Uncovering the role of microRNA-206 in Duchenne muscular dystrophy

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

Follistatin N terminus differentially regulates muscle size and fat in vivo

Delivery of follistatin (FST) represents a promising strategy for both muscular dystrophies and diabetes, as FST is a robust antagonist of myostatin and activin, which are critical regulators of skeletal muscle and adipose tissues. FST is a multi-domain protein, and deciphering the function of different domains will facilitate novel designs for FST-based therapy. Our study aims to investigate the role of the N-terminal domain (ND) of FST in regulating muscle and fat mass in vivo. Different FST constructs were created and packaged into the adeno-associated viral vector (AAV). Overexpression of wild-type FST in normal mice greatly increased muscle mass while decreasing fat accumulation, whereas overexpression of an N terminus mutant or N terminus-deleted FST had no effect on muscle mass but moderately decreased fat mass. In contrast, FST-I-I containing the complete N terminus and double domain I without domain II and III had no effect on fat but increased skeletal muscle mass. The effects of different constructs on differentiated C2C12 myotubes were consistent with the in vivo finding. We hypothesized that ND was critical for myostatin blockade, mediating the increase in muscle mass, and was less pivotal for activin binding, which accounts for the decrease in the fat tissue. An in vitro TGF-beta1-responsive reporter assay revealed that FST-I-I and N terminus-mutated or -deleted FST showed differential responses to blockade of activin and myostatin. Our study provided direct in vivo evidence for a role of the ND of FST, shedding light on future potential molecular designs for FST-based gene therapy

Overcoming Insulin Insufficiency by Forced Follistatin Expression in β -cells of db/db Mice

Diabetes poses a substantial burden to society as it can lead to serious complications and premature death. The number of cases continues to increase worldwide. Two major causes of diabetes are insulin resistance and insulin insufficiency. Currently, there are few antidiabetic drugs available that can preserve or protect β-cell function to overcome insulin insufficiency in diabetes. We describe a therapeutic strategy to preserve β-cell function by overexpression of follistatin (FST) using an AAV vector (AAV8-Ins-FST) in diabetic mouse model. Overexpression of FST in the pancreas of db/db mouse increased β-cell islet mass, decreased fasting glucose level, alleviated diabetic symptoms, and essentially doubled lifespan of the treated mice. The observed islet enlargement was attributed to β-cell proliferation as a result of bioneutralization of myostatin and activin by FST. Overall, our study indicates overexpression of FST in the diabetic pancreas preserves β-cell function by promoting β-cell proliferation, opening up a new therapeutic avenue for the treatment of diabetes