rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice

Abstract

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we demonstrate that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores dystrophin expression in the striated musculature of these animals, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vectormediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy (DMD).Miniaturized dystrophin expression cassettes that restore sarcolemmal organization of the dystrophin-glycoprotein complex can be highly functional in transgenic mice 1 . However, delivering potentially therapeutic constructs throughout the musculature of animals with preexisting muscular dystrophy using traditional methods has proven inefficient. Recently, we established that intravascular administration of recombinant adeno-associated viral vectors pseudotyped with the serotype-6 capsid (rAAV6) can transduce the striated musculature of adult mice 2 . This advance enables assessment of systemic microdystrophin delivery in animal models of disease. Historically the dystrophin-deficient mdx mouse 3 has been employed as the primary model of DMD, although this animal does not experience the severe, body-wide dystrophy that shortens lifespan by 75% in patients [4][5][6] . The robustness of mdx mice is attributed to compensatory over-expression of the dystrophin-related protein utrophin, as knockout of both dystrophin and utrophin in mice causes progressive muscle wasting, impaired mobility and premature death 5,6 . In this study, we tested the hypothesis that systemic administration of rAAV6-microdystrophin can ameliorate the pathology associated with severe muscular dystrophy in dystrophin/utrophin double-knockout (dko) mice.A single intravenous administration of ~3×10 12 vector genomes of rAAV6-microdystrophin was administered to one month-old dko mice as described previously 2 , resulting in uniform, body-wide expression of dystrophin for at least one year ( Supplementary Fig. 1 online). Because muscle deterioration leading to respiratory failure is the primary cause of death in patients with DMD 7 , we examined the effects of treatment upon mouse diaphragm (DIA) muscles 4 . Dystrophin expression in DIA muscles examined 18 weeks after treatment was widespread (Fig. 1a,b) and was associated with a significant reduction in the prevalence of smaller regenerating muscle cells compared with untreated muscles (Fig. 1c). Treatment also reduced the frequency of centrally-nucleated myofibers -a feature of muscle regeneration -by ~85% compared with the DIA muscles of untreated mice (Fig. 1d). Importantly, the DIA muscles of treated animals demonstrated more than two-fold increased normalized force-producing capacity compared with the muscles of untreated mice (Fig. 1e).Using a protocol we developed for subjecting muscles to progressively ...

Employment of Microencapsulated Sertoli Cells as a New Tool to Treat Duchenne Muscular Dystrophy

“…AAV vectors carried mini-or micro-dystrophins (i.e., different reduced parts of the DMD gene that still result in functional forms of the protein) have been used to treat animal models of DMD and DMD patients [19,27]. AAV vectors carrying minigenes have been injected in mdx mice and GRMD dogs demonstrating the efficient therapeutic effects of this treatment [28][29][30]. However, a phase I clinical trial using AAV-mini-dystrophin revealed only limited dystrophin expression and irrelevant muscle improvements due to the potential presence of AAV-neutralizing antibodies already present in humans, and the immune response against non-self-dystrophin [31], which requires this kind of approach be accompanied by immunosuppressive therapy.…”

Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction

“…10,11 We and others have reported successful improvement of the dystrophic phenotype in skeletal muscles of DMD mice models following introduction of this transgene. 12,13 To achieve transduction of an entire extremity, a limb perfusion by retrograde intravenous administration of the rAAV in larger animal models is being developed. 14 For cardiac transduction, we utilized rAAV serotype 9, which can be administered systemically with excellent cardiac tropism.…”

Comparative Analysis of Antisense Oligonucleotide Sequences for Targeted Skipping of Exon 51 During Dystrophin Pre-mRNA Splicing in Human Muscle

“…The efficacy of internally truncated dystrophins lacking an appreciable portion of the rod domain has also been demonstrated in transgenic mdx mice, and exploited to design so-called microdystrophins compatible with delivery by AAV vectors. These engineered recombinant microdystrophins have been shown to restore normal expression of the dystrophin-associated protein complex (DPC), improve sarcolemmal stability, and prevent myofiber degeneration in the mdx mouse model (Wang et al, 2000;Fabb et al, 2002;Harper et al, 2002;Gregorevic et al, 2006).…”