Expression of full-length dystrophin reverses muscular dystrophy defects in young and old mdx4cv mice

Abstract

Gene replacement therapies mediated by adeno-associated viral (AAV) vectors represent a promising approach for treating genetic diseases. However, their modest packaging capacity (~4.7 kb) remains an important constraint and significantly limits their application for genetic disorders involving large genes. A prominent example is Duchenne muscular dystrophy (DMD), whose protein product dystrophin is generated from a 11.2 kb segment of the DMD mRNA. Here, we explored methods that enable efficient expression of full-length dystrophin via triple AAV codelivery. This method exploits the protein trans-splicing mechanism mediated by split inteins. We identified a combination of efficient and specific split intein pairs that enabled the reconstitution of full-length dystrophin from 3 dystrophin fragments. We show that systemic delivery of low doses of the myotropic AAVMYO1 in mdx4cv mice led to efficient expression of full-length dystrophin in the hind limb, diaphragm, and heart muscles. Notably, muscle morphology and physiology were significantly improved in triple-AAV-treated mdx4cv mice versus saline-treated controls. This method shows the feasibility of expressing large proteins from several fragments that were delivered using low doses of myotropic AAV vectors. It can be adapted to other large genes involved in disorders for which gene replacement remains challenged by the modest AAV cargo capacity.

Keywords: Gene therapy; Genetic diseases; Genetics; Muscle biology; Skeletal muscle; Therapeutics.

Figure 1. Efficient expression of full-length dystrophin in young mdx^4cv mice.

(A) Cartoon depicting the triple AAV approach to express full-length dystrophin via PTS mediated by 2 split inteins. (B) Dystrophin structure with the split site used to insert split inteins. (C) Western blots showing full-length dystrophin expression in TA, diaphragm, and heart muscle lysates collected from WT mice and young mdx^Acv mice treated with saline- or triple-AAV (3x AAV). (D) Dystrophin protein expression normalized to GAPDH and expressed as a percentage versus WT levels. n = 5–6 samples per group. Data represent the mean ± SEM. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with Tukey’s post hoc test. Low, low dose (4 × 10¹³ vg/kg); Med, medium dose (8 × 10¹³ vg/kg).

Figure 2. Improvement of TA muscle histology with triple AAV delivery.

(A) TA cross-sections stained with H&E or Trichrome (top row, scale bars: 100 μm) or immunolabeled for dystrophin (N-terminal antibody), β-dystroglycan, γ-sarcoglycan, or laminin (lower panels, scale bars: 100 μm). Originally captured in RGB colors, image sections were inverted to white color to better visualize the staining. The original panel is presented in Supplemental Figure 1. (B) Percentage of dystrophin⁺ (Dys⁺) fibers. n = 5 samples per group; n = 600–1,000 myofibers counted per sample. (C) Percentage of myofibers with central nuclei. n = 5–6 samples per group; n = 400–700 myofibers analyzed per sample. (D) Myofiber area and (E) minimal Feret’s diameter measured on TA cross-sections. n = 5 samples per group; n >700 myofibers analyzed per sample. The average values are shown on top of the violin bars. The solid line represents the median, while the dashed lines show the quartiles. (F) The collagen area of TA muscle sections was measured using trichrome-stained cross-sections (no significance was found between the groups). n = 5 samples per group. ***P < 0.001 versus WT; ^$$$P < 0.001 versus the saline-treated group; ^###P < 0.001 versus the low-dose-treated group. The mean of each group was compared by 1-way ANOVA with Tukey’s post hoc test. Data represent the mean ± SEM.

Figure 3. Broad biodistribution of dystrophin expression detected in diaphragm and heart.

(A) Diaphragm and heart cross-sections stained with H&E or trichrome (scale bars: 100 μm), or immunolabeled for dystrophin (N-terminal antibody) or laminin (scale bars: 100 μm for diaphragms, 50 μm for hearts). The original panel (green and red colors) is presented in Supplemental Figure 2. (B) Percentage of dystrophin⁺ fibers quantified on diaphragm sections. n = 5 samples per group; n = 600–1,000 myofibers counted per sample. (C) Percentage of dystrophin⁺ cardiomyocytes. n = 5 samples per group; n = 600–1,000 cells counted per sample. (D) Myofiber area and (E) minimal Feret’s diameter measured on diaphragm cross-sections. n = 5 samples per group; n >300 myofibers analyzed per sample. The average values are shown on top of the violin bars. The solid line represents the median, and the dashed lines show the quartiles. (F) The collagen area of the diaphragm samples was measured using cross-sections stained with trichrome. ***P < 0.001 versus WT; ^$P < 0.05 and ^$$$P < 0.001 versus the saline group; ^#P < 0.0 and ^###P < 0.001 versus the low-dose group. The mean of each group was compared by 1-way ANOVA with Tukey’s post hoc test. Data represent the mean ± SEM.

