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. Author manuscript; available in PMC: 2025 Feb 1.
Published in final edited form as: Circ Genom Precis Med. 2024 Jan 30;17(1):e004305. doi: 10.1161/CIRCGEN.123.004305

AAV-mediated Delivery of Plakophilin-2a Arrests Progression of Arrhythmogenic Right Ventricular Cardiomyopathy in Murine Hearts: Preclinical Evidence Supporting Gene Therapy in Humans

Chantal JM van Opbergen 1,*, Bitha Narayanan 2,*, Chester B Sacramento 2, Katie M Stiles 2, Vartika Mishra 2, Esther Frenk 2, David Ricks 2, Grace Chen 1, Mingliang Zhang 1, Paul Yarabe 2, Jonathan Schwartz 2, Mario Delmar 1,†, Chris D Herzog 2,†, Marina Cerrone 1,†
PMCID: PMC10923105  NIHMSID: NIHMS1960192  PMID: 38288614

Abstract

Background:

Pathogenic variants in plakophilin-2 (PKP2) cause arrhythmogenic right ventricular cardiomyopathy (ARVC), a disease characterized by life-threatening arrhythmias and progressive cardiomyopathy leading to heart failure. No effective medical therapy is available to prevent and/or arrest the disease. We tested the hypothesis that AAV-mediated delivery of the human PKP2 gene to an adult mammalian heart deficient in PKP2 can arrest disease progression and significantly prolong survival.

Methods:

Experiments were carried out using a cardiac-specific, tamoxifen (TAM)-activated PKP2 knockout murine model (PKP2-cKO). The potential therapeutic, AAVrh.74-PKP2a (RP-A601), is a recombinant AAVrh.74 gene therapy viral vector encoding the human PKP2 variant A (PKP2a). AAVrh.74-PKP2a was delivered to adult mice by a single tail vein injection either before or after TAM-activated PKP2-cKO. PKP2 expression was confirmed by molecular and histopathologic analyses. Cardiac function and disease progression were monitored by survival analyses, echocardiography and electrocardiography.

Results:

Consistent with prior findings, loss of PKP2 expression caused 100% mortality within 50 days after TAM injection. In contrast, AAVrh.74-PKP2a-mediated PKP2a expression resulted in 100% survival for more than 5 months (at study termination). Echocardiographic analysis revealed that AAVrh.74-PKP2a prevented right ventricle dilation, arrested left ventricle functional decline, and mitigated arrhythmia burden. Molecular and histological analysis showed AAVrh.74-PKP2a-mediated transgene mRNA and protein expression and appropriate PKP2 localization at the cardiomyocyte intercalated disc. Importantly, therapeutic benefit was shown in mice receiving AAVrh.74-PKP2a after disease onset.

Conclusion:

These preclinical data demonstrate the potential for AAVrh.74-PKP2a (RP-A601) as a therapeutic for PKP2-related ARVC in both early and more advanced stages of disease.

Keywords: arrhythmogenic right ventricular cardiomyopathy, plakophilin 2, gene therapy, RP-A601, AAVrh.74-PKP2a, AAV, Cardiomyopathy, sudden death

Introduction

Pathogenic variants in the PKP2 gene, coding for the protein plakophilin-2 (PKP2) are the primary cause of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC) in humans, a progressive disorder with autosomal dominant inheritance.1,2 ARVC is a condition that falls within the umbrella term of arrhythmogenic cardiomyopathy (ACM), as defined in Towbin et al.3 For simplicity, in the present document we refer to PKP2-related ARVC as “PKP2-ACM.” Average age at first presentation is estimated between the second to fourth decade and the disease is symptomatic more commonly in males.2,4 Pathogenic variants in PKP2 account for 20–45% of all cases of arrhythmogenic cardiomyopathy cases, with an estimated prevalence between 1:1000 to 1:5000 in Europe and North America.1 ARVC is characterized by a high risk of life-threatening arrhythmias prior to other clinical manifestations (i.e., during the concealed phase of the disease), followed by a stage of clinically overt loss of myocardial mass and the presence of fibrofatty infiltrates.2 The cardiomyopathy phenotype presents first in the right ventricle, later progressing to a biventricular disease.1,2 ARVC is a disease without a cure. Conventional heart failure therapies can be used; however, as is the case for other cardiomyopathies, they often fail to arrest disease progression and the heart advances to end-stage failure, leaving cardiac transplant as the only option to prevent death.

