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 sequence of SEQ ID NO: 32 or 33; any one of SEQ ID NO: 4, 7, and 88; SEQ ID NO: 63; and SEQ ID NO: 75; or a polynucleotide sequence having 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 in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In specific embodiments, the PKP2a polypeptide comprises 837 amino acids. In related embodiments, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding at least 800, at least 830, at least 837, or at least 881 amino acids of PKP2b. In specific embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0237] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a PKP2a transgene; an optional WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence of SEQ ID NO: 31; any one of SEQ ID NOs: 3, 6, and 87; or a polynucleotide sequence that is 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% identical to each of the foregoing. In certain embodiments, the vector genome is packaged in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In a specific embodiment, 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 PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In a specific embodiment, the PKP2b polypeptide comprises 881 amino acids.

[0238] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a PKP2a transgene; an optional WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence of SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 3, 6, and 87; or a polynucleotide sequence that is 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% identical to each of the foregoing. In certain embodiments, the vector genome is packaged in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In a specific embodiment, 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 PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In a specific embodiment, the PKP2b polypeptide comprises 881 amino acids.

[0239] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; a PKPb transgene; an optional WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence of SEQ ID NO: 31; any one of SEQ ID NOs: 4, 7, and 88; or a polynucleotide sequence that is 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% identical to each of the foregoing. In certain embodiments, the vector genome is packaged in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In a specific embodiment, 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 PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In a specific embodiment, the PKP2b polypeptide comprises 881 amino acids.

[0240] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; a PKPb transgene; an optional WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, the polynucleotide sequence of SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 4, 7, and 88; or a polynucleotide sequence that is 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% identical to each of the foregoing. In certain embodiments, the vector genome is packaged in 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 at least 800, at least 837, or at least 830 amino acids of PKPa. In a specific embodiment, 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 PKP2b of at least 800, at least 830, at least 837, or at least 881 amino acids. In a specific embodiment, the PKP2b polypeptide comprises 881 amino acids.

[0241] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; an SV40 intron; a PKPa transgene; an optional WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, a polynucleotide sequence of SEQ ID NO: 31; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 3, 6, and 87; or a polynucleotide sequence having 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 in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In specific embodiments, the PKP2a polypeptide comprises 837 amino acids. In related embodiments, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding at least 800, at least 830, at least 837, or at least 881 amino acids of PKP2b. In specific embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0242] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; an SV40 intron; a PKPa transgene; an optional WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, a polynucleotide sequence of SEQ ID NO: 32 or 33; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 3, 6, and 87; or a polynucleotide sequence having 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 in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In specific embodiments, the PKP2a polypeptide comprises 837 amino acids. In related embodiments, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding at least 800, at least 830, at least 837, or at least 881 amino acids of PKP2b. In specific embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0243] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an MHCK7 promoter; an SV40 intron; a PKPb transgene; an optional WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, a polynucleotide sequence of SEQ ID NO: 31; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 4, 7, and 88; or a polynucleotide sequence having 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 in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In specific embodiments, the PKP2a polypeptide comprises 837 amino acids. In related embodiments, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding at least 800, at least 830, at least 837, or at least 881 amino acids of PKP2b. In specific embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0244] In certain embodiments, the vector genome comprises, in 5' to 3' order, a 5' ITR; an hTnnT2 promoter; an SV40 intron; a PKPb transgene; an optional WPRE element; a polyadenylation sequence; and a 3' ITR. The vector genome may comprise, in 5' to 3' order, a polynucleotide sequence of SEQ ID NO: 32 or 33; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 4, 7, and 88; or a polynucleotide sequence having 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 in 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 at least 800, at least 837, or at least 830 amino acids of PKP2a. In specific embodiments, the PKP2a polypeptide comprises 837 amino acids. In related embodiments, instead of a PKP2a transgene, the transgene is a full-length or substantially full-length transgene, i.e., a transgene encoding at least 800, at least 830, at least 837, or at least 881 amino acids of PKP2b. In specific embodiments, the PKP2b polypeptide comprises 881 amino acids.

[0245] In each case, the optional WPRE element may be present or absent.

[0246] In certain embodiments, the vector is AAVrh.74, and the vector genome encodes PKP2-A (a shorter isoform, predominantly expressed in the heart) operably linked to a cardiac-specific promoter with or without codon optimization (e.g., optimized for human expression and CpG island reduction). The AAVrh.74 vector is administered intravenously (IV) at a dose range of mid-E13 vg / kg to mid-E14 vg / kg, for example, for a patient population requiring a high vector load, such as adults with an average onset of symptoms at age 35.

[0247] Adeno-associated virus vector

[0248] AAV vectors useful in practicing the present disclosure can be packaged into AAV virions (viral particles) using a variety of systems, including adenovirus-based 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. Illustrative cofactor-free systems include, but are not limited to, those described in US 6,004,797; US 7,588,772; and US 7,094,604.

[0249] The AAV DNA in the rAAV genome can be from any AAV variant or AAV serotype for which a recombinant virus can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAVrh.10, including wild-type and any variants of these serotypes. For example, the production of pseudotyped rAAV is disclosed in WO 01 / 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.

[0250] In some cases, rAAV includes a self-complementary genome. As defined herein, rAAV comprising a "self-complementary" or "double-stranded" genome refers to an rAAV that has been engineered so that the coding region of rAAV is configured to form an intramolecular double-stranded DNA template, such as 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 rAAV comprising a self-complementary genome in some cases, because after infection (this transduction), rather than waiting for the synthesis of the second chain of the rAAV genome mediated by the cell, the two complementary halves of scAAV will associate to form a double-stranded DNA (dsDNA) unit ready to be replicated and transcribed immediately. It will be understood that the rAAV comprising a self-complementary genome can only accommodate about half the amount (≈2.4 kb), rather than the full coding capacity (4.7-6 kb) found in rAAV.

[0251] In other cases, the rAAV vector comprises a single-stranded genome. As defined herein, a "single-stranded" genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAV has a single-stranded DNA genome. There are some indications that, in order to achieve efficient transduction of cells, rAAV should be scAAV. However, the present disclosure contemplates rAAV vectors that may have a single-stranded genome rather than a self-complementary genome, understanding that other genetic modifications to rAAV vectors are beneficial for obtaining optimal gene transcription in target cells. 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 anterior segment in the mouse eye. PLoS ONE 12(8): e0182473(2017).

[0252] In some cases, the rAAV vector is a vector of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.10 or AAVrh.74. The production of pseudotyped rAAVs is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAVs 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 a vector of serotype AAV9. In some embodiments, the rAAV vector is a vector of serotype AAV9 and comprises a single-stranded genome. In some embodiments, the rAAV vector is a vector of serotype AAV9 and comprises a self-complementary genome. In some embodiments, the rAAV vector comprises an inverted terminal repeat (ITR) sequence of AAV2. 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.

[0253] The full-length and capsid gene sequences of most known AAVs are provided in U.S. Patent No. 8,524,446, which is incorporated herein in its entirety.

[0254] AAV vectors may comprise wild-type AAV sequences, or they may comprise one or more modifications to wild-type AAV sequences. 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 specific embodiments, the modification provides reduced immunogenicity when the AAV vector is provided to a subject.

[0255] The capsid protein of rAAV can be modified so that the rAAV is targeted to a specific target tissue of interest, such as cardiomyocytes. In some embodiments, rAAV is injected directly into the intraventricular space of a subject.

[0256] 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 is at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identical to a reference AAV2 capsid protein, such as SEQ ID NO: 76.

[0257] 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 is at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identical to a reference AAV9 capsid protein, such as SEQ ID NO: 77.

[0258] In some embodiments, the rAAV virion is an AAV6 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAV6 capsid comprises a capsid protein that is at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identical to a reference AAV6 capsid protein, such as SEQ ID NO: 78.

[0259] In some embodiments, the rAAV virion is an AAVrh.10 rAAV virion. The capsid can be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAVrh.10 capsid comprises a capsid protein that is at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identical to a reference AAVrh.10 capsid protein, such as SEQ ID NO: 79.

[0260] 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 is at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, or 100% identical to a reference AAVrh.74 capsid protein, such as SEQ ID NOs: 81-83.

[0261] The polynucleotide sequence of wild-type AAVrh.74cap is provided as SEQ ID NO: 80. The present disclosure also provides the protein sequences of AAVrh.74VP1, VP2, and VP3, including SEQ ID NOs: 81-83 and homologs or functional variants thereof.

[0262] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL(AAVrh.74VP1;SEQ ID NO:81)

[0263] TAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL(AAVrh.74VP2;SEQ ID NO:82)

[0264] MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL(AAVrh.74VP3;SEQ ID NO:83)

[0265] In some cases, 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 comprising, consisting essentially of, or further consisting of, e.g., 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 comprising, consisting essentially of, or further consisting of, for example, 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 usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74VP2 shown in SEQ ID NO:82. In some embodiments, the rAAV vector comprises a polypeptide comprising, consisting essentially of, or further consisting of, for example, 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 usually 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of AAVrh.74VP3 shown in SEQ ID NO:83.

[0266] In some embodiments, the rAAV virion is an AAV-PHP.B rAAV virion or a neurotrophic variant thereof, such as, but not limited to, those disclosed in International Patent Publication Nos. WO 2015 / 038958A1 and WO 2017 / 100671 A1. For example, the AAV capsid protein may comprise at least four consecutive amino acids from the sequence TLAVPFK (SEQ ID NO: 85) or KFPVALT (SEQ ID NO: 86), for example, inserted between the sequence encoding amino acids 588 and 589 of the AAV9 capsid protein.

[0267] The capsid can be an AAV-PHP.B capsid or a functional variant thereof. In some embodiments, the AAV-PHP.B capsid comprises a capsid protein that is at least 98%, 99%, or 100% identical to a reference AAV-PHP.B capsid protein, such as SEQ ID NO: 84.

[0268] Further AAV capsids for use in the rAAV virions of the present disclosure include those disclosed in patent publication numbers WO 2009 / 012176 A2 and WO 2015 / 168666 A2.

