Long-Term Efficacy of AAV9-U7snRNA-Mediated Exon 51 Skipping in mdx52 Mice

Philippine Aupy,Faouzi Zarrouki,Quentin Sandro,Cécile Gastaldi,Pierre-Olivier Buclez,Kamel Mamchaoui,Luis Garcia,Cyrille Vaillend,Aurélie Goyenvalle

doi:10.1016/j.omtm.2020.04.025

Abstract

Gene therapy and antisense approaches hold promise for the treatment of Duchenne muscular dystrophy (DMD). The advantages of both therapeutic strategies can be combined by vectorizing antisense sequences into an adeno-associated virus (AAV) vector. We previously reported the efficacy of AAV-U7 small nuclear RNA (U7snRNA)-mediated exon skipping in the mdx mouse, the dys−/utr− mouse, and the golden retriever muscular dystrophy (GRMD) dog model. In this study, we examined the therapeutic potential of an AAV-U7snRNA targeting the human DMD exon 51, which could be applicable to 13% of DMD patients. A single injection of AAV9-U7 exon 51 (U7ex51) induces widespread and sustained levels of exon 51 skipping, leading to significant restoration of dystrophin and improvement of the dystrophic phenotype in the mdx52 mouse. However, levels of dystrophin re-expression are lower than the skipping levels, in contrast with previously reported results in the mdx mouse, suggesting that efficacy of exon skipping may vary depending on the targeted exon. Additionally, while low levels of exon skipping were measured in the brain, the dystrophin protein could not be detected, in line with a lack of improvement of their abnormal behavioral fear response. These results thus confirm the high therapeutic potential of the AAV-mediated exon-skipping approach, yet the apparent discrepancies between exon skipping and protein restoration levels suggest some limitations of this experimental model.

Highlights

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that affects 1 out of 5,000 live male births and is caused by mutations in the dystrophin (DMD) gene
We evaluated the ability of an AAV9 (AAV serotype 9)-U7 exon 51 (U7ex51) vector to induce exon skipping and to restore expression of the full-length dystrophin (Dp427) in the exon 52-deficient mdx mouse model, in which exon has been deleted by gene targeting, causing a rupture of the reading frame and the absence of the protein.[17]
In this study, we demonstrate widespread and high levels of exon 51 skipping leading to sustained restoration of muscle dystrophin in the mdx[52] model following a single injection of the AAV9-U7ex[51] vector, confirming the therapeutic potential of the associated virus (AAV)-mediated exon skipping approach. mdx[52] mice were injected with a relatively high dose of vector (3E+13 vg of AAV9-ex51) since one of the objectives was to reach substantial levels of skipping in order to investigate the resulting levels of protein restoration and potential therapeutic benefit in mdx[52] mice

Summary

Introduction

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that affects 1 out of 5,000 live male births and is caused by mutations in the dystrophin (DMD) gene. One of the most promising therapeutic strategies for DMD aims to transform an out-of-frame mutation into an in-frame mutation This would give rise to an internally truncated but still functional protein, as observed in Becker muscular dystrophy (BMD), in which in-frame mutations may lead to a milder phenotype.[1,2]. This so-called exon-skipping strategy can be achieved using antisense oligonucleotides (ASOs) that interfere with splicing signals or regulating elements in the exon, leading to the skipping of the targeted exon at the precursor (pre-)mRNA level.[3–6]. In March 2019, 9 months into their phase 1/2 study, Sarepta Therapeutics announced very encouraging results on four DMD patients treated with AAVrh[74].MHCK7.microdystrophin, showing approximately 80% dystrophin-positive fibers.[10]

Methods

Results

Conclusion