Several hereditary diseases including Duchenne muscular dystrophy (DMD) are caused by recessive loss of function mutations in a single gene. In such cases, gene replacement therapy (gene therapy) is a promising strategy. Adeno-associated virus (AAV) is a preferred vector for gene therapy, but this strategy for treatment of DMD has faced the challenge of incompatibility of the large size of the DMD gene with a limited packaging capacity of AAV. Strategies to overcome the packaging constraints of gene therapy are evolving and Insmed is evaluating a novel RNA end-joining (REJ) technology to test the delivery of multiple synthetic DNA molecules leading to the joining of RNA fragments within a single cell. This method could result in the expression of a larger protein that could not be expressed using a traditional single AAV strategy.
Insmed is developing an AAV9-midlength-dystrophin construct that creates a 248 kilodalton (kDA) dystrophin for the treatment of DMD versus the more common ~140 kDA micro-dystrophin by utilizing REJ technology to piece together two RNA fragments within a cell.
Early research studies have established that by delivering two separate AAV’s carrying two separate synthetic DNA molecules, REJ technology is efficient in joining RNA molecules encoding fragments of the dystrophin protein in cells both in vitro and in vivo. Midlength-dystrophin expression in mdx animals is observed in a dose-dependent manner in cells transduced with a dual AAV9-mid-dystrophin construct. Robust mid-dystrophin expression is also observed in skeletal and cardiac muscle when administered to the mdx mouse model of DMD. Importantly, this mid-dystrophin expression confers biological activity, as mdx animals exhibit significant improvement in muscle strength at a range of doses, relative to mdx control mice, as measured by hindlimb muscle electrophysiology. Studies have demonstrated significant expression of mid-length dystrophin by immunohistochemistry as well as Western blotting, with no evidence of truncated or partial dystrophin fragment expression.
This work opens the possibility of developing potential improved dystrophin constructs that may have considerable therapeutic benefit in muscles affected by the dystrophic pathology experienced in patients with DMD.