Therapeutic efficacy of AAV-mediated restoration of PKP2 in arrhythmogenic cardiomyopathy

Figures

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  Fig. 1. AAV-mediated restoration of PKP2 in PKP2c.2013delC/WT mutant iPS-cell-derived CMs results in molecular rescue of desmosomal assembly. a, Graphical representation of the AAV expression cassette used in iPS-cell-derived CMs. ITR, Inverted Terminal Repeat Sequences. b, Timeline for AAV6–PKP2 transduction of iPS-cell-derived CMs. c, Representative immunoblots showing v.l.-dependent PKP2 protein levels in PKP2c.2013delC/WT mutant cells in response to AAV6-PKP2. v.l.1 = 0.5 × 103 v.g. per cell, v.l.2 = 5 × 103 v.g. per cell. Vinculin (VIN) was used as a loading control. d, Quantification of c, n = 4 technical replicates per condition. e, Representative immunoblots for PKP2, JUP, DSP, DSG2 and DSC2 in corrected and mutant iPS-cell-derived CMs upon transduction with AAV6-ctr or AAV6–PKP2. VIN was used as a loading control. f, Quantification of PKP2 protein levels. g, Quantification of JUP protein levels. h, Quantification of DSP protein levels. i, Quantification of DSC2 protein levels. j, Quantification of DSG2 protein levels. Colored dots represent distinct CM differentiation, n = 6 technical replicates and 3 biological replicates per condition. Data is presented as mean values ± s.e.m.…

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  Fig. 2. AAV-mediated restoration of PKP2 in PKP2c.2013delC/WT mutant iPS-cell-derived CMs improves sodium conduction. a, Graphical representation of the experimental workflow. b–e, Representative voltage-gated sodium currents activated by voltage steps to between −80 mV and −20 mV from a holding potential of −110 mV. Experimental groups: PKP2WT/WT + AAV6-ctr shown in b, PKP2WT/WT + AAV6-PKP2 shown in c, PKP2c.2013delC/WT + AAV6-ctr shown in d, PKP2c.2013delC/WT + AAV6-PKP2 shown in e. f, Graph showing the peak transient INa density for each experimental group. Colored dots represent distinct CM differentiation. PKP2WT/WT + AAV6-ctr: n = 39 single CMs (diff.1) + 48 single CMs (diff.2), PKP2WT/WT + AAV6–PKP2: n = 26 single CMs (diff.1) + 44 single CMs (diff.2), PKP2c.2013delC/WT + AAV6-ctr: n = 21 single CMs (diff.1) + 20 single CMs (diff.2), PKP2c.2013delC/W + AAV6–PKP2: n = 23 single CMs (diff.1) + 14 single CMs (diff.2). Data are presented as mean values ± s.e.m. Statistical significance was estimated with one-way ANOVA, Tukey correction with P value at ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and not significant (NS). P values (f): PKP2WT/WT + AAV6-ctr versus…

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  Fig. 3. PKP2c.2013delC/WT mutant EHM exhibit impaired contractile function compared to the isogenic control. a, Schematic overview of the generation of PKP2 EHM. b, Representative immunoblot for the desmosomal proteins PKP2, JUP and DSP. VIN is used as a loading control. c, Representative immunoblot for the desmosomal proteins DSC2 and DSG2. VIN is used a loading control. d, Quantification of PKP2 protein levels. e, Quantification of JUP protein levels. f, Quantification of DSP protein levels. g, Quantification of DSC2 protein levels. h, Quantification of DSG2 protein levels. Colored dots represent distinct CM differentiations, n = 6 technical replicates and 3 biological replicates per condition. Data are presented as mean values ± s.e.m. Statistical significance is derived by biological replicates and determined by unpaired, two-tailed Student’s t-test, P value at ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and not significant (NS). i, Timeline for assessing contractility of PKP2WT/WT and PKP2c.2013delC/WT EHM. j, Trendline showing % pole bending as a measure of force of contraction for PKP2WT/WT and PKP2c.2013delC/WT EHM under baseline conditions at different time points. k,…

