Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV-microdystrophin gene therapy in DMD patients.

Figure 1. Analysis of cMD1 expression by immunostaining and western-blot in muscles of GRMD dogs injected with rAAV2/8-Spc512-cMD1 by the LR route.

(a) Dystrophin immunostaining (NCL-DYSB) on transverse sections of muscle samples. Representative results are presented for healthy (WT) and untreated GRMD dogs and for four different muscles of dog LR1 sampled at the time of killing (non injected and injected forelimb, below and above the tourniquet). Scale bar, 100 μm. (b) Western-blot analysis of total proteins (50 μg) extracted from muscles samples. Representative results for the same muscles as in a are shown. GRMD myoblasts transduced with the rAAV2/8-Spc5.12-cMD1 vector were used as positive control. The blot was stained with MANEX-1011C to reveal the presence of the 427 kDa dystrophin protein (WT dog) and the 138 kDa cMD1 protein, with an anti-GAPDH antibody as a loading control. The level of cMD1-positive fibres detected by immunostaining from the same muscle samples are indicated under each panel.

Figure 2. Improvement of pathological pattern in the cMD1-expressing muscles.

After killing, two muscles (the flexor carpi ulnaris muscle and the extensor carpi radialis muscle) in each forelimb of each GRMD dog injected by the LR route either with the rAAV2/8-Spc5.12-cMD1 vector (n=4 dogs and 16 muscles analysed) or with buffer (n=3 dogs and 12 muscles analysed) were analysed. Myofibre regeneration was evaluated by immunohistochemical staining of myofibres using an antibody specific for developmental myosin heavy chain isoform. Total and endomysial fibrosis were evaluated by immunohistochemical detection of Collagen I. (a) Regeneration and fibrosis immunostaining on transverse sections of muscle samples. Representative results are presented for two muscles of dog LR1 (noninjected forelimb and injected forelimb). The levels of cMD1-positive fibres detected by immunostaining from the same muscle samples are indicated above each panel. Scale bar, 100 μm. (b) Regeneration, total fibrosis and endomysial fibrosis quantification in the total of 28 muscles analysed in the GRMD dogs injected by the LR route, either with the rAAV2/8-Spc5.12-cMD1 vector or with buffer was done using an automatic measurement of the percentage of the labelled area after selection of regions of interest. Analyses were done according to the percentage of cMD1-positive fibres of each muscle: <12% (n=20, empty symbols), between 30 and 45% (n=4, grey full symbols) and between 46 and 90% (n=4, black full symbols). Each point represents the data obtained in one muscle, and the horizontal bars represent the mean of the values obtained for each group *P<0.05 (nonparametric Kruskal–Wallis test with post hoc multiple comparison Dunn’s test).

Figure 3. NMR imaging and spectroscopy analyses of muscles of GRMD dogs injected with rAAV2/8-Spc5.12-cMD1 by the LR route.

(a) Representative example of transverse fat-saturated T2-weighted NMR image of the two forelimbs obtained in dog LR2. At 3 months after injection, signal muscle intensities were decreased and more homogeneous in the injected forelimb (**) compared with the noninjected one. (b) Representative example of ³¹P-NMR spectra of the injected (blue curve) and noninjected forelimb (red curve) of the same dog (LR2). Phosphocreatine (PCr) was increased and inorganic phosphates (Pi) and phosphodiesters (PDE) were decreased relative to ATP in the injected forelimb compared with the noninjected forelimb. (c) NMR imaging fat-saturated (FS) T2w/T1w muscle signal ratio obtained from three different muscles (ECR (extensor carpi radialis brevis), ECRl (extensor carpi radialis longus) and FCU (flexor carpi ulnaris)). The values of this index in the injected forelimb (red closed symbols) were decreased compared with the values obtained in the noninjected forelimb (open red symbols) or in untreated GRMD dogs (yellow symbols), and they were closer to the healthy dog (WT) indices (black symbols). (d) NMR spectroscopy Pi/γATP muscle signal ratios of the injected (red closed symbols) and noninjected forelimbs (open red symbols) as compared with untreated GRMD (yellow symbols) and healthy (WT) controls (black symbols).

Figure 4. Evolution of the extension strength of the wrist in the forelimbs of GRMD dogs injected with rAAV2/8-Spc5.12-cMD1 by the LR route.

The extension strength of the wrist of the forelimbs was measured using a specific torque measurement device. Three measurement sessions were performed all along the protocol: before injection, at day +45 and at day +90. (a) The evolution of the maximal torques, normalized by the animal weight, over the three different measurement sessions, was represented on each panel. Each point represents the mean value (±95% confidence interval) of the results obtained in forelimbs of healthy (WT) golden retriever dogs (n=14, grey line), in forelimbs of untreated GRMD dogs (n=6, grey dotted line), in the injected forelimb of rAAV2/8-Spc5.12-cMD1-treated GRMD dogs (n=4, dark line) and in the noninjected forelimb of the same dogs (n=4, dark dotted line). (b) Extension strength change between day 0 (before injection) and day +90. Each point represents the ratio between the maximal torque (normalized by the animal weight) obtained at day 0 and at day +90 in a same forelimb for healthy (WT) golden retriever dogs (n=14, grey full symbols), in untreated GRMD dogs (n=6, grey empty line), in the injected forelimb of rAAV2/8-Spc5.12-cMD1-treated GRMD dogs (n=4, black full symbols) and in the noninjected forelimb of the same dogs (n=4, black empty symbols). The horizontal bars represent the mean of the values obtained for each group. *P<0.05 (nonparametric Kruskal–Wallis test with post hoc multiple comparison Dunn’s test).