AAVrh.74-PKP2a (RP-A601) is a recombinant AAVrh.74 gene therapy viral vector encoding the human plakophilin 2 transcript variant A (PKP2a) being developed to treat patients with ARVC caused by pathogenic variants in PKP2. The purpose of the present study was to examine the hypothesis that AAVrh.74-mediated delivery of an exogenous PKP2a gene in the setting of PKP2 deficiency in an adult mammalian heart can arrest the progression of the arrhythmogenic and cardiomyopathic components of the disease and significantly prolong life expectancy. For this purpose, we utilized a previously characterized cardiomyocyte-specific, tamoxifen (TAM)-activated, PKP2 conditional knockout murine model (PKP2-cKO).5 Previous studies in these animals have shown that loss of PKP2 expression in adult mice leads to an arrhythmogenic cardiomyopathy of right ventricular predominance 21 days after TAM injection, a decrease in left ventricular systolic function by 28 days post-TAM, and progression to end-stage heart failure and death by the sixth week after TAM injection. As such, this animal model presents, in a compressed time span, the various stages of human disease.2,5 The purpose of the present study was to assess whether AAVrh.74-mediated delivery of PKP2a in the PKP2-cKO mouse model can arrest the progression of the arrhythmia burden and cardiomyopathy components of the disease while significantly prolonging survival. These data provide preclinical support to the notion that gene replacement therapy effectively interrupts the progression of an otherwise deadly condition, paving the way for future translation to a carefully selected human patient population that could benefit from a gene therapy approach.

Methods

An expanded methods section is provided in the Supplemental Material. The data that support the findings of this study are presented in the article and in the Supplemental Material. Additional information can be made available from the corresponding author upon reasonable request. Procedures conformed with the Guide for Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the NYU-IACUC committee [#160726–03].

Results

Initial experiments sought to establish that AAVrh.74-mediated PKP2a expression driven by a cardiac-selective human Troponin T promoter (hTnT; schematic presented in Supplemental Figure I) could prevent the ARVC phenotype in PKP2-cKO mice. All mice received a single tail vein injection of either AAVrh.74-PKP2a, at the specified dose, or formulation buffer (FB) alone. For the first set of experiments, all mice were then injected with TAM 28 days after the injection of AAVrh.74-PKP2a or FB. Four groups of male mice, 3–4 months of age were studied: 1) Control (i.e., PKP2 flox/flox Cre-negative) injected with FB; 2) PKP2-cKO (flox/flox Cre-positive) injected with FB; 3) PKP2-cKO injected with AAVrh.74-PKP2a at a dose of 3×1013 vg/kg; 4) PKP2-cKO injected with AAVrh.74-PKP2a at a dose of 6×1013 vg/kg (Supplemental Figure IIA). Hearts were collected 28 days after TAM injection (i.e., 56 days after injection of either AAVrh.74-PKP2a or FB). The presence and abundance of AAV vector DNA, transgene mRNA transcripts, and PKP2 protein in the heart lysates were determined by ddPCR, RT-ddPCR and Western blot, respectively. Cumulative results are shown in Figure 1A–C. Examples of Western blots are presented in Supplemental Figure III. Based on the ratio of mRNA to DNA copies, we found that human PKP2a expression is enriched in the heart by more than 300-fold compared to skeletal muscle and liver (Supplemental Table II). The results demonstrate efficient transduction and subsequent enriched expression of human PKP2a (hPKP2a) transgene mRNA and PKP2 protein in the heart in all AAVrh.74-PKP2a injected mice.

Figure 1.

Figure 1.

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AAVrh.74-PKP2a-mediated expression in PKP2-cKO mouse heart. Dose-dependent detection of (A) vector genomes (ddPCR), (B) transgene PKP2a mRNA (RT-ddPCR) and (C) PKP2 protein expression (Western Blot) in cardiac tissue from AAVrh.74-PKP2a injected animals relative to controls. Data are presented as Mean ± SD. Black bars, control mice treated with Formulation Buffer (FB); red bars, PKP2-cKO mice treated with FB; blue bars, PKP2-cKO mice treated with AAVrh.74-PKP2a 3 × 1013 vg/kg; purple bars, PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 1013 vg/kg. Number of mice studied noted in corresponding bars (N). Data in panels A and C passed a test for normal distribution (Shapiro-Wilk) and significance was assessed by One-way ANOVA followed by Tukey’s post-hoc analyses. For panel B, statistical significance was assessed using a non-parametric test (Kruskal-Wallis followed by Dunn’s post-hoc analyses). Considering the small number of samples per group, the assessment of significance was repeated for data in panels A and C using a non-parametric test. The results are presented in Supplemental Table I. <LoQ: Less than limit of quantitation; FB: Formulation Buffer.