[0269] Without being limited by theory, the present inventors have determined that AAV9 vectors, such as AAVrh.74 or AAVrh.10 vectors, will confer the desired cardiac tropism on the vector. Without being limited by theory, the present inventors have also determined that AAV9 vectors, such as AAVrh.74 or AAVrh.10 vectors, will provide the desired specificity to cardiac cells.

[0270] In one aspect, the present disclosure provides pharmaceutical compositions comprising the rAAV virions of the present disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0271] For the purpose of administration, optionally by injection, various solutions, such as sterile aqueous solutions, can be used. If necessary, such aqueous solutions can be buffered and the liquid diluent first made isotonic with saline or glucose. Solutions of rAAV as free acids (DNA contains acidic phosphate groups) or pharmaceutically acceptable salts can be prepared in water, suitably mixed with a surfactant such as poloxamer 188, for example at 0.001% or 0.01%. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, the sterile aqueous media employed are all readily available by standard techniques well known to those skilled in the art.

[0272] The pharmaceutical form suitable for injection includes but is not limited to sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injection solutions or dispersions. In all cases, the form is sterile and must exist in a fluid that is easy to inject. It must be stable under the conditions of manufacture and storage and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), their suitable mixtures, and vegetable oils. Suitable fluidity can be maintained by using, for example, a coating of lecithin, by maintaining the required particle size in the case of dispersions, and by using a surfactant. The effect of microorganisms can be prevented by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, for example, sugar or sodium chloride. The extended absorption of the composition for injection can be achieved by using an agent that delays absorption, such as aluminum monostearate and gelatin.

[0273] Sterile injectable solutions can be prepared by combining the rAAV in the required amount with the various other ingredients described above in a suitable solvent, followed by filtered sterilization, as desired. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those described above. In the case of sterile powders for the preparation of sterile injectable solutions, certain preparation methods include vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0274] In another aspect, the present disclosure includes a kit comprising the rAAV virions of the present disclosure and instructions for use.

[0275] In one aspect, the present disclosure provides a method for increasing PKP2 activity in a cell, comprising contacting the cell with an rAAV of the present disclosure. In another aspect, the present disclosure provides a method for increasing PKP2 activity in a subject, comprising administering an rAAV of the present disclosure to the subject. In some embodiments, the cell and / or subject lacks PKP2 messenger RNA or PKP2 protein expression levels and / or activity and / or comprises a loss-of-function mutation in PKP2. The cell can be a cardiac cell, such as a cardiomyocyte. In a specific embodiment, the subject is a mammal, such as a human.

[0276] In some embodiments, the method promotes the survival of cardiac cells, such as cardiomyocytes, in cell culture and / or in vivo. In some embodiments, the method promotes and / or restores cardiac function.

[0277] On the other hand, the present disclosure provides a method for treating a disease or condition in a subject in need thereof, comprising administering an effective amount of rAAV virions of the present disclosure to the subject. In some embodiments, the disease or condition is a cardiac disease or condition. Exemplary cardiac conditions 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 specific embodiments, the subject is a mammal such as a human having a loss-of-function mutation in the PKP2 gene. In a specific method, treatment with rAAV virions results in expression of PKP2 protein encoded by the rAAV virion in the subject, for example, in the heart or myocardial tissue of the subject. In certain embodiments, treatment with rAAV virions results in at least two times, at least five times, at least ten times or more PKP2 protein levels being detectable in the heart of the subject.

[0278] High-risk PKP2 patients include, for example, 1) carriers of protein-truncated PKP2 variants, including nonsense, frameshift, deletion, stop-gain mutations, 2) patients with ICD implantation, or 3) patients who meet HRS Class I or Class IIa criteria for ICD placement (may have an in situ ICD). In specific embodiments, the methods of the present disclosure can be used to treat any disclosed patient population.

[0279] The term "termination variant" refers to a type of nonsense mutation, for example, a termination codon and a replacement of a truncated protein with a loss of function (or a significant reduction in the degradation of the transcript and the amount of the protein due to nonsense-mediated decay). The term "termination variant" refers to a mutation that results in a mutation that results in a mutation that results in a truncated protein with a loss of function (e.g., a truncated transcript and a significant reduction in the amount of the protein). The term "termination variant" refers to a mutation that results in a mutation that results in a truncated protein with a loss of function (e.g., a truncated transcript and a significant reduction in the amount of the protein). The term "termination variant" refers to a mutation that results in a truncated protein with a loss of function (e.g., a truncated transcript and a significant reduction in the amount of the protein) ... The prevalence of high-order PKP2 (163k) * the frequency of PKP2 termination acquisition (0.28) * the frequency of PKP2 ICD (0.41) = 18.7k pts. The prevalence of high-order PKP2 (153k) * the frequency of PKP2 termination acquisition (0.28) * the frequency of PKP2 ICD (0.41) = 17.5k pts.

[0280] In a specific embodiment, the subject includes a patient with a pathogenic PKP2 variant and a clinical diagnosis of PKP2-ACM. The prevalence of ACM ranges from 1:1000 to 1:5000 (Peters S, Trümmel M, Meyners W. Prevalence of right ventricular dysplasia-cardiomyopathy in anon-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). In 2,572 ACM patients evaluated from 13 publications, an aggregated average of 32.9% had PKP2 mutations.

[0281]

[0282] Using a conservative ACM prevalence of 1:5000 and a PKP2 mutation frequency of 32.9% in ACM, the prevalence of PKP2-ACM patients in the United States and European Union is approximately 50,000 patients.

[0283] In specific embodiments, the subject comprises a PKP2 stop-gain variant mutation, including but not limited to any mutation specifically disclosed herein, while in other embodiments, the subject may comprise a different type of PKP2 mutation.

[0284] In specific embodiments, the subject may contain an implantable cardioverter-defibrillator (ICD), while in other embodiments, the subject may not contain an ICD.

[0285] In specific embodiments, the subject comprises a PKP2 stop-gain variant mutation, including but not limited to any mutation disclosed herein, while in other embodiments, the subject may comprise a different type of PKP2 mutation and an implantable cardioverter-defibrillator (ICD).

[0286] In specific embodiments, the subject treated with the disclosed vectors or according to the disclosed methods comprises a PKP2 stop-gain variant mutation and / or has an implantable cardioverter defibrillator (ICD). https: / / www.mayoclinic.org / tests-procedures / implantable-cardioverter-defibrillators / about / pac-20384692

[0287] Delivery of PKP2 protein to the heart via AAV can increase lifespan, prevent or alleviate cardiac cell degeneration, heart failure, scarring, reduced ejection fraction, arrhythmias, angina pectoris, exercise intolerance, angina pectoris (chest pain), sudden cardiac death, exertional myalgia and spasms. Delivery of PKP2 protein to the heart via AAV can show improvement or prevention of normal disease processes detected from pathological electrocardiograms, cardiac MRI, and cardiac biopsy, reduction in paroxysmal ventricular arrhythmias, reduction in sudden cardiac death, and / or reduction or absence of further development of fibrofatty deposits in the right ventricular myocardium. The methods disclosed herein can prevent the reduction of right ventricular ejection fraction (RVEF), restore and / or increase right ventricular ejection fraction.

[0288] The methods disclosed herein can provide effective biodistribution in the heart. They can result in sustained expression in all or most cardiac cells, such as cardiomyocytes. Notably, the methods disclosed herein can provide persistent expression of PKP2 protein throughout the life of the subject following administration of the AAV vector. In some embodiments, PKP2 protein expression in response to treatment continues for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 years.

[0289] The present disclosure also contemplates combination therapies. Combinations of the methods of the present disclosure with standard medical treatments (e.g., corticosteroids or topical antihypertensive drugs) and with novel therapies are particularly contemplated. In some cases, a subject may be treated with a combination of steroids and / or immunosuppressants to prevent or reduce an immune response to the administration of the rAAV described herein.

[0290] In some embodiments, the AAV vector is expressed at approximately 1×10 12 -5×10 14 vector genome (vg) or approximately 1×10 12 -6×10 14 The AAV vector is administered at a dose of about 1×10 13 -5×10 14 In some embodiments, the AAV vector is administered at a dose of about 1×10 13 -1×10 14 In some embodiments, the AAV vector is administered at a dose of about 3×10 13 -3×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 13 -3×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 13 -5×10 14 In some embodiments, the AAV vector is administered at a dose of about 5×10 13 -1×10 14 In some embodiments, the AAV vector is administered at a dose of less than about 1×10 12 vg / kg, less than about 3×10 12 vg / kg, less than about 5×10 12 vg / kg, less than about 7×10 12 vg / kg, less than about 1×10 13 vg / kg, less than about 3×10 13vg / kg, less than about 5×10 13 vg / kg, less than about 7×10 13 vg / kg, less than about 1×10 14 vg / kg, less than about 3×10 14 vg / kg, less than about 5×10 14 vg / kg, less than about 7×10 14 vg / kg, less than about 1×10 15 vg / kg, less than about 3×10 15 vg / kg, less than about 5×10 15 vg / kg or less than about 7×10 15 vg / kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAVrh.74 vector, such as 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 of the AAVrh.74 vector than the AAV9 vector ... a dose of about 1×10 13 - Approximately 1×10 14 vg / kg dose of AAV9 vector, for example, about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 or about 5×10 13 In certain embodiments, about 5×10 13 - Approximately 5×10 14 vg / kg dose of AAVrh.74 vector, for example, 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 vg / kg. In some embodiments, at least about 5×10 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 about 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 The AAV vector, such as the AAVrh.74 vector, is administered at a dose of vg / kg.

