Dual AAV Vectors for Duchenne Muscular Dystrophy Therapy

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DESCRIPTION (provided by applicant): Dystrophin gene mutations lead to Duchenne muscular dystrophy (DMD), a severe muscle disease that affected nearly all muscles in the body. A cure for DMD requires body-wide therapy. Adeno-associated virus (AAV) is currently the only viral vector that can efficiently transduce whole body muscle. For this reason, AAV has been considered as the vector-of-choice for DMD gene therapy. Despite great promise, AAV gene therapy is challenged by the small viral packaging capacity (5 kb maximal). The 11.5 kb full-length dystrophin coding sequence cannot be delivered by a single AAV vector. To overcome this obstacle, investigators have developed abbreviated micro/mini-dystrophin genes. Microgenes (< 4 kb) can fit into a single AAV but they only partially improve muscle force. The 6~8 kb minigenes can normalize muscle force but they are too big for AAV packaging. Furthermore, they cannot restore the neuronal nitric oxide synthase (nNOS) recruiting function of the full-length protein. The goals of the parental grant are (1) to identify the nNOS localization domain in the dystrophin gene and to develop novel synthetic minigenes that can restore sarcolemmal nNOS; (2) to explore dual AAV vector-mediated systemic mini-dystrophin gene therapy in the mouse model; (3) to develop systemic AAV gene transfer in a canine DMD model. Tremendous progress has been achieved since we started this project. Most remarkably, we have identified R16/17 as the nNOS anchoring domain in dystrophin. We have also generated a new ?H2-R15 minigene that restores sarcolemmal nNOS and enhances exercise performance (published in the Journal of Clinical Investigation on March 24, 2009). The loss of dystrophin leads to contraction-associated sarcolemmal injury and histopathology. However, muscle force reduction does not always correlate with histopathology. The molecular pathway(s) leading to the loss of muscle strength in DMD is currently unclear. With the support of the parental R01 grant, we recently generated preliminary data suggesting that cytosolic nNOS mislocalization and subsequent nitrosative/oxidative stress may underlie force reduction in DMD. In this revision, we propose to further explore the molecular mechanisms/consequencies of nNOS mislocalization in DMD pathogenesis and therapy. Specifically, we will test the following two hypotheses: (1) R16/17- mediated nNOS recruiting is sequence specific and phase specific; (2) nNOS mislocalization results in nitrosative/oxidative stress and S-nitrosylation of contractile proteins contributes to muscle force reduction in DMD. These studies expand our project to new directions beyond the scope of the parental grant. The findings will shed new light on our understanding of the disease process and uncover novel therapeutic avenues for DMD treatment. PUBLIC HEALTH RELEVANCE: Duchenne muscular dystrophy (DMD) is a life threatening diseases affecting a fairly large population. It is caused by dystrophin gene mutation. Interestingly, the loss of dystrophin disrupts subcellular localization of neuronal nitric oxide synthase (nNOS) in muscle cells. in the alteration of nNOS homeostasis has been suggested as a critical factor in DMD pathogenesis. In an effort to better develop DMD gene therapy, we recently identified the nNOS recruiting domain in the dystrophin gene. Here, we propose to further investigate the molecular interactions between nNOS and dystrophin. In addition, we will evaluate whether the nitrosative/oxidative stress generated by nNOS mislocalization is responsible for the force loss in DMD. These studies will advance our understanding on DMD pathogenesis and open new doors for DMD therapy.