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  Fig. 4. AAV-mediated restoration of PKP2 in PKP2c.2013delC/WT EHM leads to an increase in desmosomal and junctional protein levels, which further translates into improved contractility. a, Timeline for the AAV transduction and maturation of PKP2WT/WT and PKP2c.2013delC/WT EHM with either AAV6-ctr or AAV6–PKP2. b, Representative fluorescent image of 6-week-old EHM transduced with either AAV6-ctr (left) or AAV6–PKP2 fused with the mNeongreen fluorescent protein (right). Scale bar, 1 mm. This experiment was repeated independently three times with similar results. c, Representative immunoblots for PKP2, JUP, DSP, DSG2, NCAD and αCAT in 6-week-old PKP2WT/WT and PKP2c.2013delC/WT EHM, transduced either with AAV6-ctr or AAV6–PKP2. VIN was used as a loading control for the desmosomal proteins, whereas αTUB was used for the quantification of NCAD and αCAT. d, Quantification of PKP2 protein levels. e, Quantification of JUP protein levels. f, Quantification of DSP protein levels. g, Quantification of DSG2 protein levels. h, Quantification of NCAD protein levels. i, Quantification of αCAT protein levels. Colored dots represent distinct CM differentiations, n = 6 technical replicates and 3 biological…

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  Fig. 5. AAV-mediated restoration of PKP2 in Pkp2c.1755delA/WT pups and adult mice leads to the recovery of desmosomal and non-desmosomal components of the ID. a, Graphical representation of the AAV expression cassette used in mice, ITR=Inverted Terminal Repeat Sequences. b, Workflow followed after the intraperitoneal (IP) AAV9–PKP2 administration in Pkp2c.1755delA/WT and wild-type pups. c, Immunofluorescence on paraffin sections of mouse cardiac tissue showing localization of the exogenously introduced PKP2 in the injected hearts. DAPI, blue; ACTN2, gray; PKP2, green; MYC epitope, red; exogenous PKP2, yellow in merged image. Scale bar, 10 μm. This experiment was repeated independently 18 times (in 18 distinct mouse hearts) with the same results. d, Representative immunoblots for PKP2, MYC, JUP, DSP and DSG2 in Pkp2WT/WT and Pkp2c.1755delA/WT pups injected with either AAV9-ctr or AAV9–PKP2. VIN is used as a loading control. e, Quantification of PKP2 protein levels. f, Quantification of MYC protein levels. g, Quantification of JUP protein levels. h, Quantification of DSP protein levels. i, Quantification of DSG2 protein levels. Experimental groups: Pkp2WT/WT pups injected with AAV9-ctr n =…

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  Fig. 6. A single dose of AAV9-PKP2 in 8-week-old Pkp2c.1755delA/WT mice prevents cardiac dysfunction at 12 months of age. a, Timeline for intravenous AAV9–PKP2 administration in Pkp2c.1755delA/WT and long-term monitoring of cardiac function in adult mice. b, Immunofluorescence on paraffin sections of mouse cardiac tissue showing localization of the exogenously introduced PKP2 in the injected hearts. DAPI, blue; ACTN2, gray; PKP2, green; MYC epitope, red; exogenous PKP2, yellow in merged image. Scale bar, 10 μm. This experiment was performed independently ten times (in ten distinct mouse hearts) with the same results. c, Representative immunoblots for PKP2, MYC, JUP, DSP and DSG2 in Pkp2WT/WT and Pkp2c.1755delA/WT mice injected with either AAV9-ctr or AAV9–PKP2. VIN is used as a loading control. d, Quantification of PKP2 protein levels. e, Quantification of MYC protein levels. f, Quantification of JUP protein levels. g, Quantification of DSP protein levels. h, Quantification of DSG2 protein levels. Experimental groups: Pkp2WT/WT mice injected with AAV9-ctr n = 8, Pkp2WT/WT mice injected with AAV9–PKP2 n = 8, Pkp2c.1755delA/WT mice injected with AAV9-ctr n = 8, Pkp2c.1755delA/WT mice…

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  Extended Data Fig. 1. AAV-mediated restoration of PKP2 in PKP2c.2013delC/WT mutant iPS-cell-derived CMs does not affect the mRNA levels of desmosomal proteins. a-e, Gene expression of PKP2, JUP, DSP, DSG2 and DSC2 in Pkp2c.2013delC/WT iPS-cell-derived CMs upon transduction with either AAV6-ctr or AAV6-PKP2. As a reference, the PKP2 corrected iPS-cell-derived CM line was transduced with AAV6-ctr. Values were normalized to GUS. n = 3 biological replicates (distinct CM differentiations) and 6 technical replicates per differentiation. Data is presented as mean +/− s.e.m. Statistical significance is derived from biological replicates and is determined with one-way ANOVA (Tukey’s post-hoc test), p-value at ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and not significant (ns). Exact P-value for panel (a) is < 0.0001 for all comparisons. Source data