[0291] In some embodiments, about 1×10 12 vg / kg, about 3×10 12 vg / kg, about 5×10 12 vg / kg, about 7×10 12 vg / kg, about 1×10 13 vg / kg, about 2×10 13 vg / kg, about 3×10 13 vg / kg, about 4×10 13 vg / kg, about 5×10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg, about 5×10 14 vg / kg, about 6×10 14 vg / kg, about 7×10 14 vg / kg, about 8×10 14 vg / kg, about 9×10 14 vg / kg, about 1×10 15 vg / kg, about 3×10 15 vg / kg, about 5×10 15 vg / kg or about 7×10 15 In some embodiments, the AAV vector is administered at a dose of about 1×10 vg / kg. In some embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAVrh.74 vector, such as a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. 13 - Approximately 1×10 14 vg / kg dose of AAV9 vector, for example, about 1×1013 , about 2×10 13 , about 3×10 13 , about 4×10 13 or about 5×10 13 In certain embodiments, about 5×10 13 - Approximately 5×10 14 vg / kg dose of AAVrh.74 vector, for example, 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 vg / kg. In some embodiments, at least about 5×10 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 about 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 The AAV vector, such as the AAVrh.74 vector, is administered at a dose of vg / kg.

[0292] In some embodiments, 1×10 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、7×10 13 vg / kg、8×1013 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 some embodiments, the AAV vector is administered at a dose of about 1×10 vg / kg. In some embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAVrh.74 vector, for example, a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In some embodiments, the AAV vector is administered at a dose of about 1×10 13 - Approximately 1×10 14 vg / kg dose of AAV9 vector, for example, about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 or about 5×10 13 In certain embodiments, about 5×10 13 - Approximately 5×10 14 vg / kg dose of AAVrh.74 vector, for example, 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 vg / kg. In some embodiments, at least about 5×10 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 13vg / kg, at least about 1×10 14 vg / kg, at least about 2×10 14 vg / kg, at least about 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 The AAV vector, for example, AAVrh.74 vector, is administered at a dose of vg / kg.

[0293] In some embodiments, the AAV vector is expressed at approximately 1×10 12 -5×10 14 The AAV vector is administered systemically at a dose of vector genomes (vg) / kilogram (kg) of total body weight of the subject (vg / kg). In some embodiments, the AAV vector is administered systemically at a dose of about 1×10 13 -5×10 14 vg / kg. In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 13 -3×10 14 vg / kg. In some embodiments, the AAV vector is administered systemically at a dose of about 5×10 13 -1×10 14 vg / kg. In some embodiments, the AAV vector is administered systemically at a dose of less than about 1×10 12 vg / kg, less than about 3×10 12 vg / kg, less than about 5×10 12 vg / kg, less than about 7×10 12 vg / kg, less than about 1×10 13 vg / kg, less than about 3×10 13 vg / kg, less than about 5×10 13 vg / kg, less than about 7×10 13 vg / kg, less than about 1×10 14 vg / kg, less than about 3×10 14 vg / kg, less than about 5×10 14 vg / kg, less than about 7×10 14 vg / kg, less than about 1×10 15 vg / kg, less than about 3×10 15 vg / kg, less than about 5×10 15 vg / kg or less than about 7×10 15In some embodiments, the AAV vector is systemically administered at a dose of about 1×10 vg / kg. In some embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAVrh.74 vector, for example, a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In some embodiments, the AAV vector is administered at a dose of about 1×10 13 - Approximately 1×10 14 vg / kg dose of AAV9 vector, for example, about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 or about 5×10 13 In certain embodiments, about 5×10 13 - Approximately 5×10 14 vg / kg dose of AAVrh.74 vector, for example, 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 vg / kg. In some embodiments, at least about 5×10 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 about 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 The AAV vector, for example, AAVrh.74 vector, is administered at a dose of vg / kg.

[0294] In some embodiments, about 1×10 12 vg / kg, about 3×10 12 vg / kg, about 5×10 12 vg / kg, about 7×1012 vg / kg, about 1×10 13 vg / kg, about 2×10 13 vg / kg, about 3×10 13 vg / kg, about 4×10 13 vg / kg, about 5×10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg, about 5×10 14 vg / kg, about 6×10 14 vg / kg, about 7×10 14 vg / kg, about 8×10 14 vg / kg, about 9×10 14 vg / kg, about 1×10 15 vg / kg, about 3×10 15 vg / kg, about 5×10 15 vg / kg or about 7×10 15 In some embodiments, the AAV vector is systemically administered at a dose of about 1×10 vg / kg. In some embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAVrh.74 vector, for example, a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In some embodiments, the AAV vector is administered at a dose of about 1×10 13 - Approximately 1×10 14 vg / kg dose of AAV9 vector, for example, about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 or about 5×10 13 In certain embodiments, about 5×10 13 - Approximately 5×10 14 vg / kg dose of AAVrh.74 vector, for example, 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 2×1014 , about 3×10 14 , about 4×10 14 or about 5×10 14 vg / kg. In some embodiments, at least about 5×10 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 about 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 The AAV vector, for example, AAVrh.74 vector, is administered at a dose of vg / kg.

[0295] In some embodiments, 1×10 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、7×10 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 15vg / kg、5×10 15 vg / kg or 7×10 15 In some embodiments, the AAV vector is systemically administered at a dose of about 1×10 vg / kg. In some embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAVrh.74 vector, for example, a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In some embodiments, the AAV vector is administered at a dose of about 1×10 13 - Approximately 1×10 14 vg / kg dose of AAV9 vector, for example, about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 or about 5×10 13 In certain embodiments, about 5×10 13 - Approximately 5×10 14 vg / kg dose of AAVrh.74 vector, for example, 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 vg / kg. In some embodiments, at least about 5×10 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 about 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 The AAV vector, for example, AAVrh.74 vector, is administered at a dose of vg / kg.

[0296] In some embodiments, the AAV vector is expressed at approximately 1×10 12 -5×10 14The AAV vector is administered intravenously at a dose of vector genomes (vg) / kilogram (kg) of total body weight of the subject (vg / kg). In some embodiments, the AAV vector is administered at a dose of about 1×10 13 -5×10 14 vg / kg. In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 13 -3×10 14 vg / kg. In some embodiments, the AAV vector is administered intravenously at a dose of about 5×10 13 -1×10 14 vg / kg. In some embodiments, the AAV vector is administered intravenously at a dose of less than about 1×10 12 vg / kg, less than about 3×10 12 vg / kg, less than about 5×10 12 vg / kg, less than about 7×10 12 vg / kg, less than about 1×10 13 vg / kg, less than about 3×10 13 vg / kg, less than about 5×10 13 vg / kg, less than about 7×10 13 vg / kg, less than about 1×10 14 vg / kg, less than about 3×10 14 vg / kg, less than about 5×10 14 vg / kg, less than about 7×10 14 vg / kg, less than about 1×10 15 vg / kg, less than about 3×10 15 vg / kg, less than about 5×10 15 vg / kg or less than about 7×10 15 In some embodiments, the AAV vector is administered intravenously at a dose of about 1×10 vg / kg. In some embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAVrh.74 vector, for example, a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In some embodiments, the AAV vector is administered intravenously at a dose of about 1×10 vg / kg. 13 - Approximately 1×10 14 vg / kg dose of AAV9 vector, for example, about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 or about 5×10 13 In certain embodiments, about 5×10 13 - Approximately 5×10 14 vg / kg dose of AAVrh.74 vector, for example, about 5×1013 , 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 vg / kg. In some embodiments, at least about 5×10 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 about 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 The AAV vector, for example, AAVrh.74 vector, is administered at a dose of vg / kg.

[0297] In some embodiments, about 1×10 12 vg / kg, about 3×10 12 vg / kg, about 5×10 12 vg / kg, about 7×10 12 vg / kg, about 1×10 13 vg / kg, about 2×10 13 vg / kg, about 3×10 13 vg / kg, about 4×10 13 vg / kg, about 5×10 13 vg / kg, about 6×10 13 vg / kg, about 7×10 13 vg / kg, about 8×10 13 vg / kg, about 9×10 13 vg / kg, about 1×10 14 vg / kg, about 2×10 14 vg / kg, about 3×10 14 vg / kg, about 4×10 14 vg / kg, about 5×10 14vg / kg, about 6×10 14 vg / kg, about 7×10 14 vg / kg, about 8×10 14 vg / kg, about 9×10 14 vg / kg, about 1×10 15 vg / kg, about 3×10 15 vg / kg, about 5×10 15 vg / kg or about 7×10 15 vg / kg. In certain embodiments, the AAV vector is administered intravenously at a dose of about 1×10 13 - Approximately 1×10 14 vg / kg dose of AAV9 vector, for example, about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 or about 5×10 13 In certain embodiments, about 5×10 13 - Approximately 5×10 14 vg / kg dose of AAVrh.74 vector, for example, 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 vg / kg. In some embodiments, at least about 5×10 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 about 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 The AAV vector, for example, AAVrh.74 vector, is administered at a dose of vg / kg.

[0298] In some embodiments, 1×10 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、7×10 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 some embodiments, the AAV vector is administered intravenously at a dose of about 1×10 vg / kg. In some embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAVrh.74 vector, for example, a vector comprising a sequence disclosed in any of SEQ ID NOs: 8-15 or 89-96. In some embodiments, the AAV vector is administered intravenously at a dose of about 1×10 vg / kg. 13 - Approximately 1×10 14 vg / kg dose of AAV9 vector, for example, about 1×10 13 , about 2×10 13 , about 3×10 13 , about 4×10 13 or about 5×10 13 In certain embodiments, about 5×10 13 - Approximately 5×10 14 vg / kg dose of AAVrh.74 vector, for example, about 5×10 13 , about 6×10 13 , about 7×1013 , 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 vg / kg. In some embodiments, at least about 5×10 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 about 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 The AAV vector, for example, AAVrh.74 vector, is administered at a dose of vg / kg.

[0299] Evidence of functional improvement, clinical benefit, or efficacy in patients can be revealed by changes in New York Heart Association functional classification (NYHA class), pathological electrocardiogram, cardiac MRI, cardiac biopsy, reduction in paroxysmal ventricular arrhythmias, reduction in sudden cardiac death, and / or reduction or absence of further development of fibrofatty deposits in the right ventricular myocardium. Benefits can be observed in electrocardiographic features commonly associated with arrhythmogenic right ventricular cardiomyopathy, such as T wave inversion, prolonged S wave ascending segment, regional QRS widening, and / or paroxysmal episodes of ventricular tachycardia.

