Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the loss of dystrophin. Dystrophin is a cytoskeleton protein that anchors neuronal nitric oxide synthase (nNOS) to the sarcolemma. Without dystrophin, nNOS is lost from the muscle cell membrane. Sarcolemmal localization of nNOS is crucial for muscle health because it allows short-lived vasodilator nitric oxide (generated by nNOS) to reach the surrounding vasculature to counteract sympathetic vasoconstriction during exercise. In dystrophin-deficient mdx mice and human patients, the loss of sarcolemmal nNOS abolishes protective sympatholysis during contraction and aggravates muscle disease. We recently identified dystrophin spectrin-like repeats 16 and 17 (R16/17) as the nNOS-binding domain in mice. R16/17 is encoded by exons 42 to 45. In-frame deletion of these exons abolishes sarcolemmal nNOS expression in patients, suggesting R16/17 is also the nNOS-binding domain in human. To study the therapeutic effect of sarcolemmal nNOS, we expressed synthetic dystrophins with or without R16/17 in mdx mice and found that R16/17-containing dystrophins (but not the ones without R16/17) improved blood flow and enhanced exercise performance. The immediate next question is whether the mouse results can be translated to large mammals. Several groups have tried to tease out the relevance of nNOS to disease severity in human patients by genotype-phenotype analysis. Unfortunately, deletion mutations that destroy nNOS binding often disrupt the normal phasing of dystrophin spectrin-like repeats. Since both factors (nNOS localization and spectrin-like repeat phasing) contribute to disease severity, it has become extremely challenging (if not impossible) to separate the two effects. Indeed, the published results are controversial. We reasoned that a well-designed hemodynamic study in the canine DMD model using normally phased dystrophins (with or without the nNOS-binding domain) can yield critical insight on the therapeutic significance of sarcolemmal nNOS in large mammals. Hence, we introduced R16/17-containing dystrophin to DMD dog muscle. Unexpectedly, it did not anchor nNOS to the membrane, suggesting canine dystrophin is unique. Such species-specific difference in dystrophin has never been observed before. We hypothesize that the nNOS-binding domain is located in a different region in dog dystrophin and we further hypothesize that an adeno-associated virus (AAV) vector can be generated to deliver an nNOS-binding domain-containing canine dystrophin gene to dog muscle.
Our specific aims are (1) to localize the canine dystrophin nNOS-binding domain using a combinatory approach of plasmid/AAV gene transfer and the epitope mapping of revertant fibers; (2) to develop a dog dystrophin AAV vector that carries the canine nNOS-binding domain. This vector will be used in future studies to test whether sarcolemmal nNOS can benefit a dystrophic large mammal (affected dogs).
Disruption of dystrophin-dependent nNOS localization is a critical pathogenic mechanism in Duchenne muscular dystrophy (DMD). In this project, we will identify the nNOS-binding domain in the canine dystrophin gene. Our study will pave the way for studying the therapeutic benefit of the nNOS-binding domain in the symptomatic dog model of DMD in the future.
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| Kodippili, Kasun; Hakim, Chady H; Pan, Xiufang et al. (2018) Dual AAV Gene Therapy for Duchenne Muscular Dystrophy with a 7-kb Mini-Dystrophin Gene in the Canine Model. Hum Gene Ther 29:299-311 |
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| Zhao, Junling; Kodippili, Kasun; Yue, Yongping et al. (2016) Dystrophin contains multiple independent membrane-binding domains. Hum Mol Genet 25:3647-3653 |
| Duan, Dongsheng (2016) Dystrophin Gene Replacement and Gene Repair Therapy for Duchenne Muscular Dystrophy in 2016: An Interview. Hum Gene Ther Clin Dev 27:9-18 |