Immunofluorescence images acquired from fixed sections of heart tissue are shown in Figure 2. Panel A shows the immunolocalization of native PKP2 in the heart of a control (Cre-negative, TAM-injected) mouse. The image shows a clear immunoreactive PKP2-positive signal, visualized as well-defined plaques that align perpendicular to the direction of the fibers, as expected for an intercalated disc protein (see arrows in Fig. 2A). Panels B and C show cardiac sections of PKP2-cKO mice euthanized 28 days post-TAM and previously injected (28 days pre-TAM) either with FB (panel B) or with FB + AAVrh.74-PKP2a (panel C). Immunolabeled PKP2 signal is notably absent in the heart from PKP2-cKO mice injected with FB alone (panel B). However, dense PKP2 signals oriented perpendicular to the fiber orientation are present in the heart that received the AAVrh.74-PKP2a therapy (panel C; arrows). These results were confirmed in 4 mice per group. Overall, the data show that the exogenous PKP2 gene was transcribed and translated, and the expressed protein was properly localized to the subcellular domain expected for the native PKP2 protein.

Figure 2.

Figure 2.

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Cardiac expression of PKP2 in AAVrh.74-PKP2a treated PKP2-cKO mice. Representative images of immunofluorescence staining for plakophilin2 (PKP2; in red) and nuclei (DAPI; in blue) in hearts from (A) control mice treated with Formulation Buffer (FB), (B) PKP2-cKO mice treated with FB, and (C) PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 1013 vg/kg. Mice were treated with FB or AAVrh.74-PKP2a 28 days before Tamoxifen injection. White arrowheads in panels A and C highlight PKP2 localization at the intercalated disc. FB: Formulation Buffer. Scale bar panels on the left = 150 μm, Scale bar panels on the right = 50 μm.

We examined whether expression of the exogenous protein prevented the cardiomyopathic phenotype. Assessment of cardiac function in vector treated mice by echocardiography was performed 28-days post-TAM injection. Figure 3 shows echocardiographic images (panel A) and cumulative data (panels B-C) obtained from hearts of mice injected with FB alone (control, black bar; PKP2-cKO, red bar), or with AAVrh.74-PKP2a (PKP2-cKO + 3×1013 vg/kg AAVrh.74-PKP2a, blue; PKP2-cKO + 6×1013 vg/kg AAVrh.74-PKP2a, purple bar). As in Figure 2, AAVrh.74-PKP2a or FB were injected 56 days before recording, and 28 days prior to TAM injection (Supplemental Figure IIA). As expected, loss of PKP2 expression caused a significant drop in the left ventricular ejection fraction (LVEF; red dotted line and red bar in panel B) and an increase in right ventricular (RV) area (red dotted line and red bar in panel C). In contrast, AAVrh.74-mediated expression of hPKP2a mitigated or prevented the loss of contractile function in the LV and the increase in RV area in a dose-dependent manner.

Figure 3.

Figure 3.

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Cardiac contractility in AAVrh.74-PKP2a treated PKP2-cKO mice. (A) Representative images of left (LV) and right ventricular (RV) echocardiography to assess contractility in control and PKP2-cKO mice treated with Formulation Buffer (FB), or in PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 1013 vg/kg, 28 days before Tamoxifen injection. Measurements were collected 28 days post-Tamoxifen injection. B) Quantification of left ventricular ejection fraction (LVEF) measured by long axis B-mode echocardiography and C) right ventricular (RV) area measured by modified long axis B-mode echocardiography, as described in Cerrone et al.5 Data are presented as Mean ± SD. Black bars, control mice treated with FB; red bars, PKP2-cKO mice treated with FB; blue bars, PKP2-cKO mice treated with AAVrh.74-PKP2a 3 × 1013 vg/kg; purple bars, PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 1013 vg/kg. Data distribution passed a test of normality (Shapiro-Wilk and Kolmogorov-Smirnov) and significance was evaluated by One-way ANOVA followed by Tukey’s post-hoc analyses. LVID: Left ventricular internal diameter, LVEF: Left ventricular ejection fraction, RV: Right ventricle, FB: Formulation Buffer.

