A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant adenoassociated virus pseudotype 6 vectors. The inclusion of vascular endothelium growth factor/vascular permeability factor, to achieve acute permeabilization of the peripheral microvasculature, enhanced tissue transduction at lower vector doses. This technique enabled widespread muscle-specific expression of a functional micro-dystrophin in the skeletal muscles of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy. We propose that these methods may be applicable for systemic delivery of a wide variety of genes to the striated muscles of adult mammals.Human mortality and quality of life are significantly affected by diseases of the striated musculature. Genetic treatments that are being developed for conditions such as heart disease, aging-associated muscle wasting and the muscular dystrophies have been limited by an inability to achieve widespread and efficient gene transfer to the heart and dispersed skeletal muscles of an adult organism 1-4 . For example, anesthesia, invasive surgery and hazardous cofactors are required to transduce varying fractions of the cardiomyocyte population efficiently 1,5 . Similarly, the transfer of genes to the muscles of individual limbs using various vectors requires either direct injection of individual muscles, or complex surgical procedures performed under anesthesia to distribute vectors via the circulation 2-4,6-10 . Here we describe a simple and highly efficient method to transfer genes systemically to the cardiac and skeletal muscles of adult mammals. This approach uses intravenous administration of recombinant adeno-associated virus pseudotype 6 (rAAV6-pseudotyped vectors) 11 , which are extremely effective at transducing skeletal muscles after intramuscular injection 12 .Correspondence should be addressed to J.S.C. (jsc5@u.washington.edu).. 4 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Medicine website. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Medicine website for details). NIH Public Access RESULTS Systemic transduction of skeletal muscles by rAAV6First, we examined the potential for systemic gene transfer after intravenous administration of rAAV6 vectors at the whole-body level in young adult (6-8 wk) C57Bl/10J mice (Fig. 1). The muscles of mice examined 11 days after administration through the tail vein of ~2 × 10 11 vector genomes of rAAV6 vector containing a CMV-lacZ expression cassette, did not show obvious exogenous β-galactosidase (...

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 ...

Systemic delivery of recombinant adeno-associated virus (rAAV) 6 vectors mediates efficient transduction of the entire striated musculature, making this an attractive strategy for muscle gene therapy. However, owing to widespread transduction of non-muscle tissues, optimization of this method would benefit from the use of muscle-specific promoters. Most such promoters either lack high-level expression in certain muscle types or are too large for inclusion in rAAV vectors encoding microdystrophin. Here, we describe novel regulatory cassettes based on enhancer/promoter regions of murine muscle creatine kinase (CK) and alpha-myosin heavy-chain genes. The strongest cassette, MHCK7 (770 bp), directs high-level expression comparable to cytomegalovirus and Rous sarcoma virus promoters in fast and slow skeletal and cardiac muscle, and low expression in the liver, lung, and spleen following systemic rAAV6 delivery in mice. Compared with CK6, our previous best cassette, MHCK7 activity is approximately 400-, approximately 50-, and approximately 10-fold higher in cardiac, diaphragm, and soleus muscles, respectively. MHCK7 also directs strong microdystrophin expression in mdx muscles. While further study of immune responses to MHCK7-regulated microdystrophin expression is needed, this cassette is not active in dendritic cell lines. MHCK7 is thus a highly improved regulatory cassette for experimental studies of rAAV-mediated transduction of striated muscle.

Adeno-associated virus-based vector (AAV)-mediated gene delivery has been successful in some animal models of human disease such as the mdx mouse model of human Duchenne muscular dystrophy (DMD). However, recent evidence of immune-mediated loss of vector persistence in dogs and humans suggests that immune modulation might be necessary to achieve successful long-term transgene expression in these species. We have previously demonstrated that direct intramuscular injection of AAV2 or AAV6 in wild-type random-bred dogs resulted in a robust immune response to capsid or capsid-associated proteins. We now demonstrate that a brief course of immunosuppression with a combination of anti-thymocyte globulin (ATG), cyclosporine (CSP), and mycophenolate mofetil (MMF) is sufficient to permit long-term and robust expression of a canine micro-dystrophin (c-micro-dys) transgene in the skeletal muscle of a dog model for DMD (canine X-linked muscular dystrophy, or cxmd dog) and that its expression restored localization of components of the dystrophin-associated protein complex at the muscle membrane. This protocol has potential applications to human clinical trials to enhance AAV-mediated therapies.

Vectors based on recombinant adeno-associated viruses (rAAV) have emerged as tools of choice for gene transfer to skeletal muscle. rAAV vectors demonstrate efficient, safe, and stable transduction. Multiple serotypes of AAV exist, but vectors based on serotype 2 (rAAV2) are the most thoroughly characterized and frequently employed. Here, we characterize transduction of the skeletal musculature using rAAV vectors pseudotyped with serotype 6 capsid proteins (rAAV6). We demonstrate that rAAV6 vectors can efficiently transduce the skeletal musculature of mice at levels >500-fold higher than is achievable with rAAV2 vectors and can readily saturate individual muscles following direct injection. Further, rAAV6 vectors are capable of transducing the diaphragm and intercostal muscles of mice after a simple injection into the intrathoracic cavity and are capable of widespread transduction throughout the musculature of mice injected in the intraperitoneal space as newborn pups. These results demonstrate that rAAV6 vectors hold great potential for use in gene delivery protocols targeting the skeletal musculature.