Myotonic dystrophy is the most common form of muscular dystrophy in adults, and is characterized by a wide range of clinical features including myotonia (muscle hyperexcitability), progressive myopathy, cardiac conduction defects, hyperinsulinemia, and neuropsychiatric impairment. The multi-systemic manifestations of DM1 are attributable to dominant negative effects of a CTG repeat expansion expressed from the myotonic dystrophy protein kinase gene (DMPK) as part of the 3'untranslated region (3'-UTR) of the mRNA. The goal of the proposed experimental plan is to further develop RNA interference (RNAi) technologies to reduce the DM1 RNA containing the repeat expansion. Initial efforts to establish conditions for systemic delivery of short-hairpin RNAs (shRNAs) to muscles bodywide in a reporter mouse, the ROSA26 mouse, resulted in a level of expression that was effective in knocking down targeted ?-galactosidase expression in cardiac and skeletal muscle. However, toxicity was encountered in some cell types due to overexpression of the shRNAs which saturated the natural microRNA pathways necessary for cell viability. New versions of our current sequence targets for DM1 will incorporate microRNA-based design strategies to produce RNAi sequences that are fully processed from precursors and expressed at a level that is both efficacious and non-toxic, and can be expressed in a tissue-specific manner. The microRNA-based vectors will be compared to shRNAs that are expressed from muscle-specific promoters at a lower level than previously seen with strong RNA polymerase III promoters. Tests of these RNAi expression cassettes in the HSALR mouse model of DM are expected to establish conditions appropriate for systemic delivery by AAV6 and to establish the feasibility of therapeutic RNAi for DM. Importantly, modulating mutant RNA expression by using target sequences with different levels of expression knockdown should reveal the level of reduction necessary to eliminate muscle pathology. These approaches could result in a body-wide alleviation of dystrophic pathology in the HSALR mouse and provide clues for the adaptation of these methods to target the human DM1 gene DMPK. The success of this approach may lead to a treatment in humans for DM1 and would offer a viable approach for treating other dominantly inherited disorders, regardless of whether RNA or protein is pathogenic. We propose to develop a method that will target and inactivate the cell factor that causes myotonic dystrophy (DM). DM is a disease mainly of muscle that causes uncontrolled stiffening. Our therapeutic agent will be engineered to target muscle to eliminate the disease effects in mice with the disease to establish conditions to treat the disease in humans.
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