Cardiac fibrosis is a common feature in PKP2 deficient hearts. As shown in Figures 4A–C, visualization and quantification of the extent of collagen abundance in the ventricular free walls revealed the presence of extensive fibrosis in PKP2-cKO animals (red bars), which was significantly mitigated by AAVrh.74-PKP2a administration (blue and purple bars), particularly at the higher dose (purple bar).

Figure 4.

Figure 4.

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Cardiac fibrosis in AAVrh.74-PKP2a treated PKP2-cKO mice. (A) Upper panel; representative images of Masson’s trichrome staining of longitudinal heart sections of control and PKP2-cKO mice treated with Formulation Buffer (FB) and PKP2-cKO mice treated with AAVrh.74-PKP2a, 28 days before Tamoxifen injection. Bottom panel; High contrast mask of the same sections emphasizing collagen deposition in blue. Scale bar = 1 mm for all images. Hearts were extracted 28 days post-Tamoxifen injection. (B & C) Quantification of the percentage of left ventricular fibrosis (B) and right ventricular fibrosis (C) in hearts of the four different groups. Data are presented as Mean ± SD. Black bars, control mice treated with FB; red bars, PKP2-cKO mice treated with FB; blue bars, PKP2-cKO mice treated with AAVrh.74-PKP2a 3 × 1013 vg/kg; purple bars, PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 1013 vg/kg. Data distribution passed a test for normality (Shapiro-Wilk and Kolmogorov-Smirnov tests). Assessment of statistical significance was performed using One-way ANOVA followed by Tukey’s post-hoc analyses. Considering the small number of samples per group, we also assessed significance via a non-parametric test (Kruskal-Wallis test, followed by Dunn’s post-hoc analysis). Data are presented in Supplemental Table I. FB: Formulation Buffer.

The data presented in the previous figures demonstrated that AAVrh.74-PKP2a treatment prior to TAM-mediated knockout of PKP2 could mitigate the development of the cardiomyopathic phenotype in the PKP2-cKO mice. To evaluate whether AAVrh.74-PKP2a could arrest the progression of the ARVC phenotype when delivered after tamoxifen mediated disease induction, we studied PKP2-cKO mice injected with AAVrh.74-PKP2a 7 or 14 days after TAM injection (Supplemental Figure IIB) at doses of 6 × 1013 vg/kg or 2 × 1014 vg/kg. In addition to echocardiography analyses, the mice were followed for long-term survival. As illustrated by the Kaplan Meier curve in Figure 5, PKP2-cKO animals injected with FB only, died between 30 and 50 days after TAM injection, consistent with previous reports.5 In contrast, all but one of the mice injected with AAVrh.74-PKP2a survived for 5 months (155 days post-TAM), at which time the animals were euthanized to examine protein expression and cardiac structure. Furthermore, as shown by the representative images and cumulative data in Figures 6A and 6B, trichrome staining analyses revealed that the percent of the free wall of the LV and of the RV occupied by collagen in animals injected with the higher dose of AAVrh.74-PKP2a (2×1014 vg/kg, orange bar) was similar (though trending toward higher values) when compared to that observed in control animals injected with FB (black bar). The abundance of collagen in hearts from mice that received the lower dose of AAVrh.74-PKP2a (6×1013 vg/kg, purple bar) was higher than in control animals. Importantly, a comparison to collagen abundance in PKP2cKO animals at the same time point was not possible because of the early lethality in that group. However, the abundance of collagen in the AAVrh.74-PKP2a treated animals was clearly less than what was observed in PKP2-cKO mice treated with FB at 28 days post-TAM (as presented by the red dotted line in Fig. 6B) as well as previously published data on fibrosis in this model.5 Finally, cumulative data obtained from echocardiographic analysis of hearts from mice injected with AAVrh.74-PKP2a 7 days or 14 days after TAM are presented in Figure 6C. Hearts from FB-injected PKP2-cKO mice presented a drastic reduction in LVEF (left in Fig. 6C) and an increase in RV area (right in 6C) 28 days after TAM injection (compare red bar to black bar in Fig. 6C), consistent with previous results.5 These changes were mitigated by AAVrh.74-PKP2a, even when injected 14 days after TAM, and the beneficial effects persisted up to 5 months after TAM injection, the longest time point evaluated.