[0300] In some embodiments, the method prevents or reduces a decrease in left ventricular ejection fraction percentage (LVEF%), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject having or at risk of a disease or condition associated with or caused by loss of function in PKP2.

[0301] In some embodiments, the method prevents or reduces a decrease in left ventricular fractional shortening percentage (FS%), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject having or at risk of a disease or condition associated with or caused by loss of function in PKP2.

[0302] In some embodiments, the method prevents or reduces right ventricular area (RV area) in square millimeters (mm 2 ), optionally preventing or reducing the increase by about 50%, about 60%, about 70%, about 80%, about 90% or about 100% compared to the increase observed in an untreated subject having or at risk of developing a disease or condition associated with or caused by loss of function in PKP2.

[0303] In some embodiments, the method prevents or reduces a decrease in right ventricular velocity time integral (RVVTI) (mm / sec) in millimeters / second, optionally by about 50%, about 60%, about 70%, about 80%, about 90% or about 100% compared to the decrease observed in an untreated subject having or at risk of a disease or condition associated with or caused by loss of function in PKP2.

[0304] In some embodiments, the method prevents or reduces an increase in left or right ventricular fibrosis, optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject having or at risk of a disease or condition associated with or caused by loss of function in PKP2.

[0305] An effective dose of the composition can be administered by standard routes in the art, including but not limited to systemic, topical, direct injection, intravenous, and intracardial administration. In some cases, administration includes systemic, topical, direct injection, intravenous, and intracardial injection. Administration can be performed by cardiac catheterization.

[0306] In some embodiments, the present disclosure provides local administration and systemic administration of effective doses of rAAV and compositions of the present disclosure. For example, systemic administration can be administration into the circulatory system so that the whole body is affected. Systemic administration includes parenteral administration by injection, infusion or implantation. The administration routes of the compositions disclosed herein include intravenous ("IV") administration, intraperitoneal ("IP") administration, intramuscular ("IM") administration, intralesional administration or subcutaneous ("SC") administration, or implantation of a slow-release device (e.g., a mini-osmotic pump, a reservoir formulation, etc.). In some embodiments, the methods of the present disclosure include administering the AAV vector of the present disclosure or its pharmaceutical composition thereof by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardial, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoresis or intracranial administration.

[0307] Specifically, the administration of the rAAV of the present disclosure can be achieved by using any physical method that can deliver the rAAV recombinant vector to the target tissue of the animal. Administration includes, but is not limited to, injection into the heart.

[0308] In some embodiments, the methods of the present disclosure include intracardiac delivery. A dedicated cannula, catheter, syringe / needle can be used for infusion using an infusion pump. Administration can include delivering an effective amount of rAAV virions or a pharmaceutical composition comprising rAAV virions to the heart. These can be achieved by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, ​​intracardial, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, iontophoresis or intracranial administration. The compositions of the present disclosure can also be administered intravenously.

[0309] The therapeutic methods disclosed herein can reduce and / or prevent one or more symptoms, including but not limited to ventricular hypertrophy, ventricular tachycardia, exercise intolerance, angina, and reduced RVEF. The benefits of AAV-mediated PKP2 overexpression can be demonstrated by increased survival and a reduction in 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 greater right ventricular velocity time integral (interval) compared to PKP cKO formulation buffer control animals).

[0310] Electrophysiological evidence of functional benefits of AAV-mediated PKP2 protein delivery can be demonstrated by mitigating disease-associated disrupted calcium dynamics in affected cardiomyocytes, most notably by measurements of L-type calcium flux, sarcoplasmic reticulum calcium leak, diastolic calcium leak, and standard measures of calcium transients in affected (e.g., PKP2-deficient) cardiomyocytes (e.g., time to peak amplitude and relaxation time constant). Histological analysis can reveal the benefits of AAV-mediated PKP2 overexpression by a reduction in the appearance of disease-associated collagen deposition in various regions of the heart, including the ventricles (e.g., by trichrome staining). Additional benefits can also be revealed by assessing cardiomyocyte ventricular proteins involved in calcium signaling pathways, as measured by increased (i.e., normalized) relative levels of Casq2 and / or Trdn and / or Cav 1.2 and / or AnkB and / or RyR2.

[0311] Effects of rAAV administration

[0312] In some embodiments, administration of a rAAV of the present disclosure can have a beneficial effect on a subject.

[0313] life

[0314] In some embodiments, administration of a rAAV of the present disclosure increases the lifespan of a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline.

[0315] In some embodiments, administration of a rAAV of the disclosure increases 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 a rAAV of the disclosure or to baseline.

[0316] In some embodiments, administration of a 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%, about 10% to about 25%, about 15% to about 20%, about 20% to about 35%, about 10% to about 40%, about 15% to about 20%, about 20% to about 35%, about 10% to about 50%, about 10% to about 15%, about 15% to about 20%, about 20% to about 35%, about 10% to about 25%, about 15% to about 20%, about 20% to about 35%, about 1 ... From about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, from about 95% to about 100%, from about 100% to about 200%, from about 200% to about 300%, from about 300% to about 400%, or from about 400% to about 500%.

[0317] Ejection fraction

[0318] In some embodiments, administration of a rAAV of the present disclosure limits a decrease in ejection fraction in a subject, restores and / or increases ejection fraction in a subject, compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure limits a decrease in ejection fraction in a subject, restores and / or increases ejection fraction in a subject over time.

[0319] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0320] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0321] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases the ejection fraction in a subject 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.

[0322] In some embodiments, administration of the rAAV of the present disclosure limits the decrease in ejection fraction 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% over time.

[0323] In some embodiments, administration of a rAAV of the present disclosure restores and / or increases 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%, about 10% to about 15%, about 10% to about 15%, about 15% to about 16%, about 17% to about 18%, about 19% to about 20%, about 20% to about 25%, about 25% to about 26%, about 20% to about 27%, about 20% to about 28%, about 20% to about 29%, about 30% to about 30 ... From about 100% to about 150%, from about 15% to about 20%, from about 20% to about 35%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%.

[0324] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases ejection fraction 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 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%.

[0325] In some embodiments, administration of a rAAV of the present disclosure limits a decrease in ejection fraction to 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 10%, or less than about 10% to less than about 10%. 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%.

[0326] In some embodiments, administration of the rAAV of the present disclosure limits the decrease in ejection fraction in a subject to 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 From about 15% to less than about 20%, from about 20% to less than about 35%, from about 25% to less than about 30%, from about 30% to less than about 35%, from about 35% to less than about 40%, from about 40% to less than about 45%, from about 45% to less than about 50%, from about 50% to less than about 55%, from about 55% to less than about 60%, from about 60% to less than about 65%, from about 65% to less than about 70%, from about 70% to less than about 75%, from about 75% to less than about 80%, from about 80% to less than about 85%, from about 85% to less than about 90%, from about 90% to less than about 95%, or from about 95% to about 100%.

[0327] Left ventricular ejection fraction (LVEF)

[0328] In some embodiments, administration of the rAAV of the present disclosure prevents a decrease in LVEF in a subject, restores, and / or increases LVEF 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 limits a decrease in LVEF in a subject, restores, and / or increases LVEF in a subject over time.

[0329] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0330] In some embodiments, administration of a rAAV of the present disclosure limits a 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 a rAAV of the present disclosure or to baseline.

[0331] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases LVEF in a subject 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.

[0332] In some embodiments, administration of the rAAV of the present disclosure limits the decrease in LVEF 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% over time.

[0333] In some embodiments, administration of a rAAV of the present disclosure restores and / or increases 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%, about 15% to about 16%, about 17% to about 18%, about 19% to about 20%, about 20% to about 25%, about 20% to about 26%, about 20% to about 27%, about 20% to about 28%, about 20% to about 29%, about 30% to about 31%, about 30% to about 32%, about 30% to about 33%, about 30% to about 34%, about 30% to about 35%, about 30% to about 36%, about 30% to about 37%, about 30% to about 38%, about 30% to about 39%, about 30% to about 31%, about 30% to about 39%, about 30% to about 31%, about 30% to about 31%, about 30% to about 3 % to about 15%, 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%.

[0334] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases LVEF in a subject over time 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 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%.

[0335] In some embodiments, administration of a rAAV of the present disclosure limits a decrease in LVEF to 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%, compared to a subject not administered a 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%.

[0336] In some embodiments, administration of the rAAV of the present disclosure limits a decrease in a subject's LVEF over time to 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 From about 15% to less than about 20%, from about 20% to less than about 35%, from about 25% to less than about 30%, from about 30% to less than about 35%, from about 35% to less than about 40%, from about 40% to less than about 45%, from about 45% to less than about 50%, from about 50% to less than about 55%, from about 55% to less than about 60%, from about 60% to less than about 65%, from about 65% to less than about 70%, from about 70% to less than about 75%, from about 75% to less than about 80%, from about 80% to less than about 85%, from about 85% to less than about 90%, from about 90% to less than about 95%, or from about 95% to about 100%.

[0337] Right ventricular ejection fraction (RVEF)

[0338] In some embodiments, administration of the rAAV of the present disclosure prevents a decrease in the subject's RVEF, restores, and / or increases the subject's RVEF, 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 limits a decrease in the subject's RVEF over time, restores, and / or increases the subject's RVEF.

[0339] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0340] In some embodiments, administration of a rAAV of the present disclosure limits a 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 a rAAV of the present disclosure or to baseline.

[0341] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases RVEF in a subject over time 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%.

[0342] In some embodiments, administration of the rAAV of the present disclosure limits the decrease in RVEF 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%, less than about 75%, less than about 80%, less than about 85%, less than about 90%, less than about 95%, or less than about 100% over time.

[0343] In some embodiments, administration of a rAAV of the present disclosure restores and / or 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%, about 10% to about 15%, about 15% to about 16%, about 17% to about 18%, about 19% to about 20%, about 20% to about 25%, about 25% to about 26%, about 25% to about 27%, about 25% to about 28%, about 20% to about 29%, about 30% to about 30 ... From about 100% to about 150%, from about 15% to about 20%, from about 20% to about 35%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%.

