UCLA

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder with no cure. Patients with DMD typically have out-of-frame mutations in the DMD gene, which leads to lack of dystrophin protein. However, there is an allelic, milder disease, Becker muscular dystrophy (BMD), where in-frame mutations allow for some production of an internally deleted dystrophin, which is at least somewhat functional. Thus, a therapeutic strategy is to restore the DMD reading frame, thereby making a DMD mutation into a BMD mutation and likely leading to a milder clinical phenotype. Gene editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) 9 system would allow for permanent gene reframing. Here we describe development of a CRISPR/Cas9 gene editing platform which deletes DMD exons 45-55, creating an in-frame mutation associated with a very mild disease course in BMD patients. This region also encompasses a hotspot of approximately half of all DMD patient mutations, meaning this single platform would be therapeutically relevant for a large percentage of patients.

We first demonstrate in vitro applicability of our CRISPR/Cas9 platform in DMD human induced pluripotent stem cells where we show restored dystrophin protein and function in cardiac and skeletal muscle cells in vitro and after engraftment in vivo. An alternative route of delivery besides cell therapy is direct delivery of CRISPR/Cas9 to muscle in vivo. To that end, we created a novel humanized dystrophic mouse model containing an out-of-frame DMD gene that we can target with our CRISPR/Cas9 platform, which is human sequence specific. We demonstrate restored dystrophin protein after application of our CRISPR/Cas9 platform through electroporation, viral delivery, and nanoparticle delivery in vivo. Although we have achieved highest efficiency of dystrophin generation using adeno-associated virus (AAV) as a delivery vehicle, this work also provides proof-of-principle development of novel nanoparticles as an alternative means to AAV to deliver gene editing platforms to muscle in vivo. Non-viral delivery strategies may be advantageous due to their potential to evade an immune response and achieve stem cell targeting. Future work will continue to improve both approaches for effective direct delivery of our CRISPR platform, which reframes the DMD gene for a large percentage of Duchenne patients.