Duchenne muscular dystrophy (DMD) is an X-linked disorder that affects 1:5000 live male births in the US each year. DMD is caused by loss-of-function mutations in the DYSTROPHIN (DMD) gene that results in severe muscle wasting, loss of ambulation by age 12, cardiac arrhythmia, and death in early adulthood due to respiratory failure. While there are several novel strategies (such as exon-skipping compounds to create partially-functional Dystrophin protein) in clinical trials, none of them deal with the secondary signaling pathways and subsequent muscle pathogenesis that occurs in DMD. There is growing evidence that genetic modifiers of DMD have a significant impact on the outcomes and severity of DMD-related pathologies in patients. These DMD genetic modifiers have the potential to serve as novel therapeutic entry points for the treatment of diseases. Previously my laboratory identified miR-486 as a muscle-enriched microRNA that is decreased in expression in DMD patient skeletal muscle biopsies. In this proposal, we will test the hypothesis that miR-486 and its target (DOCK3) play a significant role in the regulation of skeletal muscle. We have strong data that demonstrates that miR-486 expression levels decrease and an mRNA target (DOCK3) increases corresponding with the degree of dystrophic pathology.
In Aim 1, we will characterize the functional role of miR-486 on skeletal and heart muscles via characterizing muscle performance and cardiac remodeling in miR-486 knockout (KO) mice on normal and mdx5cv backgrounds.
Aim 2 focuses on identifying the consequences of manipulating miR-486 and Dock3 expression on key downstream signaling pathways including PTEN/AKT and Rac1/RhoA using a novel Dock3 conditional muscle knockout mouse. Additionally, we will identify novel miR-486 target genes using Ago2-sequencing (CLIP-seq) strategy to identify in vivo skeletal muscle targets of miR-486. Lastly, Aim 3 focuses on using transient miR-486 overexpression strategies (Adeno-associated viral vectors and engineered exosomes) delivered intramuscularly and systemically to improve dystrophic symptoms at key developmental time points in mdx5cv mice. Our combinatorial use of DMD patient muscle cell lines, miR-486 KO and Dock3 conditional muscle KO mice, and experience evaluating genetic and drug compounds in mouse models of human diseases will allow us to rigorously evaluate the use of a microRNA (miR-486) overexpression as a novel therapy for the treatment of DMD.
The major focus of this proposal is the characterization and manipulation of a microRNA regulator (miR-486) of Duchenne muscular dystrophy (DMD), and one of its target mRNAs (DOCK3). We will manipulate miR-486 and Dock3 expression levels, characterize their muscle phenotypes, and characterize their effects on key downstream signaling cascades including PTEN/AKT and Rac1/RhoA signaling. Finally, we will explore whether miR-486 overexpression (via Adeno-associated viral vectors/AAV or engineered exosomes) can block the progression of dystrophin-deficient disease muscle pathology.