Figure 5.

Figure 5.

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Survival following AAVrh.74-PKP2a injection in PKP2-cKO mice at 7- or 14-days post-Tamoxifen. Kaplan Meier curve depicting the long-term survival of PKP2-cKO mice following AAVrh.74-PKP2a (6 × 1013 vg/kg, 7 days post-Tamoxifen or 2 × 1014 vg/kg, 14 days post-Tamoxifen) administration compared to control and PKP2-cKO mice injected with Formulation Buffer (FB).

Figure 6.

Figure 6.

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Disease progression in PKP2-cKO mice upon AAVrh.74-PKP2a treatment at 7- or 14-days post-Tamoxifen. (A) Upper panel; representative images of Masson’s trichrome staining of longitudinal heart sections of control mice treated with Formulation Buffer (FB) and PKP2-cKO mice treated with AAVrh.74-PKP2a, 2 × 1014 vg/kg 14 days post-Tamoxifen. Hearts were extracted 5 months post-Tamoxifen injection. Bottom panel; High contrast mask of the same sections emphasizing collagen deposition in blue. Scale bar = 1 mm for all images. (B) Quantification of the percentage of left ventricular fibrosis (left panel) and right ventricular fibrosis (right panel) by Masson’s trichrome staining of longitudinal heart sections in control mice treated with FB and PKP2-cKO mice treated with AAVrh.74-PKP2a, 7 days (6 × 1013 vg/kg) or 14 days (2 × 1014 vg/kg) post-Tamoxifen. Hearts were extracted 5 months post-Tamoxifen. Red dashed line indicates mean value of percent fibrosis recorded in PKP2-cKO mice 28 days post-Tamoxifen, injected with FB (as reported in Figure 4). Data passed the Shapiro-Wilk and Kolmogorov-Smirnov tests for normal distribution. Statistical significance was evaluated using One-way ANOVA followed by Tukey’s post-hoc analyses. (C) Quantification of left ventricular ejection fraction (LVEF; left panel) and right ventricular area (RV area; right panel) across time in PKP2-cKO mice treated with AAVrh.74-PKP2a, 7 or 14 days post-Tamoxifen with the dose indicated at the bottom of the bars. Echocardiography was performed at 28 days and at 5 months post-Tamoxifen. Echocardiography for control and PKP2-cKO mice treated with FB was performed at 28 days post-Tamoxifen only. Data are presented as Mean ± SD. Black bars; control mice treated with FB; red bars; PKP2-cKO mice treated with FB, purple bars; PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 1013 vg/kg, orange bars; PKP2-cKO mice treated with AAVrh.74-PKP2a 2 × 1014 vg/kg. Data from the treated groups were compared to PKP2cKO-FB. Data on Left Ventricular Ejection Fraction (left side of Panel C) passed the Shapiro-Wilk and Kolmogorov-Smirnov tests for normal distribution. Significance was evaluated using One-way ANOVA followed by Tukey’s post-hoc analysis. Data on Right Ventricular Area (right side of Panel C) did not pass the normality test. Significance was evaluated by Kruskal-Wallis followed by Dunn’s post-hoc analyses. FB: Formulation Buffer.

Previous studies have documented that a bolus injection of isoproterenol (ISO) 3 mg/kg leads to premature ventricular contractions in anesthetized PKP2-cKO mice 21 days after TAM injection.5,6 Therefore, we used the ISO challenge protocol to determine whether AAVrh.74-PKP2a delivered 14 days after TAM can mitigate arrhythmia burden in PKP2-cKO animals. Representative ECG traces are shown in Figure 7A. FB-injected PKP2-cKO animals presented multiple premature ventricular contractions (PVCs), and 6 out of 10 animals showed more than 100 PVCs within the 30 minutes of recording after ISO injection (Fig.7B) with a total count of ~300 PVCs on average (Fig.7C). In contrast, both parameters of arrhythmia burden were drastically reduced by administration of AAVrh.74-PKP2a at both doses tested (6 × 1013 vg/kg and 2 × 1014 vg/kg purple and orange bars, respectively, in Figure 7B&C). Overall, our data show that gene therapy is a viable way of limiting the impact of loss of the native PKP2 gene on cardiac mechanical and electrical functions, with no overt toxicity at clinically applicable doses.

Figure 7.