[0344] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases RVEF in a subject over time 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 16% to about 17%, about 18% to about 20%, about 19% to about 21%, about 22% to about 23%, about 24% to about 25%, about 26% to about 27%, about 28% to about 29%, about 30% to about 31%, about 32% to about 33%, about 34% to about 35%, about 36% to about 37%, about 38% to about 39%, about 40% to about 50 ... From about 15% to about 20%, from about 20% to about 35%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%.

[0345] In some embodiments, administration of a rAAV of the present disclosure limits a decrease in RVEF to 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 10%, or less than about 10% to less than about 10%. 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%.

[0346] In some embodiments, administration of the rAAV of the present disclosure limits the decrease in RVEF in a subject over time to 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 1 From about 5% to less than about 20%, from about 20% to less than about 35%, from about 25% to less than about 30%, from about 30% to less than about 35%, from about 35% to less than about 40%, from about 40% to less than about 45%, from about 45% to less than about 50%, from about 50% to less than about 55%, from about 55% to less than about 60%, from about 60% to less than about 65%, from about 65% to less than about 70%, from about 70% to less than about 75%, from about 75% to less than about 80%, from about 80% to less than about 85%, from about 85% to less than about 90%, from about 90% to less than about 95%, or from about 95% to about 100%.

[0347] Right ventricular (RV) area

[0348] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in RV area in a subject, restores RV area in a subject, and / or reduces RV area in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure prevents an increase in RV area in a subject, restores RV area in a subject, and / or reduces RV area in a subject over time.

[0349] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in, restores, 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 a rAAV of the present disclosure or to baseline.

[0350] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in RV area, restores and / or reduces RV area 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 20%. %, about 10% to about 15%, 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%.

[0351] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in, restores, and / or reduces RV area in a subject over time 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%.

[0352] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in RV area, restores, and / or reduces RV area 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%, or about 15% to about 16%. to about 15%, 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%.

[0353] Left ventricular (LV) area

[0354] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in, restores, and / or reduces LV area in a subject compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure prevents an increase in, restores, and / or reduces LV area in a subject over time.

[0355] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in, restores, 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 a rAAV of the present disclosure or to baseline.

[0356] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in LV area, restores and / or reduces LV area 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 20%. %, about 10% to about 15%, 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%.

[0357] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in, restores, and / or reduces LV area in a subject over time 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%.

[0358] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in LV area, restores and / or reduces LV area 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%, or about 15% to about 16%. to about 15%, 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%.

[0359] Right ventricular (RV) velocity time integral (VTI)

[0360] In some embodiments, administration of a rAAV of the present disclosure limits a decrease in, restores, and / or increases a subject's RV VTI compared to a subject not administered a rAAV of the present disclosure or to a baseline. In some embodiments, administration of a rAAV of the present disclosure limits a decrease in, restores, and / or increases a subject's RV VTI over time.

[0361] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0362] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0363] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases RV VTI in a subject over time 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%.

[0364] In some embodiments, administration of the rAAV of the present disclosure limits the reduction in RV VTI 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% over time.

[0365] In some embodiments, administration of a rAAV of the present disclosure restores and / or increases 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%, about 15% to about 20%, about 20% to about 30%, about 15% to about 20%, about 20% to about 30%, about 10% to about 25%, about 15% to about 2 ... 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%.

[0366] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases RV VTI in a subject over time 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 30% to about 50%, about 10% to about 20%, about 30% to about 5 ... 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%.

[0367] In some embodiments, administration of a rAAV of the present disclosure limits a reduction in RV VTI to 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%, less than about 20 From about 100% to about 150% of the total weight of the product, about 100% to about 200% of the total weight of the product may be present in the form of: 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%.

[0368] In some embodiments, administration of the rAAV of the present disclosure limits a reduction in RV VTI in a subject to 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%, less than about 20% to less than 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 to less than about 95% to about 100%.

[0369] Left ventricular (LV) velocity time integral (VTI)

[0370] In some embodiments, administration of the rAAV of the present disclosure limits a decrease in LV VTI in a subject, restores LV VTI in a subject, and / or increases LV VTI 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 limits a decrease in LV VTI in a subject, restores LV VTI in a subject, and / or increases LV VTI in a subject over time. Thus, the methods disclosed herein can be used to limit a decrease in LV VTI in a subject in need thereof, restore LV VTI in a subject, and / or increase LV VTI in a subject, e.g., a subject in need thereof, e.g., a subject with ACM such as ARVC or ARVD.

[0371] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0372] In some embodiments, administration of a rAAV of the present disclosure limits the reduction 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 a rAAV of the present disclosure or to baseline.

[0373] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases LV VTI in a subject 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.

[0374] In some embodiments, administration of the rAAV of the present disclosure limits the reduction in LV VTI 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% over time.

[0375] In some embodiments, administration of a rAAV of the present disclosure restores and / or increases 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%, about 15% to about 20%, about 20% to about 30%, about 15% to about 20%, about 20% to about 30%, about 10% to about 15 ...10% to about 15 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%.

[0376] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases LV VTI in a subject over time 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%. 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%.

[0377] In some embodiments, administration of a rAAV of the present disclosure limits a reduction in LV VTI to 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%, 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%.

[0378] In some embodiments, administration of the rAAV of the present disclosure limits a reduction in LV VTI in a subject to 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%, 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%.

[0379] Left ventricular (LV) fibrosis

[0380] In some embodiments, administration of the 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 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 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, e.g., a subject in need thereof, e.g., a subject with ACM such as ARVC or ARVD.

[0381] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0382] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis 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%, compared to a subject not administered a rAAV of the present disclosure or to baseline. , about 10% to about 15%, 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%.

[0383] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis in a subject 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.

[0384] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in LV fibrosis and / or reduces LV fibrosis 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%. to about 15%, 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%.

[0385] Right ventricular (RV) fibrosis

[0386] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in RV fibrosis in a subject and / or reduces RV fibrosis 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 RV fibrosis in a subject 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 in a subject in need thereof and / or reduce RV fibrosis in a subject in need thereof, e.g., a subject in need thereof, e.g., a subject with ACM such as ARVC or ARVD.

[0387] In some embodiments, administration of a 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 a subject not administered a rAAV of the present disclosure or to baseline.

[0388] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in RV fibrosis and / or reduces RV fibrosis 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%, compared to a subject not administered a rAAV of the present disclosure or to baseline. , about 10% to about 15%, 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%.

[0389] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in RV fibrosis in a subject and / or reduces RV fibrosis in a subject 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.

[0390] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in RV fibrosis in a subject and / or reduces RV fibrosis in a subject over time 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%. to about 15%, 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%.

[0391] Premature ventricular contractions (PVCs)

[0392] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in PVCs in a subject and / or reduces PVCs 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 PVCs in a subject and / or reduces PVCs in a subject over time. Thus, the methods disclosed herein can be used to prevent an increase in PVCs in a subject in need thereof and / or reduce PVCs in a subject in need thereof, e.g., a subject in need thereof, e.g., a subject with ACM such as ARVC or ARVD.

[0393] In some embodiments, administration of a rAAV of the present disclosure prevents an increase 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% compared to a subject not administered a rAAV of the present disclosure or to baseline.

[0394] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in PVC and / or reduces PVC 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 10% to about 25%, about 15% to about 20%, about 10% to about 25%, about 15% to about 25 ... 10% to about 15%, 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%.

[0395] 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 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%.

[0396] 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 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 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%.

[0397] Nonsustained ventricular tachycardia (NSVT)

[0398] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in NSVT in a subject and / or reduces NSVT 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 in a subject and / or reduces NSVT in a subject. Thus, the methods disclosed herein can be used to prevent an increase in NSVT in a subject in need thereof and / or reduce NSVT in a subject in need thereof, e.g., a subject in need thereof, e.g., a subject with ACM such as ARVC or ARVD.

[0399] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0400] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in NSVT and / or reduces NSVT 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%, compared to a subject not administered a rAAV of the present disclosure or to baseline. From about 10% to about 15%, from about 15% to about 20%, from about 20% to about 35%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%.

[0401] 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 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%.

[0402] 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 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 10%. 5%, 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%.

[0403] Ventricular tachycardia (VT)

[0404] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in VT in a subject and / or reduces VT in a subject compared to a subject not administered a rAAV of the present disclosure or to baseline. In some embodiments, administration of a rAAV of the present disclosure prevents an increase in VT in a subject and / or reduces NSVT in a subject over time.

[0405] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline. Thus, the methods disclosed herein can be used to prevent an increase in VT in a subject in need thereof and / or reduce VT in a subject in need thereof, e.g., a subject in need thereof, e.g., a subject with ACM such as ARVC or ARVD.

[0406] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in VT and / or reduces VT 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 10% to about 25%, about 15% to about 20%, about 10% to about 25%, about 15% to about 20%, about 10% to about 25%, about 15% to about 25 ... 0% to about 15%, 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%.

[0407] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in VT and / or reduces VT in a subject over time 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%.

[0408] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in VT and / or reduces VT 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% over time. , 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%.

[0409] Ectopic beats

[0410] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats 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 ectopic beats and / or reduces ectopic beats in a subject over time. Thus, the methods disclosed herein can be used to prevent an increase in ectopic beats in a subject in need thereof and / or reduce ectopic beats in a subject in need thereof, e.g., a subject in need thereof, e.g., a subject with ACM such as ARVC or ARVD.

[0411] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0412] In some embodiments, administration of a rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats 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 15% to about 20%, about 16% to about 21%, about 17% to about 22%, about 18% to about 23%, about 19% to about 24%, about 25% to about 26%, about 27% to about 28%, about 29% to about 30%, about 30% to about 30%, about 30% to about 30%, 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 From about 10% to about 15%, from about 15% to about 20%, from about 20% to about 35%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%.

[0413] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats in a subject over time 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%.

[0414] In some embodiments, administration of the rAAV of the present disclosure prevents an increase in ectopic beats and / or reduces ectopic beats in a subject over time 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 10%, or about 10% to about 10%. 5%, 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%.

