379. MicroRNA-29 and Micro-Dystrophin Combinatorial Therapy Suppresses Fibrosis and Restores Function to mdx/utrn+/− Mice
Authors: Kristin Heller, Eric Meadows, Sarah Lewis, Josh Mendell, Jerry Mendell, Louise Rodino-Klapac
Journal: Molecular Therapy (2016)

Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency resulting in muscle loss and progressive muscle weakness and fibrotic scarring. Muscle fibrosis impairs blood flow and excludes endomysial derived constituents hampering muscle repair and regeneration. Irrespective of the success of gene restoration (molecular or pharmacologic) functional improvement is limited without reduction of muscle fibrosis. miR-29c regulates collagen levels making it an ideal candidate for decreasing muscle fibrosis. miR-29c levels are reduced in DMD and our goal is to develop an anti-fibrotic therapy by overexpressing miR-29c with adeno-associated virus (AAV) mediated delivery in combination with micro-dystrophin to improve membrane stability. We injected scAAVrh.74.CMV. miR-29c alone, co-delivered with rAAVrh.74.MCK. micro-dystrophin, and rAAVrh.74.MCK. micro-dystrophin alone by intramuscular injection (IM) into the left gastrocnemius (GAS) muscle of 3 month old mdx/utrn+/- mice, a DMD mouse model. GAS muscle was analyzed 3 months post-injection to assess collagen accumulation by Sirius Red staining and subsequent quantification with ImageJ. Additional outcomes included miR-29c and collagen transcript levels, force measurements in the GAS muscle, fiber diameter measurements and western blot analysis for proteins involved in muscle regeneration (MyoD, Myogenin). Analogous to DMD tissue, we demonstrated a significant reduction in miR-29c levels in mdx/utrn+/− muscle correlated with increased fibrosis measured by Sirius red staining. Following 3 months of treatment with scAAV. miR-29c alone, there was a significant reduction in fibrosis (treated-23.5%±1.3 vs. untreated-27.8% ±0.6, p<0.01) in the GAS muscle. When co-delivered with micro-dystrophin we see further reduction in collagen (41%) by Sirius red staining along with significantly reduced mRNA levels of Col1A, Col3A, fibronectin and TGF-β levels. We observed an increase in specific and absolute force in the muscle treated with miR-29c alone compared to the untreated limb, which when combined with micro-dystrophin led to absolute and specific force that were not significantly different than wild-type (miR-29c treated-204.7±11.7 vs. untreated-151.6±14.5 vs. combined-244.2±6.6 vs. wild type-313.1±40.69 p<0.01). We also observed a significant increase in gastroc weight in those muscles that were co-treated. Demonstration of increased fibrosis and decreased miR-29c expression in the mdx/utrn+/− mice and dystrophin-deficient patients validates the mouse model as representative of the human disease. Initial results using AAV. miR-29c as an anti-fibrotic therapy suggest that there is beneficial effect with reduction in collagen levels, a key contributor in fibrosis. Moreover, when combined with micro-dystrophin to improve membrane stability, miR-29 upregulation normalized muscle force. These data provide rationale for overexpression of miR-29c to reduce fibrosis along with dystrophin replacement as a potential treatment for DMD.