Figure 4. Improvement of TA and diaphragm contractile properties.

(A) TA muscle–specific force development measured in situ following sciatic nerve stimulation. n = 6–7 mice per group. (B) Muscle force drop measured following a mechanical injury induced by 20% stretching beyond the optimal L0. n = 6–7 mice per group. (C) Specific force development measured in vitro using diaphragm strips from mdx^4cv mice treated with saline or triple AAVMYO1 or from age-matched WT mice. n = 5–7 mice per group. Data represent the mean ± SEM. *P < 0.05 and ***P < 0.001 versus WT; ^$P < 0.05, ^$$P < 0.01, and ^$$$P < 0.001 versus the saline-treated group. One-way ANOVA with Tukey’s post hoc test.

Figure 5. Expression of full-length dystrophin restores TA muscle defects in old and severely affected mdx^4cv mice.

(A) Diaphragm specific force recorded in vitro using isolated muscle trips. n = 5–6 mice per group. (B) Maximal specific force developed by TA muscles at L0 following sciatic nerve stimulation. n = 5–7 mice per group. (C) Muscle force measured after eccentric contraction injury induced by 15% lengthening beyond L0. n = 5–7 mice per group. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Tukey’s post hoc test. Data represent the mean ± SEM. (D) Western blots showing full-length dystrophin expression in TA muscle lysates collected from WT mice and old mdx^4cv mice treated with saline- or triple AAV, but lower levels were detected in diaphragm samples of the same animals.

Figure 6. Partial correction of TA muscle histology with triple AAV treatment.

(A) General muscle morphology of TA and diaphragm muscle cross-sections stained with H&E or trichrome (right columns, scale bars: 100 μm). Dystrophin expression is shown across muscle sections immunolabeled with antibodies against dystrophin or laminin (left columns, scale bars: 100 μm). The original staining (green and red) is displayed in Supplemental Figure 3. (B) Percentage of dystrophin⁺ fibers quantified on TA sections. n = 5–6 samples per group; n = ~2,000 myofibers counted per sample. (C) Myofiber area and (D) minimal Feret’s diameter determined from TA cross-sections. n = 5 samples per group; n = 1,000 myofibers analyzed per sample. The average values are shown on top of the violin bars. The solid line represents the median, and the dashed lines show the quartiles. (E) Percentage of centrally nucleated myofibers quantified using TA sections stained with H&E. (F) The fibrosis area was measured using TA cross-sections stained with trichrome. **P < 0.01 and ***P < 0.001 versus WT; ^$$$P < 0.001 versus the saline group, by 1-way ANOVA with Tukey’s post hoc test. Data represent the mean ± SEM.

Figure 7. High levels of full-length dystrophin rescue mdx^4cvcardiac defects at the late stage of the disease.

(A) Rate pressure product (RPP) at baseline (BL) or at different time points under high-workload (HWL) conditions. (B) Increase of +dP/dT_(max) during high-workload challenge compared with baseline. Data in A and B represent the mean ± SEM. (C) Protein expression in heart tissue analyzed by Western blotting. (D) General morphology of the heart tissue assessed with H&E and trichrome staining (top rows, scale bars: 200 μm). Dystrophin expression and distribution were determined by immunolabeling using anti-dystrophin and anti-laminin antibodies (bottom rows, scale bar: 50 μm). (E) The percentage of cardiomyocytes expressing dystrophin was determined by dystrophin immunostaining. (F) The collagen percentage present in heart sections was quantified using trichrome staining. *P < 0.05, **P < 0.01, and ***P < 0.001 versus WT; ^$$$P < 0.001 versus the saline-treated group. One-way ANOVA with Tukey’s post hoc test. Data represent the mean ± SEM.