[0415] intercalary plate

[0416] In some embodiments, administration of the rAAV of the present disclosure limits the reduction of, restores, and / or increases intercalated discs in a subject compared to a subject not administered the rAAV of the present disclosure or to a baseline. In the myocardium of the heart, connections between adjacent cells are formed by intercalated discs. To achieve heartbeat, intercalated discs are highly specialized and allow for the coordinated function of cardiomyocytes. In some embodiments, administration of the rAAV of the present disclosure limits the reduction of, restores, and / or increases intercalated discs in a subject over time. Thus, the methods disclosed herein can be used to limit the reduction of, restore, and / or increase intercalated discs in a subject in need thereof, such as a subject in need thereof, such as a subject with ACM such as ARVC or ARVD.

[0417] In some embodiments, administration of a 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 a rAAV of the present disclosure or to baseline.

[0418] In some embodiments, administration of a rAAV of the present disclosure limits the reduction in 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 a rAAV of the present disclosure or to baseline.

[0419] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases intercalated discs in a subject over time 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%.

[0420] In some embodiments, administration of the rAAV of the present disclosure limits the reduction of intercalated discs 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% over time.

[0421] In some embodiments, administration of a rAAV of the present disclosure restores and / or increases intercalated discs 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 10% compared to a subject not administered a rAAV of the present disclosure or to baseline. to about 15%, 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%.

[0422] In some embodiments, administration of the rAAV of the present disclosure restores and / or increases intercalated discs in a subject over time 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 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%.

[0423] In some embodiments, administration of a rAAV of the present disclosure limits the reduction in intercalated discs to 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%, compared to a subject not administered a 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%.

[0424] In some embodiments, administration of the rAAV of the present disclosure limits the reduction of intercalated discs in a subject over time to 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 From about 100% to about 100%, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, from about 100% to about 200% more, Example

[0425] Example 1: In vitro testing of adeno-associated virus vectors

[0426] Prepare the AAV vector described herein (having 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., those vectors with structures shown in SEQ ID NO: 15 or 99) and used to transduce CHO-Lec2 cells, and the expression level of PKP2 was assessed by Western blotting ( Figures 5A-5B ; Figure 5A The lanes in the SDS-PAGE gel provided correspond to Figure 5B Surprisingly, the MHCK7 promoter elicited robust expression of PKP2 in cardiomyocytes ( Figure 5A 1 and 3 in Figure 5), whereas the hTnnT2 promoter ("hTnT") produced negligible PKP2 levels above background under these assay conditions ( Figure 5A Compared with AAV9 serotype vectors, AAVrh.74 serotype induced higher PKP2 expression ( Figure 5A Lanes 3 and 4 in Figure 5).

[0427] These results indicate that AAV9 and AAVrh.74 vectors can be effectively used to express PKP2 in cardiomyocytes and that the MHCK7 promoter is superior to the hTnnT2 promoter when only this in vitro assay is used to assess the relative levels of PKP2 expression.

[0428] Example 2: In vivo efficacy of adeno-associated viral vectors

[0429] As described in Cerrone et al., Nat Comm., 2017, evidence of the benefits of AAV-mediated PKP2 overexpression was determined using a cardiomyocyte-specific, tamoxifen-activated PKP2 knockout mouse line (αMHC-Cre-ER(T2) / Pkp2 fl / fl; referred to as "PKP2-cKO"). This mouse model allows for controlled onset of PKP2 expression loss, restricting PKP2 loss to adult myocytes, and triggering a progression of molecular and functional events leading to arrhythmogenic cardiomyopathy (ACM) with an initial right ventricular-dominant course, but involving both the left and right ventricles in this mouse. 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 a RV-predominant arrhythmogenic cardiomyopathy that includes hallmark functional, molecular, and structural markers consistent with the ACM disease phenotype.

[0430] PKP2-cKO mice were injected with tamoxifen, resulting in myocyte-specific knockout of PKP2. 3 × 10 13 vg / kg of AAV vectors (described below) were injected into mice. Four weeks later, myocyte-specific knockout of PKP2 was induced by treating the mice with tamoxifen. The vector genomes tested were:

[0431] 5'ITR; MHCK7 promoter (with its enhancer element); SV40 intron; Kozak sequence; PKP2a transgene; WPRE (x); hGH polyadenylation sequence; 3'ITR (structure shown in Figure 1B SEQ ID NO: 89); and

[0432] 5'ITR; hTnnT2 promoter (with exon 1); Kozak sequence; PKP2a transgene; WPRE(x); hGH polyadenylation sequence; 3'ITR (structure shown in Figure 2 for example, SEQ ID NO: 13 or 97).

[0433] Each vector genome was tested in either an AAV9 serotype vector or an AAVrh.74 serotype vector.

[0434] Twenty-eight days after tamoxifen treatment (i.e., 56 days after AAV treatment), various physiological parameters of mice were assessed essentially as described in Cerrone et al., NatComm, 2017, or using standard methods known in the art. Left ventricular ejection fraction percentage (LVEF%) ( Figure 6 ), left ventricular fractional shortening percentage (FS%) ( Figure 7 ), right ventricular area (RV area) in square millimeters (mm 2 )( Figure 8 ), right ventricular velocity time integral RV VTI (mm / sec) ( Figure 9 ) and the degree of fibrosis ( Figures 10A-10B ) to assess the efficacy of treating the disease. These measures are appropriate functional and structural indicators to evaluate the potential efficacy of AAV-mediated overexpression of PKP2 in cardiomyocytes, as they are some of the key parameters that indicate ACM in human disease. Normally, the right ventricle is normal with slightly more fibrosis (irrelevant to the disease), and this is further exacerbated in the cKO model due to the lack of PKP2. Progressive deterioration of these parameters was observed within 28 days after injection of tamoxifen, because injection of tamoxifen results in myocyte-specific knockout of the PKP gene. Western blot was used to detect PKP2 levels to demonstrate in vivo expression of the transgene ( Figures 11A-11B ). 28 days after tamoxifen injection (i.e., AAV (dose 3×10 13 Gel images of PKP2 detected in the heart 56 days after injection of vg / kg of AAV9-MHCK7-PKP2a, AAV9-hTnT-PKP2a, AAVrh.74-MHCK7-PKP2a, or AAVrh.74-hTnT-PKP2a ( Figure 11A ) and quantified (normalized to GAPDH) (quantified from the top gel of panel A obtained at the same time) ( Figure 11B ).

[0435] Evidence of a reduced disease phenotype was observed following AAV9-mediated PKP expression and AAVrh.74-mediated PKP expression, to varying degrees. 13vg / kg) using a pre-treatment paradigm (AAV injection 4 weeks before tamoxifen induction of PKP cKO). Given the cardiac tropism of AAVrh.74 and the biological effects of AAVrh.74-mediated PKP2 overexpression observed in this model (e.g., LVEF%, FS%, and right ventricular area), the optimization of the AAVrh.74 dose in combination with the appropriate promoter (MHCK7 or hTnnT2) was also tested.

[0436] In the next set of experiments, 6 × 10 13 vg / kg, and for AAV9-hTnT-PKP2a, 1×10 13 vg / kg. Cardiac parameters were assessed as in the previous examples 28 days after tamoxifen treatment and 56 days after AAV treatment. Left ventricular ejection fraction percentage (LVEF%) ( Figure 12A ), left ventricular fractional shortening percentage (FS%) ( Figure 12B ), right ventricular area (RV area) in square millimeters (mm 2 )( Figure 13A ), right ventricular velocity time integral RV VTI (mm / sec) ( Figure 13B ) and the degree of fibrosis ( Figures 14A-14B ) to evaluate the efficacy of treating diseases. Western blot was used to detect PKP2 to demonstrate the protein level expressed in vivo ( Figures 15A-15B ). 28 days after tamoxifen injection (i.e., AAV (dose 1×10 13 vg / kg of AAV9-hTnT-PKP2a or a dose of 6×10 13 Gel image of PKP2 detected in the heart 56 days after injection of vg / kg AAVrh.74-hTnT-PKP2a) ( Figure 15A ) and quantified (normalized to GAPDH) (quantified from the top gel of panel A obtained at the same time) ( Figure 15B ).

[0437] In other analyses, samples were collected from control (Cre negative) mice injected with formulation buffer (control-FB; n = 4 mice), PKP2 cKO mice injected with formulation buffer (PKP2 cKO-FB; n = 4 mice), or PKP2-cKO mice injected with AAVrh.74-PKP2a (PKP2 cKO-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 sections. Fluorescence intensity maps were collected from stretches of 5 to 8 myometriums deemed suitable for alignment for analysis. Figure 31A An example of adult ventricular tissue stained with an AnkB antibody is shown (Cerrone et al 2017). The red line indicates the range of fluorescence intensity as shown in the right panel. Two parameters were collected: mean intensity and peak depth (defined as the range between peaks and valleys; see Figure 31A , right panel). Representative images obtained under the three conditions tested are shown in Figure 31B The values ​​of each intensity map were averaged and considered as one data point. Multiple regions were drawn for each tissue section, and data were collected separately from samples of the right ventricle (RV) or left ventricle (LV). The results collected from the LV and the results from the RV are shown in Figure 31C and Figure 31D Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc analysis, and statistical significance (p-values) are indicated at the top of the bars. The results indicate that re-expression of PKP2 in myocytes of PKP2cKO hearts reverses the previously reported characteristic molecular phenotype, namely, decreased AnkB expression.

[0438] Similarly, tissue sections were immunostained for desmin ( Figures 32A-32C ; Immunostaining method is the same as described for PKP2; Desmin antibody is the same as used in Pérez-Hernández et al. 2022; PMID: 35959657). Representative examples and results are shown in Figures 32A-32C In this case, the broad distribution of data affected statistical power, but the increased intensities obtained from PKP2 cKO cardiomyocytes (see Pérez-Hernández et al. 2022; PMID: 35959657) Figure 8 ) and statistically significant restoration of signal intensity in AAV-treated mice was confirmed, accompanied by a trend toward restoration of peak depth in RV and LV samples. The data showed that two molecular features of PKP2 cKO hearts, decreased AnkB and increased desmin, were reversed in hearts treated with the AAV-delivered PKP2 transgene.

