MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin
Kristin N. Heller, … , Jerry R. Mendell, Louise R. Rodino-Klapac
Kristin N. Heller, … , Jerry R. Mendell, Louise R. Rodino-Klapac
Published May 4, 2017
Citation Information: JCI Insight. 2017;2(9):e93309. https://doi.org/10.1172/jci.insight.93309.
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Research Article Muscle biology Therapeutics

MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin

Abstract

Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency resulting in progressive muscle weakness and fibrotic scarring. Muscle fibrosis impairs blood flow, hampering muscle repair and regeneration. Irrespective of the success of gene restoration, functional improvement is limited without reducing fibrosis. The levels of miR-29c, a known regulator of collagen, are reduced in DMD. Our goal is to develop translational, antifibrotic therapy by overexpressing miR-29c. We injected the gastrocnemius muscle with either self-complementary AAV.CMV.miR-29c or single-stranded AAV.MCK.micro-dystrophin alone or in combination in the mdx/utrn+/– mouse, a DMD mouse model. Treatment of 3-month-old mdx/utrn+/– mice with AAV.miR-29c showed a reduction in collagen and increased absolute and specific force compared with untreated animals, but neither parameter reached WT levels. Combinatorial gene delivery in 3-month-old mdx/utrn+/– mice further decreased fibrosis, and showed a reduction of transcript levels for Col1A, Col3A, fibronectin, and Tgfb1. In addition, absolute and specific force was normalized and equivalent to WT. However, protection against eccentric contraction fell short of WT levels at this time point. When this same mouse model was treated with miR-29c/micro-dystrophin combinatorial therapy at 1 month of age, there was complete normalization of specific and absolute force and protection against eccentric contraction–induced injury was comparable to WT. These findings highlight the potential for miR-29c as an important addition to the armamentarium for translational gene therapy, especially when used in combination with micro-dystrophin in DMD.

Authors

Kristin N. Heller, Joshua T. Mendell, Jerry R. Mendell, Louise R. Rodino-Klapac

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Figure 7

Early treatment with AAV.miR-29c/micro-dystrophin combination therapy is more effective at reducing fibrosis and extracellular matrix component expression.

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Early treatment with AAV.miR-29c/micro-dystrophin combination therapy is...
(A) Picrosirius red staining of gastrocnemius (GAS) muscle from WT, untreated, AAV.miR-29c–, AAV.micro-dystrophin–, and AAV.miR-29c/AAV.micro-dystrophin–treated mice injected at 4 to 5 weeks of age taken out 12 weeks after injection. Original magnification, ×20. (B) Quantification of picrosirius red staining shows cotreated muscle had a 51.1% reduction in collagen compared with untreated GAS muscle (n = 4–6 per group except n = 12 untreated); 1-way ANOVA. (C) qRT-PCR confirms an increase in miR-29c transcript levels in the treated cohorts (n = 3–5 for all groups except n = 7 untreated); 1-way ANOVA. Semiquantitative qRT-PCR shows a significant reduction in Col1A1 and Col3A1 (D and E), Fbn (F), and Tgfb1 (G) levels in the AAV.miR-29c/AAV.micro-dystrophin–treated muscle compared with the contralateral limb and each of the single therapies. (D–G) (n = 4–6 per group except n = 9 untreated); 1-way ANOVA. All data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.