507. AAV-Mini-Agrin for Laminin-alpha2 Deficient CMD

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Laminin alpha2 (merosin)-deficient congenital muscular dystrophy (CMD) (MDC1A) is characterized by severe muscle wasting, premature death in early childhood, and lack of effective treatment. The laminin alpha2 gene (around 10kb) is too large to fit into adeno-associated viral (AAV) vectors. Alternatively, a mini-agrin, which has similar function as the laminin alpha2, is utilized as the therapeutic gene. Our previous study with AAV1-mini-agrin gene therapy in dyw/dyw mice showed dramatic therapeutic effects in muscle pathology, but weak on peripheral neuropathy. The objective of the current study was to see whether the nerve pathology in MDC1A mice could be improved by mini-agrin using the AAV9 vector, which has superior ability to transduce nervous system.AAV9-mini-agrin (2 × 10^11vg/pup) was injected into 2 to 3 days old homozygous dyw/dyw pups via the temporal vein. The gene therapy treatment significantly improved mouse body weight (15 ± 45g in treated mice vs. 10.±.09g of untreated homozygous mice at 11 weeks of age, n=6, p< 0.001) and quadrupled life span (41.±0.04 wks of average life span in treated group vs. 10.18±.12 wks in untreated group, n= 9 to 24, p<0.0001, Fig a). In tests for motor function, untreated dyw/dyw mice were hypoactive in an open field and had overt deficits in a marble-burying assay. AAV9-mini-agrin gene therapy led to significant increases in locomotion (p=0.0373) and a marked rescue of marble-burying (Fig b. p=0.0068), an indication of both nerve and muscle functions. Gene therapy also resulted in a significant improvement in nerve pathology vs. the untreated controls shown by toluidine blue staining with more myelinated nerve fibers, and partial restoration of the basal lamina shown by electron microscopy in the treated mice. More importantly, the treated dyw/dyw male and female mice become fertile, which has never been seen before. Genotyping of the unborn pups confirmed that they were all homozygous dyw/dyw mice. This indicates that mini-agrin can partially replace the function of laminin alpha-2 in MDC1A and achieve therapeutic effects in muscle pathology, peripheral neuropathy, and fertility. This study represents a significant advancement in AAV-mediated gene therapy for MDC1A. View Large Image | Download PowerPoint SlideFig. Life span extension and functional improvement by the treatment. a. Both AAV9-CMV-mini-agrin and AAV9-CB-mini-agrin treatment resulted in significant improvement in mouse survival. n = 9 to 24, p < 0.0001. b. Treatment led to an overt increase in marble-burying. n=5, * p< 0.01.