150. Further Development of an Allele-Specific Gene Silencing Strategy to Correct a Dominant-Negative Mutation Causing Collagen VI-Related Muscular Dystrophy

Abstract

Collagen VI-related congenital muscular dystrophies (COL6-RD), caused by mutations in any of the three genes coding for collagen type VI (COL6A1, COL6A2, COL6A3), underlie a spectrum of disorders ranging from severe life-threatening early onset Ullrich muscular dystrophy via intermediate phenotypes to the milder Bethlem type, typically presenting with generalized muscle weakness, proximal joint contractures, and respiratory failure. At present, there are no treatment options available for individuals affected with these diseases. The fact that the majority of COL6-RD cases are carriers of inherited or de novo dominant mutations, acting as dominant-negative, poses a challenge for the development of targeted therapies. In contrast to gene replacement, allele-specific silencing has the potential to treat these disorders, as it would convert the dominant-negative state into a clinically asymptomatic haploinsufficient state. Therefore, in the laboratory we aim at exploring targeted RNA interference (RNAi) approaches as a potential therapeutic approach for dominant COL6-RD. We have previously demonstrated the allele-specificity and efficacy of siRNA oligos to downregulate the expression of a mutant COL6A3 transcript in vitro in patient-derived fibroblasts. In preparation of in vivo testing we have now extended our study to cells isolated from a mouse model of the disorder, which carries the most frequent dominant-negative mutation, a deletion of exon 16 on Col6a3. We transfected a series of small interfering RNA (siRNA) oligonucleotides into Col6a3del16+/- fibroblast cells, isolated from skin and muscle. To assay efficacy we used outcome measures such as unsaturated PCR, quantitative RT-PCR, and immunofluorescence. In addition, we performed in vivo electroporation of siRNA oligonucleotides in FDB muscles of Col6a3del16+/- mice. We found that siRNA oligonucleotides were effective in Col6a3del16+/- cells, to specifically knockdown the expression from the mutant allele and to restore the production of a collagen VI matrix in culture. This study provides further proof-of-concept of the use of RNAi as a potential treatment for COL6-RD.