Scalable Non-viral Gene Delivery for Neuromuscular Diseases

The most common adult-onset muscular dystrophy, myotonic dystrophy type 1 (DM1), is caused by the expansion of an unstable CTG-repeat (50-4000 copies) in the myotonic dystrophy protein kinase (DMPK) gene. Expression of non-coding nucleotide repeats of DMPK mRNA leads to toxic DMPK mRNA in muscle cells triggering wide-ranging clinical features of DM, like muscle hyperexcitability (myotonia), progressive skeletal muscle weakness and degeneration (myopathy), cardiac conduction defects, and cataracts. There is no effective treatment for this devastating disorder. We propose to test natural cell-derived nanovesicles to modulate toxic DMPK mRNA selectively and efficiently to improve safety and decrease costs compared to expensive risk-prone viral-mediated gene therapy. In Phase 1 of this SBIR project, we aim to validate interfering RNA-loaded nanovesicles in vitro by screening up to 15 different siRNAs targeting unique regions of the DMPK gene, identify the optimal constructs that reduce toxic DMPK mRNA in human induced pluripotent stem cells (iPSCs) derived from myotonic dystrophy patients, and ultimately guide the local and systemic studies in an animal model of disease proposed in Phase 2. In Phase 2, we propose to generate IND-enabling data for a commercial product. Once a lead nanovesicle construct is identified in Phase 1, we will compare the efficacy of nanovesicle delivery in a DM1 mouse model following intramuscular (IM) injections in comparison to AAV delivery of DMPK RNAi. The resulting data will inform the nomination of a development candidate (DC) for systemic dose-finding and safety studies in a GLP setting. Altogether, the experiments proposed here are aimed to advance preclinical technology for nanovesicle DMPK RNAi as a viable therapy for myotonic dystrophy patients. If successful, this project can serve as a benchmark for neuromuscular diseases with similar gain-of-function mutations.