[0439] These results demonstrate that both AAV9 and AAVrh.74 can provide benefit in relevant animal models of PKP2-associated ACM, also known as arrhythmogenic right ventricular dysplasia (ARVD) or arrhythmogenic cardiomyopathy (ACM). Additionally, AAV vectors harboring either the MHCK7 promoter or the hTnnT2 promoter have been shown to be effective in treating PKP2-associated diseases.

[0440] Example 3: In vivo efficacy of adeno-associated viral vectors

[0441] After evaluating the in vivo effects of various AAV-PKP2 vector constructs at different doses in the PKP-cKO model of ACM, the AAVrh.74-hTnT-PKP2a vector construct ( Figure 2 ; e.g., SEQ ID NO: 13 or 97) for further R&D and clinical development. This is based on the robust functional and anatomical benefits, the safety profile observed to date, and the potential to avoid the immune response to the AAV9 capsid observed in clinical trials in adults using high doses delivered intravenously.

[0442] To further evaluate and characterize the potential benefits of the lead vector AAVrh.74-hTnT-PKP2, further experiments were performed. Specifically, experiments were performed to assess the extent to which benefits in this ACM model could be observed after disease onset using the PKP2-cKO mouse model. An example of an experimental model and timeline is shown in Figure 16A -C. Animals initially received a tamoxifen injection and then received an intravenous injection of various doses of AAVrh.74-hTnT-PKP2 or control formulation buffer (FB) after -28, +7, or +14 days. Treatment groups across the various studies included control animals, AAV9-hTnT-PKP2, and various doses of AAVrh.74-hTnT-PKP2 as described below (see figures and figure legends for specific doses used in individual experiments):

[0443] WT (Cre-): formulation buffer (control);

[0444] PKP2-cKO: formulation buffer (negative control);

[0445] PKP2-cKO:AAV9-hTnT-PKP2:3E13vg / kg;

[0446] PKP2-cKO: AAVrh.74-hTnT-PKP2: 6E13vg / kg; and

[0447] PKP2-cKO:AAVrh.74-hTnT-PKP2:2E14vg / kg.

[0448] The expression cassette of the vector is Figure 2 The expression cassettes shown in . Echocardiography was performed 21 days, 28 days and 5 months after tamoxifen, according to each experiment.

[0449] The experimental results using the delayed AAVrh.74-hTnT-PKP2a injection paradigm in the PKP2-cKO model are shown in Figure 17-2 0 and Figure 27-2 9. We found that the previously defined “effective dose” of AAVrh.74-hTnT-PKP2, 6E13 vg / kg, resulted in 100% survival at an a priori defined time point, 5 months after tamoxifen, compared to 100% mortality in PKP2-cKO control animals at day 50 ( Figure 17 A “high dose” of AAVrh.74-hTnT-PKP2a (2E14 vg / kg) resulted in a 90% survival rate, with one animal death found to be unrelated to AAV-hTnT-PKP2a. All animals injected with AAVrh.74-hTnT-PKP2 were found to have preserved ejection fraction (EF) at 28 days. Figure 18A ), shorten the score( Figure 18B ) and right ventricular area, which persisted to 5 months ( Figure 19A Importantly, AAVrh.74-hTnT-PKP2a administered after disease onset (i.e., 14 days after tamoxifen) resulted in a significant reduction in the isoproterenol-induced premature ventricular contractions typically observed as a consequence of PKP2 loss in this mouse model as well as in ACM patients ( Figures 20A-20C ). Figure 21A -B shows the quantification of collagen percentage in the left ventricle 5 months after tamoxifen based on cardiac trichrome histological staining ( Figure 21A ) and right ventricle ( Figure 21B ) degree of fibrosis.

[0450] The presence and abundance of AAV vector DNA, transgene mRNA transcripts, and PKP2 protein in the heart were determined by ddPCR, RT-ddPCR, and Western blotting of heart lysates, respectively. Cumulative results are shown in Figure 22 and Figure 23A - C. The results show efficient transduction and subsequent expression of human PKP2a (hPKP2a) transgenic mRNA and PKP2 protein in the hearts of all mice injected with AAVrh.74-PKP2a.

[0451] Immunofluorescence images obtained from fixed heart tissue sections are shown in Figure 24A -C. Figure 24AImmunolocalization of native PKP2 in the heart of a control (Cre-negative, TAM-injected) mouse is shown. As expected for intercalated disc protein, the image shows a clear immunoreactive PKP2-positive signal, which is visualized as well-defined patches aligned perpendicular to the direction of the fibers ( Figure 24A (arrow in the middle). Figure 24B and Figure 24C Shown are the cases of patients euthanized 28 days after TAM and treated with FB ( Figure 24B ) or FB+AAVrh.74-PKP2a( Figure 24C )-injected heart sections of PKP2-cKO mice. There was a clear lack of immunolabeled PKP2 signals in the hearts of PKP2-cKO mice injected only with FB ( Figure 24B However, in hearts treated with AAVrh.74-PKP2a, there was a dense PKP2 signal oriented perpendicular to the fiber orientation ( Figure 24C ; Arrows). These results were confirmed in 4 mice per group. The data show that the exogenous PKP2 gene is transcribed and translated, and the expressed protein is correctly localized to the subcellular domain expected for the native PKP2 protein.

[0452] The ability of exogenous protein to prevent the cardiomyopathy phenotype was also examined. Cardiac function was assessed by echocardiography 28 days after TAM injection in treated mice. Figure 25A -C shows the results of FB treatment with FB alone (control, first bar from the left; PKP2-cKO, second bar from the left) or with AAVrh.74-PKP2a (PKP2-cKO + 3 × 10 13 vg / kg AAVrh.74-PKP2a, third from the left; PKP2-cKO+6×10 13 Echocardiographic images of the mouse heart injected with vg / kg AAVrh.74-PKP2a (fourth from the left) ( Figure 25A ) and cumulative data ( Figure 25B and Figure 25C ).like Figure 25A As in Figure ​-C, AAVrh.74-PKP2a or FB were injected 56 days before recording and 28 days before TAM injection. As expected, loss of PKP2 expression resulted in a decrease in left ventricular ejection fraction (LVEF; Figure 25B The dashed line and the second line in the figure) decreased significantly, as did the right ventricular (RV) area ( Figure 25C In contrast, AAVrh.74-mediated hPKP2a expression attenuated or prevented the loss of systolic function in the LV and the increase in RV area in a dose-dependent manner.

[0453] Cardiac fibrosis is a common feature of PKP2-deficient hearts. Figure 26A -C, visualization and quantification of collagen abundance in the ventricular free wall revealed extensive fibrosis in PKP2-cKO animals (second bar from the left), which was significantly alleviated by AAVrh.74-PKP2a administration (third and fourth bars from the left), especially at higher (6 × 10 13 Notably, a dose-related reduction in cardiac fibrosis was observed in both the left and right ventricles of animals injected with AAVrh.74-PKP2a.

[0454] The data presented in the previous figures indicate that AAVrh.74-PKP2a treatment prior to TAM-mediated PKP2 knockout can attenuate the development of the cardiomyopathy phenotype in PKP2-cKO mice. To evaluate whether AAVrh.74-PKP2a can prevent the progression of the ARVC phenotype when delivered after tamoxifen-mediated disease induction, 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, the mice were followed up for long-term survival. Figure 27 As shown in the Kaplan-Meier curves in Figure 3, PKP2-cKO animals injected with FB alone died between 30 and 50 days after TAM injection. In contrast, all but one of the mice injected with AAVrh.74-PKP2a survived for 5 months (155 days after TAM), at which time the animals were sacrificed for examination of protein expression and cardiac structure.

[0455] In addition, if Figure 28A As shown in representative images and cumulative data in Figure 2B, trichrome staining analysis revealed that the expression of AAVrh.74-PKP2a at a higher dose (2 × 10 14 vg / kg, third bar from the left) was similar (although trending toward higher values) to that observed in control animals injected with FB (first bar from the left). 13 vg / kg, second bar from the left) was higher in the hearts of mice treated with TAM than in control animals. Importantly, due to early lethality in this group, comparison with collagen abundance in PKP2-cKO animals at the same time point was not possible. However, collagen abundance in treated animals was less than that documented by historical data from PKP2-cKO mice at 42 days after TAM injection (close to the time of their death, as per Figure 27Finally, the cumulative data obtained from echocardiographic analysis of the hearts of mice injected with AAVrh.74-PKP2a 7 or 14 days after TAM are shown in Figure 5. Figure 28C -D. Consistent with previous results, hearts from FB-injected PKP2-cKO mice showed a dramatic decrease in LVEF 28 days after TAM injection ( Figure 28C ) and increase in RV area ( Figure 28D )(Will Figure 28C -D (compare the second bar from the left with the first bar). These changes were attenuated 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.

[0456] Previous studies have documented that a bolus injection of 3 mg / kg of isoproterenol (ISO) resulted in premature ventricular contractions in anesthetized PKP2-cKO mice 21 days after TAM injection. Therefore, an ISO challenge protocol was used to determine whether AAVrh.74-PKP2a delivered 14 days after TAM could reduce the arrhythmia burden in PKP2-cKO animals. Representative ECG traces are shown in Figure 29A -B. PKP2-cKO animals injected with FB developed multiple PVCs, and 6 of 10 animals developed more than 100 PVCs within 30 minutes of recording after ISO injection ( Figure 29C ), with an average total count of approximately 300 PVCs ( Figure 29D In contrast, the two tested doses (6×10 13 vg / kg and 2×10 14 vg / kg, in Figure 29C -D) administration of AAVrh.74-PKP2a drastically reduced both parameters of arrhythmia burden.