Figure 7.

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Isoproterenol-induced arrhythmias in PKP2-cKO hearts treated with AAVrh.74-PKP2a. (A) Representative electrocardiogram (ECG) traces from PKP2-cKO mice treated with (a) Formulation buffer (FB) and (b) AAVrh.74-PKP2a 6 × 1013 vg/kg, 14 days post-Tamoxifen injection. B) Percentage of mice that presented with >100 premature ventricular contractions (PVCs) after isoproterenol (ISO). C) Number of ISO-induced ectopic beats in PKP2-cKO mice treated with FB or AAVrh.74-PKP2a. The black-dashed line represents the cut-off value of 100 PVCs. Data in B & C was quantified over a period of 30 minutes after ISO injection and ECGs were recorded 21 days post-Tamoxifen. Data presented as Mean ± SD. Red bars, PKP2-cKO mice treated with FB; purple bars, PKP2-cKO mice treated with AAVrh.74-PKP2a 6 × 1013 vg/kg; orange bars, PKP2-cKO mice treated with AAVrh.74-PKP2a 2 × 1014 vg/kg. Data did not pass the normality test. Determination of statistical significance was performed using the Kruskal Wallis test, followed by Dunn’s post-hoc analysis. PVC: Premature ventricular contraction, FB: Formulation Buffer.

Discussion

The present study provides experimental evidence indicating that delivery of the human PKP2 gene into cardiomyocytes following administration of an AAVrh.74-PKP2a vector by a single intravenous injection, can arrest the progression of an otherwise lethal arrhythmogenic cardiomyopathy of right ventricular predominance caused by loss of expression of the native PKP2 gene. Our results demonstrate that PKP2 gene therapy can drastically improve clinical outcomes in an animal model of ARVC. The results enable the potential for a cautious pursuit of studies that can determine the utility of PKP2-based gene therapy for human patients affected with ARVC consequent to PKP2 deficiency.

Early evidence that exogenous genes can be introduced into cardiac cells via viral particles with the purpose of affecting cardiac electrophysiology was provided by Leor and colleagues7 and later, by Donahue et al.8 Those attempts highlighted both the potential of the methodological principle and the hurdles associated with it. Bongianino et al. were first to demonstrate that AAV-mediated gene delivery can restore function in an animal model of an inheritable arrhythmia disease,9 a demonstration later expanded upon by Liu et al.10 These two proof-of-principle studies focused on very rare non-RyR2 mutations causative of Catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently though, the group of Ackerman has implemented a dual method to repress expression of an endogenous -mutated- gene while expressing a gene with a native coding sequence.11,12 Their experiments, using cellular models of long QT syndrome have been successful, and provide a solid and encouraging framework for future implementation of this approach in the patient population. Of note, this method is a major breakthrough for the potential of gene therapy under conditions in which the mutated protein acts as a dominant negative component that disrupts function. Clinically-relevant conditions resulting from strict loss-of-function mutations would not require repression of the native -mutated- gene for the replacement therapy approach to be successful. Any potentially translated protein fragment produced from a truncated variant of PKP2 is very likely to be degraded and therefore unable to localize and/or remain in the intercalated disc,13 such that there is low likelihood of negative competition against the therapeutic protein.

Strong support for the hypothesis that AAV-mediated gene therapy may confer benefit in cardiomyopathy can be found in translational studies for Danon disease, an X-linked autophagic vacuolar myopathy, in which LAMP2B gene transfer has been found to improve metabolic and physiologic function in a preclinical animal model14 and has subsequently been found in clinical trials to result in confirmed transgene protein expression in the heart with improvements in key cardiac biomarker and functional measures in this patient population.15

In the adult mammalian heart, PKP2 is densely localized in the intercalated disc, where it integrates into structural complexes necessary for intercellular adhesion (desmosomes and area composita).16–18 Our data show that this precise localization is retained for the AAV-mediated expression of the PKP2 protein, thus indicating that trafficking mechanisms necessary for adequate localization of PKP2 to its functional site remain available for the exogenous protein. Although detailed future studies will be necessary, it is important to note that we did not observe diffuse or erroneous cellular localization of PKP2 in AAVrh74-injected PKP2-cKO mouse myocytes. The latter suggests that, at least at the doses injected, the cellular machinery was not overwhelmed by the presence of the exogenous gene and/or associated therapeutic hPKP2a protein expression.

