Research ArticleGene Therapy

Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A

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Science Translational Medicine  27 Nov 2019:
Vol. 11, Issue 520, eaat6072
DOI: 10.1126/scitranslmed.aat6072
  • Fig. 1 Molecular and histological validation of the dKO mouse model.

    (A) Diagram of the genetic modification present in the C3 KO and Blaj mice. (B) mRNA expression of Calpain 3 and Dysferlin in mice according to their genotype. The measures were performed on gluteus, gastrocnemius, and soleus muscle. Statistical analyses were performed using nonparametric Kruskal-Wallis test and the post hoc multiple comparisons Dunn’s test (n = 4 to 6). *P < 0.05; ***P < 0.001. (C) Western blot of calpain 3 and dysferlin in skeletal muscle extracts according to the mouse genotype. (D) Histological sections of skeletal muscles (psoas, quadriceps, and TA) from each genotype. Scale bars, 50 μm. BLAJ, B6.A-Dysfprmd/J; BDC HOHE, Dysf−/−Capn3+/−−; BDC HOHO, Dysf−/−Capn3−/−.

  • Fig. 2 Evaluating calpain 3 gene transfer in the Dysf−/−Capn3−/− dKO model.

    (A) The expression cassette used in this study includes a human desmin promoter, a chimeric β-globin intron, the mfa calpain 3 coding sequence fused with a V5 tag, and a simian SV40 polyadenylation signal. In addition, a tandem miR-208a target sequence was included in the 3′UTR of the vector (rAAV-C3+miRT). (B) Schematic representing design of the in vivo experiment (n = 4 to 7). IM, intramuscular; D, days. (C) Calpain 3 mRNA expression obtained by ddPCR in skeletal muscle extracts. Statistical analyses were performed using nonparametric Kruskal-Wallis test and the post hoc multiple comparison Dunn’s test (n = 4 to 7). **P < 0.01. (D) Representative gluteus muscle sections stained by hematoxylin phloxine saffron (HPS), Sirius red (SR), myosin heavy chain developmental form (MHCd), and CD11b. Scale bars, 150 μm. (E) Quantitative RT-PCR (qRT-PCR) quantification of genes representative of several dystrophic features: Cd11b for macrophage infiltrates, myogenin for regeneration, and collagen VI (Col6a) for fibrosis in muscles of Blaj, BDC HOHO, and injected BDC HOHO. The data are expressed as abundance normalized by Rplp0. Statistical analyses were performed using nonparametric Kruskal-Wallis test and the post hoc multiple comparisons Dunn’s test (n = 4 to 7) *P < 0.05; **P < 0.01. (F) Radar graph showing recovery score for each dystrophic markers. Values are expressed relative to those of BlaJ mice (set to 100%) and BDC HOHO (0%). Individual data are presented in fig. S1B.

  • Fig. 3 NHP biodistribution study and transgene expression.

    (A) Study design (n = 2 to 3). IV, intravenous injection. (B) Determination of vector copy number (VCN) per diploid genome in a range of tissues after systemic injection of rAAV-C3 or rAAV-C3+miRT sequences, represented by heat maps with a linear color table from red (high) to yellow (low). The values presented in the upper heat map for heart and skeletal muscle correspond to the mean of six different heart sections and the mean of all limb muscles, respectively, whose individual values are indicated in the lower heat maps. Li, liver; Sp, spleen; iLN, inguinal lymph nodes; mLN, mesenteric lymph nodes; pLN, popliteal lymph nodes; Hrt, heart; Ki, kidney; Lu, lung; Sk.mu, skeletal muscle; Te, testis; SpCO, spinal cord; FrBr, frontal brain section; PaBr, parietal brain section; OcBr, occipital brain section. (C) Expression of transgenic calpain 3 in a range of Macaca skeletal muscles (n = 2 to 3). Transgene expression was measured by RT-ddPCR and is presented in copies per microgram of RNA normalized to Rplp0. Temp, temporalis; Bif, biceps femoris; Tri, triceps brachii; Dia, diaphragm; Pso, psoas; Qua, quadriceps; GA, gastrocnemius; Sol, soleus. (D) Detection of transgenic calpain 3 using PLA. Red dots correspond to a specific amplification due to proximity ligation of two antibodies detecting epitopes present in our transgenic protein, specifically CAPN3 and V5 antibodies. Colabeling with γ-sarcoglycan (green) was performed to visualize muscle fibers, and 4′,6-diamidino-2-phenylindole (DAPI) is shown in blue. Inset: PLA negative control. Scale bars, 25 μm. (E) Sections of biceps femoris for all primates stained with HPS. Scale bars, 100 μm.

