Membrane repair is a fundamental cell survival process in large, irreplaceable, or frequently injured cell typessuch as muscle cells. We have reported that the skeletal muscle protein dysferlin is defective in Miyoshimyopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and that dysferlin accelerates musclemembrane repair via a calcium-triggered mechanism that invokes binding to annexins and the formation ofintracellar vesicular aggregates. It thus seems likely that MM and LGMD2B are a consequence of aberrantskeletal muscle membrane repair. We now propose to define domains of dysferlin that are critical for thisrepair process and determine whether truncated forms of 'mini-dysferlin' can rescue the MM/LGMD2Bphenotype. Our study has five Specific Aims: (1) Generate and characterize transgenic mice that over-express full-length and truncated dysferlin. Hypothesis: expression of supra-normal levels of full-length ortruncated dysferlin can rescue the dystrophic phenotype in a dysferlin-deficient model. (2) Characterize thebiochemical properties of truncated dysferlin. Hypothesis: A biochemical analysis of full-length and truncateddysferlin proteins will identify domains retaining functional properties of the full-length protein. (3)Characterize membrane repair by mini-dysferlin proteins. Hypothesis: mini-dysferlin proteins retainingfunctional domains will augment membrane repair in dysferlin-deficient muscle cells. (4) Intravenously delivercandidate AAV8-packaged mini-dysferlin genes. Hypothesis: systemic delivery of mini-dysferlin genes willgive rise to dissemination of these genes in muscle and enhanced membrane repair. (5) Analyze zebrafishfor the presence of dysferlin and for phenotypes when dysferlin homologues are down-regulated.Hypothesis: Dysferlin-deficiency will produce a myopathic phenotype in zebrafish and it will be possible toanalyze the impact of replacement of partial or full forms of dysferlin in the zebrafish. Methods: We will usea variety of techniques to define functional domains of dysferlin and will then analyze the rescue of thedysferlin-deficient phenotypes by full length and truncated dysferlin delivered to muscle in vivo via transgenicmice and systemically administered AAV8. Significance: This study willl (1) enhance our understanding ofthe molecular biology of sarcolemmal membrane repair in skeletal muscle; (2) provide insight into thefunctional domains of dysferlin; (3) determine the feasibility of developing mini-dysferlins for rescue ofmuscle phenotypes resulting from dysferlin deficiency; and (4) develop pilot data on the efficacy ofsystemically administered viral gene therapy for MM and LGMD2B.
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