Western blot analysis revealed the persistence of a light PKP2-immunoreactive band in heart lysates obtained from PKP2-cKO mice. This result, consistent with data previously published,5 is likely consequent to the fact that PKP2 is also expressed in non-myocyte cells in the heart such as in epicardial cells19. In accordance with the latter, we did not detect PKP2 in cardiac myocytes in histological sections of hearts from PKP2-cKO mice injected with formulation buffer alone, though PKP2 expression was observed in the AAV-injected animals (our Figure 2).

The murine model utilized in the present study has allowed us to learn about the importance of PKP2 in the heart, and its role in maintaining structural and functional homeostasis. Yet, we readily acknowledge that this is an imperfect model of disease. In fact, one could argue that none of the murine or cell-based models of desmosomal deficiency accurately recapitulate all aspects of ARVC. All of these models are, however, useful as entry points in the exploration of disease mechanisms and for exploration of potential therapeutic strategies. Of importance is the absence of native protein in the PKP2-cKO mice versus the clinical setting in which patients often harbor a single-allele mutation of PKP2. This raises the question of whether adding copies of the full-length gene will overcome the functional deficiency associated with the presence of a pathogenic PKP2 variant in the genome of the patient. Though a firm answer will have to await an actual clinical trial, studies in experimental models provide a reason for optimism. Indeed, there is no evidence indicating that PKP2 mutant proteins can exert dominant negative functions, and murine models in which a PKP2 mutation has been knocked-in strongly suggest that the resulting phenotype is consequent to the lack of the normal allele, and not to a dominant effect of the mutation per se.13,20 Additionally, a majority of mutations underlying the clinical disorder are truncating variants21,22, unlikely to generate deleterious aberrant proteins. Overall, the data suggest that PKP2-related ARVC is a loss-of-function disease and exogenous introduction of the full-length gene would have the potential to address the loss of function. Importantly, AAV-mediated delivery of the PKP2 gene in a normal murine heart did not lead to a pathogenic phenotype,23 thus addressing concerns about whether excessive expression of PKP2 may have adverse consequences in the treated patient population.

An additional limitation in our study is the fact that we only modify PKP2 expression in cardiac myocytes and yet, in humans, the PKP2 mutation would be present in all PKP2-expressing cells. Importantly, in humans, the heart is the only affected organ. It is theoretically possible that non-myocyte cardiac-resident (or cardiac-interacting) cells may play a role in ARVC pathogenesis. Nonetheless, animal models of desmosomal deficiency have not provided strong evidence for the potential of other cell types to generate the full cardiomyopathic and arrhythmogenic phenotypes, and studies on human induced pluripotent stem cell-derived epicardial cells24 have not been confirmed in disease models involving fully differentiated adult hearts. Furthermore, even the inflammatory component of the disease, originally thought of as consequence of the interaction with non-myocyte cells, has been shown to originate, at least in part, in the myocytes themselves.25,26 Overall, the data indicate that PKP2 deficiency in fully-differentiated cardiomyocytes is a necessary condition for the disease phenotype.

All limitations notwithstanding, our results support the notion that gene therapy may be effective to treat PKP2-related ARVC in appropriately selected patients. It is likely that a future clinical trial will exclude patients with antibodies against the AAV serotype used to deliver the gene.27 In the present study we used an adeno-associated virus vector of serotype rh.74 (AAVrh.74). This vector, originally isolated from rhesus monkeys, effectively transduces myocytes.28 Recent results (albeit preliminary in the context of all populations) suggest that pre-existing antibodies to AAVrh.74 may be present at a relatively low prevalence in the human population.29 More data will be necessary to determine if the use of AAVrh.74 increases the pool of candidates for gene therapy, based on serotype and immunogenic potential. Yet, the successful results with AAVrh.74 in the present preclinical study support its implementation in a future clinical trial.

We observed a functional dose-dependence, whereby 3 × 1013 vg/kg of AAVrh.74 PKP2a restored protein expression, but full functional restoration required a higher dose. It is possible that the protein synthesis and trafficking of the exogenous PKP2 is less effective versus that of the native gene and that, although a lower dose may yield an identifiable Western Blot band and immunofluorescence-detected spots at the intercalated disc, protein production may still be below the level of critical abundance necessary to completely restore function. Notably, the identified effective doses are consistent with those that can be clinically administered. Furthermore, the dose required to mitigate the disease phenotype in this homozygous mouse model may require transgene protein levels exceeding those required to ameliorate the disorder arising from mono-allelic mutations. Based on the data from this study and additional formal safety/toxicology studies, it is generally anticipated that clinically relevant doses for AAVrh74-PKP2a, will be in the high 1013 vg/kg or low 1014 vg/kg range.

