Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients

Abstract

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

Figure 1

Vector biodistribution in the injected forelimb and at distance. (a) Vector copy number per diploid genome in each muscle of the injected forelimb of each rAAV-injected dog at euthanasia. A total of 13 muscles were analyzed in the injected limb. Each point represents the vector copy number per diploid genome in each muscle of the injected limb. Empty symbols were used for the groups of dogs injected with 40% of the limb volume (groups 1, 2, and 3), and full symbols were used for the groups of dogs injected with 20% of the limb volume (groups 4, 5, and 6). The small horizontal bars represent the mean of the values obtained for each dog. No signal was detected (<0.004 vg/dg) in the muscles of the control dogs. CV, coefficient of variation within the injected forelimbs. (b) Vector copy number per diploid genome in different tissues of the different groups of dogs at euthanasia. For each dog, the analysis was performed on 13 muscles of the injected forelimb, on 13 muscles of the noninjected forelimb, on 20 muscles at distance (muscles of hind limbs (n = 2), muscles of the body (n = 15), heart (n = 2) and diaphragm), on spleen (n = 1), on testis (n = 2), on kidney (n = 1), on liver (n = 3), on draining lymph nodes (n = 2), and on other lymph nodes at distance (n = 5). Each bar of the histogram represents the mean (with standard deviations) of the results obtained in the different dogs of a same group. Striped bars were used for the groups of dogs injected with 40% of the limb volume (groups 1, 2, and 3). Full bars were used for the groups of dogs injected with 20% of the limb volume and a flow rate of 10 ml/minute (groups 4 and 5), and punctuated bars were used for the groups of dogs injected with 20% of the limb volume and a flow rate of 35 ml/minute (group 6). No signal was detected (<0.004 vg/dg) in the tissues of the control dogs (data not shown).

Figure 2

Dystrophin messenger analysis in muscles. Representative results are presented for one dog of each injected dose: (a) 5^E12 vg/kg: D1, (b) 1^E13 vg/kg: D3, and (c) 5^E13 vg/kg: D7. The dystrophin messenger was detected by nested RT-PCR from exon 3 to 10 in muscles sampled at euthanasia (m. FCR = muscle flexor carpi radialis; m. FDS = muscle flexor digitorum superficialis; diaphragm and heart). The PCR products (910 bp and 778 bp fragments) detected in healthy (WT) dog correspond to mRNA variants with or without the alternative exon 9. The PCR products (791 bp and 659 bp fragments) detected in the Golden Retriever Muscular Dystrophy (GRMD) dogs before injection (bef. inj.) correspond to mRNA variants lacking either exon 7 or exons 7 and 9, respectively. After injection, the two new bands (436 bp and 304 bp fragments) detected in some muscles correspond to in-frame mRNAs lacking exons 6 to 8 and 6 to 9, respectively. The copy numbers of vector genome per diploid genome detected by Q-PCR on gDNA from the same tissue sample are indicated under each panel. No skipping was detected in the muscles of the control dogs.

Figure 3

Analysis of dystrophin expression in muscles by immunostaining and western-blot analysis. (a) Dystrophin immunofluorescence (NCL-DYS2 monoclonal antibody) on transverse sections of muscle samples (muscle digitorum extensor communis) obtained at sacrifice from a healthy (WT) dog, a control Golden Retriever Muscular Dystrophy (GRMD) dog (C1) and of three GRMD dogs injected with different doses of rAAV (5^E12 vg/kg: D1. 1^E13 vg/kg: D4 and 5^E13 vg/kg: D14). Scale bar = 500 µm. (b) Western-blot analysis of total proteins (100 µg) extracted from muscles of dog 15 (m. FCR = muscle flexor carpi radialis; m. FDS = muscle flexor digitorum superficialis; m. FLU = muscle flexor lateralis ulnaris; m. pectoralis and m. deltoideus). The blot was stained with NCL-DYS1 or NCL-DYS2 monoclonal antibodies (the two antibodies having different levels of sensitivity) to reveal the presence of the novel ~405 kDa dystrophin band. Muscle from a control GRMD dog was used as negative control. Muscle from a healthy (WT) dog was used as positive control, and revealed the presence of the 427 kDa normal dystrophin band. Different quantities (25–100 µg) of this protein extract were loaded, to allow a semiquantification of the signals obtained in the injected dog. A Ponceau S red staining was used to validate protein loadings.

Figure 4

Improvement of pathological pattern in the dystrophin-expressing muscles. Two muscles per dog were analyzed (the flexor carpi ulnaris muscle and the extensor carpi radialis muscle) according to their percentage of Dys+ positive fibers: <3% (n = 38), between 3 and 33% (n = 8), between 34 and 66% (n = 18) and >66% (n = 12). *P < 0.05. (a) Evaluation of myofiber regeneration. Regeneration was quantified by immunohistochemical staining of myotubes using an antibody specific for developmental myosin heavy chain isoform and automatic measurement of the percentage of the labeled area. (b) Evaluation of endomysial fibrosis in the muscles. Endomysial fibrosis was quantified by immunohistochemical revelation of collagen I, and automatic measurement of the percentage of the labeled area after selection of regions of interest. (c) Evaluation of total fibrosis in the muscles. Total fibrosis was quantified by immunohistochemical revelation of collagen I, and automatic measurement of the percentage of the labeled area.

Figure 5

NMR imaging and spectroscopy analyses of muscles. (a) Representative example of transverse fat-saturated T2-weighted NMR image of the two forelimbs of one dog of group 3 (D8). The vector was injected in the left forelimb and imaging was performed 3 months after injection. Signal muscle intensities were decreased and more homogeneous in the injected forelimb (left) compared to the noninjected one (right). (b) Representative example of ³¹P NMR spectra of the injected (left) and noninjected forelimb (right) of the same dog (D8). Phosphocreatine (PCr) was increased and phosphodiesters (PDE) were decreased relative to ATP in the treated forelimb compared to the untreated forelimb. (c) NMR imaging T2w/T1w muscle signal ratios of dogs injected with a small volume (20% of the limb volume), obtained from three different muscles. ECR, extensor carpi radialis brevis; ECRl, extensor carpi radialis longus; FCU, flexor carpi ulnaris). The values of this index in the injected limbs (red closed symbols for high dose and blue closed symbols for intermediate dose) were decreased compared to the noninjected ones (open red and blue symbols) and controls (yellow symbols) or nontreated Golden Retriever Muscular Dystrophy (GRMD) reference population (white symbols), and they were closer to the normal dog indices (black symbols). (d) NMR spectroscopy PDE/PCr muscle signal ratios of the dogs injected with high (red symbols), intermediate (blue symbols) and low vector doses (grey symbols), with large volume (40% of the limb volume) or small volume (20% of the limb volume) as compared to GRMD controls.

Figure 6

Relative strength changes over the protocol time in the tested forelimb, related to dystrophin expression in the stimulated muscles. The tested forelimbs were classified according to their level of dystrophin-positive fibers: less than 1% (n = 30), 1–40% (n = 21), and more than 40% (n = 14).