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  Extended Data Fig. 2. PKP2 restoration in iPS-cell-derived CMs harboring the pathogenic variant PKP2 c.1845C < T, leads to a recovery of JUP and DSP protein levels. a, Representative immunoblots for PKP2, JUP and DSP in PKP2c.1845C<T/WT iPS-cell CMs upon transduction with AAV6-ctr or AAV6-PKP2. VIN was used as a loading control. b-d, Quantification of (a), n = 3 biological replicates (distinct differentiations) and 6, 3 and 3 technical replicates per differentiation respectively. Data is presented as mean +/− s.e.m. Statistical significance is derived from biological replicates and is determined with one-way ANOVA (Tukey’s post-hoc test) with p-value at ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and not significant (ns). P-values (PKP2c.1845C<T/WT+ AAV6-ctr vs PKP2c.1845C<T/WT+ AAV6-PKP2): (b) = 0.016, (c) = 0.0366, (d) = 0.0016. Source data

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  Extended Data Fig. 3. The PKP2 mutant EHM exhibit similar structural and intercellular junction organization as its isogenic control. a, Representative brightfield images of 6-week-old mutant and isogenic control EHM under baseline. Scale bar is at 1mm. b, On the left side: representative overview immunofluorescent staining of a paraffin-embedded EHM section. Scale bar is at 500μm. On the right side: representative high magnification fluorescent images of the same EHM section to show cardiomyocyte and fibroblast regions within the tissue. DAPI = blue, VIM = magenta, ACTN2 = green. Scale bar is at 20μm. c, Gene expression of ACTN2, VIM, DCN and NPPB on the mutant tissues compared to the isogenic control. Data is normalized to the housekeeping gene GUS. Colored dots represent distinct CM differentiations, n = 6 technical replicates and 3 biological replicates per condition. Data is presented as means +/− s.e.m. Statistical significance is derived from biological replicates and is determined by unpaired, two-tailed Student t-test, p-value at *****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and not significant (ns). ACTN2: P-value = 0.0077, VIM: P-value < 0.0001, DCN: P-value = 0.1033, NPPB: P-value…

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  Extended Data Fig. 4. PKP2c.2013delC/WT EHM do not exhibit an altered beating frequency, contraction velocity and relaxation velocity compared to control. a-c, Trendlines showing beating frequency (Hz), contraction velocity and relaxation velocity in PKP2 WT/WT and PKP2c.2013delC/WT EHM at different timepoints after transduction with either AAV6-PKP2 or AAV6-ct, n = 3 biological replicates (three distinct EHM batches). d-f, Graphs displaying beating frequency (Hz), contraction velocity and relaxation velocity in PKP2 WT/WT and PKP2c.2013delC/WT EHM after transduction with either AAV6-PKP2 or AAV6-ctr, on day 42 of maturation, n = 3 biological replicates (three distinct EHM batches), batch 1 = 17 isogenic control and 19 mutant tissues, batch 2 = 32 isogenic control and 27 mutant tissues and batch 3 = 11 isogenic control and 30 mutant tissues. Data is presented as means +/− s.e.m. Statistical significance is derived by biological replicates and is determined by unpaired, two-tailed Student t-test p-value at ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and not significant (ns). Source data

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  Extended Data Fig. 5. AAV-PKP2 treatment does not alter desmocollin levels in PKP2c.2013delC/WT EHM. a, Representative immunoblots for DSC2 on PKP2 mutant and isogenic control EHM upon transduction with either AAV-ctr or AAV-PKP2. b, Quantification of (a). Colored dots represent distinct batches of differentiation, n = 4 technical replicates and 3 biological replicates. Data is presented as means +/− s.e.m. Statistical significance is derived by biological replicates and determined by one-way ANOVA (Tukey’s post-hoc test) with p-value at ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, and not significant (ns). P-value = 0.0821. Source data

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  Extended Data Fig. 6. AAV9 transduces cardiomyocytes in vivo with high efficiency and with minor ectopic expression. a, Immunofluorescence on cardiac sections of mice transduced with AAV9-tdTOM to facilitate transduction efficiency assessment. Nuclei = DAPI, ACTN2 = green, tdTOM = magenta. Scale bar is at 20μm. b, Quantification of (a), n = 5 hearts, 10 regions per heart were assessed. c-d, Immunoblot for MYC and PKP2 on protein lysates of different mouse tissues. VIN was used as a loading control. e, Quantification of (d), n = 5 mice per condition. Data is presented as means +/− s.e.m. Source data