ARVC is a disease with incomplete penetrance.2 While significant progress has been made in risk stratification of patients suspect of ARVC,30–32 it remains unpredictable whether or when the disease will present in an asymptomatic gene carrier.2 As such, any future implementation of gene therapy in PKP2 pathogenic variant carriers will likely not be in an asymptomatic individual as a preventive measure, but as a therapeutic procedure for a patient with an overt disease. In that regard, we note that gene replacement in our model was effective even when the AAVrh.74-PKP2a was injected 14 days post-TAM, a time point at which no PKP2 can be detected in the cardiomyocytes and an increase in RV area can already be detected by echocardiography (see5). It is also important to highlight that reintroduction of the PKP2 gene arrested the progression of the cardiomyopathy but did not revert the functional damage. This is consistent with the notion that gene therapy in a cardiomyopathy associated with loss of muscle mass (such as ARVC) would not be expected to restore the muscle mass but only to prevent further loss as the disease progresses.

Recent studies have highlighted the importance of an inflammatory and innate immune response in PKP2-deficient hearts and myocytes.25,33 We speculate that as the AAVrh.74-mediated delivery of PKP2 arrests disease progression, it may also arrest the inflammatory process, but, additional experiments will be necessary to address this important question.

The present study focused on PKP2, the desmosomal gene most commonly associated with a gene-positive arrhythmogenic cardiomyopathy of right ventricular predominance. Recent studies suggest that the disease process consequent to variants in other desmosomal genes may follow a course different from that of PKP2, making it attractive to move to a nomenclature system in which the affected gene is represented in the name of the disease (e.g., a desmoplakin cardiomyopathy34). Along those lines and to emphasize the heavy arrhythmogenic component that is often present in patients with PKP2 pathogenic variants, we propose to use the term plakophilin-2 arrhythmogenic cardiomyopathy (PKP2-ACM) as an entity of its own, one that, among the desmosomal cardiomyopathies, offers a feasible target for gene therapy.

In conclusion, we have conducted proof-of-principle preclinical studies to determine the potential for gene therapy in the setting of an arrhythmogenic cardiomyopathy associated with PKP2 loss of function in the murine heart. Our results indicate that a single intravenous injection of AAVrh.74-PKP2a delivering the PKP2 gene leads to the production and proper localization of PKP2 in adult cardiac myocytes, arrests the progression of the disease, and drastically shifts survival, from a condition leading to 100% lethality to 100% survival in the treated population. Our data represent one component of the necessary body of evidence to support implementation of gene therapy in a carefully selected fraction of patients. While the treatment was well tolerated and effective in the experimental model, data from extensive preclinical safety studies are also needed to ensure safe and successful implementation in the treatment of patients in need.

Supplementary Material

004305 - Supplemental Material

Sources of Funding:

Partly supported through a sponsored research agreement between Rocket Pharmaceuticals, Inc. and NYU Grossman School of Medicine.

Nonstandard Abbreviations and Acronyms

AAV

adeno-associated virus vector

AAVrh.74

adeno-associated virus vector of serotype rh.74

ACM

arrhythmogenic cardiomyopathy

ARVC

arrhythmogenic right ventricular cardiomyopathy

CPVT

catecholaminergic polymorphic ventricular tachycardia

FB

formulation buffer

hTnT

human Troponin T

ISO

isoproterenol

LV

left ventricle

PKP2

plakophilin-2

PKP2a

plakophilin 2 transcript variant A

PKP2-ACM

plakophilin-2 arrhythmogenic cardiomyopathy

PKP2-cKO

cardiac-specific, tamoxifen-activated deletion of PKP2

PVC

premature ventricular contraction

RV

right ventricle

SD

standard deviation

TAM

tamoxifen

Footnotes

Disclosures: There are no past, present or future royalty obligations for MD, MC, GC, MZ and CJMvO. BN, CBS, DR, KMS, VM, EF, PY, JS and CDH are employees of Rocket Pharmaceuticals, Inc and as such receive a salary and stock options.

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Supplementary Materials

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