[0457] Safety and toxicology studies in mice revealed consistent and adequate vector DNA biodistribution. Transgene mRNA was enriched 10-100-fold in the heart relative to all other organs (including the liver). Clinical and histopathological analyses revealed that AAVrh.74-PKP2a was expressed in mice up to and including 3×10 14 The dose of vg / kg was well tolerated and safe in multiple studies.

[0458] Example 4: Phase 1 clinical trial

[0459] A multicenter dose-escalation study is being conducted in PKP2-associated ACM. Subjects may include those with mutations that result in truncated PKP2 expression, such as "correct stop gain" variant mutations. Subjects may also have ICD.

[0460] Key inclusion / exclusion criteria included: age ≥18 years; diagnosis of ACM, PKP2 gene mutation, clinically significant arrhythmia burden, and preserved LV function.

[0461] It is expected that treatment with the PKP2 gene therapy vectors disclosed herein (e.g., AAV9 or AAVrh.74) will result in improvements in one or more endpoints. Expected endpoints include, but are not limited to:

[0462] Primary: Safety (frequency and severity of AEs / SAEs)

[0463] Secondary: Pivotal exploratory / preliminary efficacy

[0464] Myocardial PKP2 expression and vector copy number

[0465] PVC reduction on serial Holter, other parameters that influence LTVA risk based on the ARVC risk calculator, or any parameters

[0466] Exploratory endpoints

[0467] VT / VF monitoring (ECG / Holter / AICD)

[0468] Reduced incidence of ICD shocks

[0469] Improved RV function (MRI / echo)

[0470] LV ejection fraction (MRI / Echo)

[0471] Blood biomarkers: BNP, NT-proBNP, troponin I

[0472] Kansas City Cardiomyopathy Questionnaire

[0473] Stable LGE on MRI

[0474] Left or right atrial volume

[0475] Additional arrhythmia-related endpoints

[0476] Physiological, cardiac-related, and quality-of-life assessments

[0477] Additional biomarkers

[0478] Example 5: In vivo expression of PKP2 in non-human primates (NHPs)

[0479] Studies were performed in non-human primates (NHPs) to demonstrate that the vectors disclosed herein result in expression in the heart of large mammals.

[0480] NHPs were intravenously administered a "low" or "high" dose of AAVrh.74-PKP2a (see Figure 2 , such as SEQ ID NO: 13 or 97) or formulation buffer (FB; vehicle control). The low dose corresponds to 8×10 13 vg / kg, the high dose corresponds to 3×10 14 vg / kg. The AAVrh.74-PKP2a vector delivers an expression cassette comprising a polynucleotide comprising:

[0481] a polynucleotide sequence encoding PKP2 isoform a of SEQ ID NO: 1;

[0482] The human TnnT2 promoter sequence (with exon 1) of SEQ ID NO: 32, wherein the promoter sequence is operably linked to a polynucleotide sequence encoding PKP2a;

[0483] and polyA sequences.

[0484] Three months after a single intravenous administration of AAVrh.74-PKP2a vector, animals were humanely sacrificed and heart tissue was collected. Expression of human PKP2a transgenic mRNA and PKP2a protein in heart tissue was determined by RT-PCR, immunostaining, and Western blotting.

[0485] like Figure 30A As shown in Figure 3-D, while NHPs administered FB showed endogenous PKP2a expression in the heart, NHPs administered AAVrh.74-PKP2a showed increased PKP2a mRNA and protein levels compared to controls. These data demonstrate that transgenic PKP2a is detected across multiple assay levels in the nonhuman primate heart. Figure 30A -B is for PKP2 protein control FB ( Figure 30A ) or “low-dose” treated animals ( Figure 30B Moderate levels of endogenous PKP2 immunolabeling were observed at the intercalated discs and junctional gaps between cardiomyocytes in animals treated with FB control (see, e.g., Figure 30A This is in stark contrast to the strong and higher frequency of PKP2-positive immunolabeling observed at the intercalated discs of “low-dose” treated animals (see e.g. Figure 30B (the hollow triangle in the figure). Figure 30C and Figure 30D Depicted are quantification of PKP2 protein and transgenic mRNA (hPKP2a) by Western blotting in the ventricles of nonhuman primate hearts following intravenous infusion of "low" or "high" doses of AAVrh.74-PKP2a or FB (control).

[0486] These data demonstrate that, following intravenous administration, the AAVrh.74-PKP2a vector transduces and expresses a potential therapeutic transgene in cardiac tissue of larger mammals.

[0487] Safety and toxicology studies in nonhuman primates (NHPs) revealed adequate vector DNA biodistribution. Clinical and histopathological analyses revealed that AAVrh.74-PKP2a was expressed in NHPs up to and including 3×10 14 vg / kg dose was well tolerated and safe in multiple studies.

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Claims

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

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

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

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

6. The polynucleotide of claim 5, wherein the MHCK7 promoter is 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% identical to SEQ ID NO:

31.

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

8. The polynucleotide of claim 7, wherein the hTNNT2 promoter is 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% identical to SEQ ID NO:

32.

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, wherein optionally the hTNNT2 promoter and exon 1 together are 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% identical to SEQ ID NO:

32.

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

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

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

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 WPRE(x).

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

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

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

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

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

1.

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

20. The polynucleotide of claim 19, wherein the PKP2 is 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% identical to SEQ ID NO:

2.

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

22. The polynucleotide of any one of claims 1 to 21, wherein the polynucleotide sequence encoding PKP2 is 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% identical to SEQ ID NO:

3.

23. The polynucleotide of any one of claims 1 to 21, wherein the polynucleotide sequence encoding PKP2 is 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% identical to SEQ ID NO:

4.

24. The polynucleotide of any one of claims 1 to 23, wherein 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.

25. The polynucleotide of any one of claims 1 to 24, wherein the polynucleotide comprises 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. The polynucleotide of any one of claims 1 to 25, wherein the polynucleotide comprises 4.0 kb to 4.6 kb, 4.0 kb to 4.5 kb, or 4.0 kb to 4.4 kb, or wherein the polynucleotide comprises 4.0 kb to 4.3 kb, 4.0 kb to 4.2 kb, or 4.0 kb to 4.1 kb.

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

28. The polynucleotide of any one of claims 1 to 27, wherein the polynucleotide is 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% identical to any one of SEQ ID NOs: 8-15, 89-96 or 97-102.

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. The polynucleotide of claim 29, wherein the ITR is an AAV2 ITR and / or the ITR is 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% identical to any one of SEQ ID NOs: 20-26.

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

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 is 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% identical 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 is 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% identical 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 is 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% identical to SEQ ID NO:

78.

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

40. The vector of claim 39, wherein the rAAV vector comprises a capsid protein that is 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% identical to SEQ ID NOs: 81-83.

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

42. The method of claim 41, wherein the disease or condition is heart disease.

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

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

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

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

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

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

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

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

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 wherein the subject has an implantable cardioverter-defibrillator (ICD).

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

53. The method of any one of claims 41 to 52, wherein the administration increases PKP2 expression by at least about 5%.

54. The method of any one of claims 41 to 52, wherein the administration increases PKP2 expression by at least about 30%.

55. The method of any one of claims 41 to 52, wherein the administration increases PKP2 expression by at least about 70%.

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

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

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

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

60. The method of any one of claims 41 to 59, wherein the method treats and / or prevents the disease or disorder.

61. The method of any one of claims 41 to 60, wherein the method comprises administering an effective amount of the vector.

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. 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. The method of any one of claims 41 to 63, wherein the subject has a mutation resulting in an amino acid substitution selected from the group consisting of Arg490Trp, Asp26Asn, Thr50-Val51SerfsX60, Arg79X, Tyr86X, Gln133X, Val406SerfsX3, Tyr616X, Trp676X, Cys796Arg, Cys796E, Tyr807X, Glu62Lys, S688P, Trp848X, Y86X, V406X, Y616X, W848X, and Y807X relative to a human PKP2 gene encoding human PKP2 having the sequence of SEQ ID NO:

2.

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

66. The method of any one of claims 41 to 65, wherein the method comprises administering to the subject approximately 1×10 11 vector genomes to approximately 1×10 13 vector genomes of the vector, administering to the subject approximately 1×10 12 vector genomes to approximately 1×10 14 vector genomes of the vector, or administering to the subject about 1×10 13 vector genomes to approximately 1×10 15 vector genomes of said vector, Optionally, wherein the vector is AAV9, optionally comprising 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, optionally comprising 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 the subject approximately 1×10 13 to about 1×10 14 vector genomes of the vector, and Optionally, wherein the vector is AAV.rh.74, optionally comprising 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, optionally comprising 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 the subject approximately 5×10 13 to about 5×10 14 The vector comprises a plurality of vector genomes.

67. A pharmaceutical composition comprising the carrier of any one of claims 31-40.

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

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

70. The vector of any one of claims 31-40, for use in treating a disease or disorder, optionally according to a method according to any one of claims 41-66.

71. A polynucleotide comprising a polynucleotide sequence that is 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% identical to any one of SEQ ID NOs: 12-15, 89-92 or 97-99 or any one of SEQ ID NOs: 8-11, 93-96 or 100-102.

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

73. The polynucleotide of claim 72, wherein the MHCK7 promoter is 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% identical to SEQ ID NO:

31.

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

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

76. The polynucleotide of claim 75, wherein the PKP2 isoform A is at least 80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NO:

1.

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

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

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

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

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

86. The method of any one of claims 81-85, wherein the method prevents or reduces a decrease in left ventricular ejection fraction percentage (LVEF%), optionally by about 50%, about 60%, about 70%, about 80%, about 90% or about 100% 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 method prevents or reduces a decrease in left ventricular fractional shortening percentage (FS%), optionally by about 50%, about 60%, about 70%, about 80%, about 90% or about 100% compared to the decrease observed in untreated subjects identified as having a mutation in the PKP2 gene.

88. The method according to any one of claims 81-87, wherein the method prevents or reduces right ventricular area (RV area) in square millimeters (mm 2 ), optionally preventing or reducing the increase by about 50%, about 60%, about 70%, about 80%, about 90% or about 100% compared to the increase observed in untreated subjects identified as having a mutation in the PKP2 gene.

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

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