Figure 5. Analysis of cMD1 expression by immunostaining and western-blot in muscular biopsies of GRMD dogs injected with rAAV2/8-Spc512-cMD1 by the IV route.

(a–c) Dystrophin immunostaining (NCL-DYSB) on transverse sections of muscle samples. Representative results are presented for two treated GRMD dogs injected with different doses of the rAAV2/8-Spc5.12-cMD1 vector by the IV route: dog IV2, injected with 1 × 10¹⁴ vg kg⁻¹ (a) and dog IV7, injected with 2 × 10¹³ vg kg⁻¹ (b). A healthy (WT) dog is also presented as control (c). For the treated dogs, different muscle samples were obtained after surgical biopsies performed before injection, at 3.5 months post injection, at 8 months post injection and at 14 months post injection (only for Dog IV2 for this latter time point). The level of cMD1-positive fibres is indicated above each panel, as well as the number of vector genomes per diploid genomes detected by qPCR in the same muscle sample. Scale bar, 100 μm. (d) Western blot analysis of total proteins (50 μg) extracted from muscles samples. Representative results are presented for two other treated GRMD dogs, injected with different doses of the rAAV2/8-Spc5.12-cMD1 vector by the IV route: dog IV4, injected with 1 × 10¹⁴ vg kg⁻¹ and dog IV6, injected with 2 × 10¹³ vg kg⁻¹. Then, 50 μg of total proteins extracted from GRMD myoblasts transduced with the rAAV2/8-Spc5.12-cMD1 vector were used as positive control, as well as 25 to 75 μg of total proteins extracted from a skeletal muscle of a WT dog. The blot was stained with MANEX-1011C to reveal the presence of the 427 kDa dystrophin protein (WT dog) and the 138 kDa cMD1 protein, with an anti-GAPDH antibody as a loading control. The level of cMD1-positive fibres detected by immunostaining as well as the number of vector genomes per diploid genomes detected by qPCR from the same muscle samples are indicated under each panel.

Figure 6. Improved clinical status of GRMD dogs injected with rAAV2/8-Spc5.12-cMD1 by the IV route.

(a) The global clinical score was determined weekly and expressed as a percentage of a healthy dog score (100%) in untreated GRMD dogs (n=9, red lines) and GRMD dogs injected intravenously with rAAV2/8-Spc5.12-cMD1 at 2 × 10¹³ vg kg⁻¹ (n=3, light grey dotted lines) or 1 × 10¹⁴ vg kg⁻¹ (n=5, dark grey lines). For each dog, the line represents a tendency curve (mobile means order 3) built to show the score evolution. (b) The global clinical scores obtained in each dog at 6 months (left panel) and 9 months (right panel) of age were individually plotted for untreated GRMD dogs (n=9, red symbols), GRMD dogs injected intravenously with rAAV2/8-Spc5.12-cMD1 at 2 × 10¹³ vg kg⁻¹ (n=3, grey symbols) or 1 × 10¹⁴ vg kg⁻¹ (n=5, black symbols). Dead animals were reported with a clinical score at 0%. The horizontal bars represent the mean of the values obtained for each group. **P<0.01 (nonparametric Kruskal–Wallis test with post hoc multiple comparison Dunn’s test).

Figure 7. Improved gait quality of GRMD dogs injected with rAAV2/8-Spc5.12-cMD1 by the IV route.

The global gait quality was determined twice a month using Locometrix and analysed by a discriminant analysis of seven accelerometric variables. The curves represent the evolution of the mean gait index with 95% confident intervals (shaded areas) in healthy dogs (n=9, including animals from retrospective cohorts, green curve), untreated GRMD dogs (n=25, including animals from retrospective cohorts, red curve) and GRMD dogs injected intravenously with rAAV2/8-Spc5.12-cMD1 at 2 × 10¹³ vg kg⁻¹ (n=3, light grey curve) or 1 × 10¹⁴ vg kg⁻¹ (n=5, dark grey curve). F1 and F2 represent the two axes used to plot data during discriminant analysis. An additional axis corresponding to the age in months was also calculated and represented. The discriminant analysis (see details in the Methods section) allows a statistical evaluation to test the probability that the gait of the treated animals was similar (P>0.95) to those of healthy dogs or of untreated GRMD dogs. The result of this statistical evaluation was represented as a colour code, using a green plot when the gait was found similar to those of healthy dogs, a red plot when similar to those of untreated GRMD dogs and yellow when the gait was considered as intermediate between healthy and untreated GRMD dogs.