University of North Carolina at Chapel Hill Graduate School
Doi
Abstract
Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder for which there is no cure. It is caused by a defect in the dystrophin gene, which encodes an important structural and regulatory protein at the muscle membrane. In DMD, the absence of dystrophin protein renders the muscle fragile and susceptible to damage. Patients gradually lose muscle mass and die prematurely from cardiac or respiratory complications. Current treatments are palliative and do not address the underlying cause Gene therapies that replace or correct mutated genes have shown promise for DMD. Recombinant adeno-associated viruses (rAAVs) are popular gene delivery vehicles because of their non-pathogenic nature and ability to establish long-term and efficient gene transfer. Still, restoring dystrophin is challenging and cannot completely alleviate motor deficits. While DMD is caused by a single gene defect, many secondary disease mechanisms are involved, such as ischemia and fibrosis. Thus, a strategy addressing multiple pathological mechanisms may be beneficial. MicroRNAs (miRs) are small, regulatory RNA molecules that inhibit target gene expression. A skeletal muscle-restricted microRNA, miR-206, is highly upregulated in dystrophic muscle. Although its role in DMD is unclear, several miR-206 targets have shown benefit for DMD, including vascular endothelial growth factor A (VEGFA) and utrophin. Counteracting miR-206 thus presents a viable treatment for DMD. The goal of this study was to determine if downregulation of miR-206 would increase therapeutic gene expression, inhibiting secondary disease mechanisms and improving dystrophic symptoms. I demonstrated that a rAAV carrying antisense sequences against miR-206, AAV-anti-miR-206, can ameliorate motor deficits in dystrophic mdx mice. To understand its therapeutic mechanism, I focused on two prominent disease pathways. Functional ischemia is a major contributor to the dystrophic phenotype and exacerbates muscle damage. Decreasing miR-206 appears to increase proangiogenic VEGFA expression, improving vascularization in mdx muscle. Also, overexpression of utrophin, a dystrophin paralog, can improve membrane stability and impede DMD progression. I observed increased utrophin in mdx muscle with miR-206 reduction, along with improved pathology, reduced fibrosis and delayed disease progression. Altogether, this study characterizes a novel therapeutic strategy for DMD and sheds light on a contributing factor in secondary pathology.Doctor of Philosoph
