Dystrophin threshold level necessary for normalisation of nNOS, iNOS and RyR1 nitrosylation in GRMD dystrophinopathy

Christel Gentil (1) , Caroline Le Guiner (2) , Sestina Falcone (1) , Jean-Yves Hogrel (1) , Cecile Peccate (3, 1) , Stéphanie Lorain (1) , Sofia Benkhelifa-Ziyyat (1) , Lydie Guigand (4, 5) , Marie-Françoise Montus (6) , Laurent Servais (1) , Thomas Voit (1) , France Pietri-Rouxel (1)

1 Institut de Myologie
2 Inserm UMR 1089 - Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques
3 Centre de recherche en myologie
4 Atlantic Gene Therapies
5 PAnTher - Physiopathologie Animale et bioThérapie du muscle et du système nerveux
6 Généthon

Currently, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine a dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial was recently conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger. Here, we exploited the rAAV8-U7snRNA transduced GRMD muscle samples, well-characterized for their percentage of dystrophin-positive fibers, in the aim to define a threshold of dystrophin rescue necessary for normalization of the status of the neuronal nitric oxide synthase mu (nNOSµ), the inducible nitric oxide synthase (iNOS), and the ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for an efficient contractile function. Results showed that the restoration of dystrophin in 40% of muscle fibers is needed to decrease the abnormal cytosolic nNOSµ expression and to reduce the overexpression of iNOS, these two parameters leading to a reduction of the NO level into the muscle fiber. Furthermore, the same percentage of dystrophin-positive fibers of 40 % was associated with the normalization of the RyR1 nitrosylation status and to a stabilization of the RyR1/calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for a proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology.

Domaines: Génétique; Médecine humaine et pathologie

Christel Gentil, Caroline Le Guiner, Sestina Falcone, Jean-Yves Hogrel, Cecile Peccate, et al.. Dystrophin threshold level necessary for normalisation of nNOS, iNOS and RyR1 nitrosylation in GRMD dystrophinopathy. Human Gene Therapy, 2016, 27 (9), pp.712-726. ⟨10.1089/hum.2016.041⟩. ⟨hal-01340106⟩