DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.207 AAV.U7 technology for two mutational hotspots of the DMD gene (∼6%) results in efficient exon skipping, protein restoration and force improvement

Abstract

Mutations in the DMD gene cause two disorders, the severe Duchenne muscular dystrophy (DMD), associated with no protein expression and the milder Becker muscular dystrophy (BMD) associated with the production of an internally truncated but semi-functional protein. Genotype-phenotype correlation of DMD and BMD prompted a promising therapeutic strategy named exon skipping that intends to convert a DMD to a BMD phenotype. This strategy makes use of antisense oligonucleotides (AONs) to hide exon definition. This should transform an out-of-frame DMD reading frame into an in-frame one, leading into expression of an internally truncated but functional protein. AONs are promising but have two major drawbacks: 1. stability which required repeated injection; 2. limited penetration in heart. Alternatively, an antisense sequence can be carried by a small nuclear RNA (U7snRNA) and delivered using a viral vector such as adeno-associated virus (AAV). This approach (AAV.U7) has been demonstrated by several group including ours, to overcome AON's drawbacks. In this study, we focused on skipping of exon 2 and exon 44, two mutational hotspots of the DMD gene as their skipping could be beneficial to ∼12% of patients. Our objective was to evaluate the efficiency of an AAV.U7 exon skipping strategy in these hotspot mutations both in vitro and in vivo. For in vitro studies, we used skin punches from several DMD patients and transdifferentiated them into myotubes. Using those cells, we showed that our constructs efficiently mediated exons 2 or 44 skipping (at least ≥50%) and induced expression of a slightly truncated dystrophin. For in vivo studies, we used either a new mouse model carrying an exon 2 duplication (referred to as Dup 2 mice) or more recently a new humanized DMD mouse model that is missing human exon 45 disrupting reading frame of hDMD. This new model (referred to as hDMD/mdx del45 mice) does not express mouse and human dystrophins and presents a dystrophic muscle pathology in multiple muscles. In vivo, following intramuscular or intravenous injection, we observed an efficient exon 2 or 44 skipping (at least ≥50%) and expression of a truncated dystrophin (at least ≥40%). Muscle strength using different tests was improved (by ∼50% compared to an untreated mouse). These AAV.U7- could help ∼12% of DMD patients. One of those vectors is being evaluated in a clinical trial.