  • Fig. 4 Cardiac expression and histological analyses after systemic delivery of calpain 3 in NHP.

    (A) Expression of transgenic calpain 3 per microgram of RNA in a range of heart sections measured by RT-ddPCR. Calpain 3 expression was normalized to Rplp0 and compared to the quantity of detected expression in a noninjected primate (total heart lysate). Cam, left atrium from mitral valve to aorta; Cas, central part of both atria including pulmonary valve and aorta; Cat, right atrium from vena cava to tricuspid valve; CX1-3, three sections taken between the apex and the top part of the ventricles. (B) Detection of the calpain 3 transgene using PLA. Red dots correspond to a specific amplification due to proximity ligation of two antibodies detecting epitopes present in our transgenic protein, specifically CAPN3 and V5 antibodies. Colabeling with γ-sarcoglycan was performed to visualize fibers (green). Scale bar, 25 μm. (C) Quantification of PLA. A.U., arbitrary unit. (D) Cardiac sections (CX3) of all injected primates stained by HPS or Sirius red. Scale bars, 100 μm.

  • Fig. 5 CAPN3 binding sites on titin.

    (A) Known binding sites of calpain 3 on the titin protein. Three sites are located in N2A/PEVK region and one in the M-line at the C-terminal part. Position of the primers used for each amplicon is drawn below the corresponding exons. (B) RT-PCR of the CAPN3 binding sites on mouse, primate, and human cardiac and skeletal muscles (exons 101 to 107 and 106 to 112 in the N2A domain, exons 114 to 137 for the domain in the upstream region of the PEVK domain, and exons 362 to 364 in the M-line). The experiments were performed in triplicate. (C) Percent spliced-in index (PSI) between mouse (Mmu), primate (Mfa), and human (Hsa) titin in the PEVK region (exons 114 to 137).

  • Fig. 6 Effect of AAV-mediated expression of calpain 3 in RBM20 KO mice.

    (A) RT-PCR performed on cardiac samples. The amplified regions corresponding to the four known binding sites are shown. (B) Survival curve of CAPN3-injected animals (WT and RBM20 KO) over a 30-day test period (n = 3 to 7). At the last time point (D30), all animals were sacrificed. (C) Transgenic CAPN3 expression in cardiac tissue was quantified by RT-qPCR (copies per microgram of RNA). Data were normalized to Rplp0. rAAV-injected mice (Inj.) versus phosphate-buffered saline (PBS) (NI). Statistical analyses were performed using nonparametric Kruskal-Wallis test and post hoc multiple comparisons Dunn’s test (n = 3 to 4). **P < 0.01. (D) Western blot of calpain 3 in cardiac tissue. Whole calpain 3 is expected at 94 kDa (black arrow), and its cleaved products are expected at ~55 to 65 kDa (bracket) according to the antibody used. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the amount of protein loaded. rAAV-injected mice versus PBS. WT skeletal muscle (TA) was used as the positive control. (E) Heart sections were stained with Sirius red to highlight fibrotic areas. Representative examples of the entire sections as well as ×2 magnification of specific areas are shown. rAAV-injected mice versus PBS. Scale bars, 100 μm. (F) Sirius red quantification. Amount of staining is expressed in percentage of the total tissue section. rAAV-injected mice versus PBS is shown. Statistical analyses were performed using nonparametric Kruskal-Wallis test and post hoc multiple comparisons Dunn’s test (n = 3 to 7). **P < 0.01. (G) Fibronectin was quantified by RT-qPCR. Results were normalized to Rplp0 and compared to a PBS-injected WT condition. rAAV-injected mice (Inj.) versus PBS (NI). Statistical analyses were performed using nonparametric Kruskal-Wallis test and post hoc multiple comparisons Dunn’s test (n = 3 to 5). *P < 0.05 and **P < 0.01.

  • Fig. 7 Effect of AAV-mediated expression of calpain 3 in DelMex5 mice.

    (A) RT-PCR was performed using primers located in exons flanking each known C3 binding site on titin using extracts from heart of DelMex5 mice and skeletal muscle of DelMex5 and WT mice. (B) Survival curve of DelMex5 mice over a 30-day period after CAPN3 injection (n = 3 to 6). All mice were sacrificed at day 30. Control mice for this experiment are shown in Fig. 6B. (C) Expression of CAPN3 mRNA after systemic injection, quantified by RT-qPCR (copies per microgram of RNA), normalized to Rplp0 (rAAV-injected mice versus PBS). Statistical analyses were performed using nonparametric Kruskal-Wallis test and the post hoc multiple comparisons Dunn’s test (n = 3 to 4). *P < 0.05. (D) Western blot of calpain 3 in cardiac tissue. rAAV-injected mice versus PBS. (E) Heart sections were stained with Sirius red to highlight areas of fibrosis. Representative images of entire sections as well as ×2 magnification of one area are shown. rAAV-injected mice versus PBS. Scale bars, 100 μm. (F) Sirius red quantification. The amount of staining is expressed in percentage of the total tissue section. rAAV-injected mice versus PBS. Statistical analyses were performed using nonparametric Kruskal-Wallis test and post hoc multiple comparisons Dunn’s test (n = 3 to 6). *P < 0.05. (G) Fibronectin was quantified by RT-qPCR. Results were normalized to Rplp0 and compared to a PBS-injected WT condition. rAAV-injected mice versus PBS. Statistical analyses were performed using nonparametric Kruskal-Wallis test and post hoc multiple comparisons Dunn’s test (n = 3 to 7). (H) Western blot of calpain 3 in muscle tissue. Whole calpain 3 is expected at 94 kDa (black arrow), and cleavage products are expected at ~55 to 65 kDa (bracket). GAPDH was used to normalize the amount of protein loaded. rAAV-injected mice versus PBS. WT skeletal muscle (TA) was used as a positive control. (I) Stained HPS muscle sections. rAAV-injected mice versus PBS.

  • Table 1 Evaluation of different serum cardiac biomarkers at D−4, D+15, and D+30.

    CK, creatine kinase; AST, aspartate aminotransferase; LD, lactate dehydrogenase; NT-proBNP, N-terminal pro b-type natriuretic peptide. No substantial difference was observed between the pre- and post-injection conditions.

    NumberDayCK (μkat/liter)LDH (μkat/liter)Myoglobin (ng/ml)Troponin T (ng/ml)NT-proBNP (ng/ml)
    PercevalD−47.685.0134.932.34.5
    With mirD1510.669.979.992.36,0
    D3012.895.7921.112.82.9
    MerlinD−411.174.7356.361.20.4
    With mirD1517.048.3299.021.20.7
    D3015.836.3145.271.32.2
    KaradocD−416.628.1635.461.54.8
    With mirD1520.5912.4784.541.85.1
    D3046.5710.03226.71.83.6
    LancelotD−416.465.327.371.54.8
    Without mirD157.036.2671.561.33,0
    D3014.855.2460.221,05,0
    MordredD−410.584.7377.467.84.3
    Without mirD155.677.576.965.32.9
    D3017.966.7857.083.73.8
  • Table 2 Measurements of cardiac miRNA expression.

    Cardiac miRs (miR-208a and miR-499) and control miR (miR-16 and miR-93) were measured in the serum of injected primates. CT values were averaged over two technical replicates. N/A: not available; ND, not detected.


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Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/520/eaat6072/DC1

    Materials and Methods

    Fig. S1. Supplementary data for the dKO experiments.

    Fig. S2. Supplementary data for the primate experiments.

    Fig. S3. Proposed model of calpain 3 activation.

    Table S1. Screening of primates for AAV9 seronegativity.

    Table S2. Antibody response against the AAV9 serotype on sera (preimmune and sacrifice).

    Table S3. Blood parameters of primates.

    Table S4. Summary of echocardiogram analysis before and after systemic injection in primates.

    Data file S1. Individual subject-level data.

    References (4043)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Supplementary data for the dKO experiments.
    • Fig. S2. Supplementary data for the primate experiments.
    • Fig. S3. Proposed model of calpain 3 activation.
    • Table S1. Screening of primates for AAV9 seronegativity.
    • Table S2. Antibody response against the AAV9 serotype on sera (preimmune and sacrifice).
    • Table S3. Blood parameters of primates.
    • Table S4. Summary of echocardiogram analysis before and after systemic injection in primates.
    • References (4043)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual subject-level data.

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