908. Design and Optimization of U7snRNAs for Skipping of Exon 51 in DMD: Promising Tools for Future Clinical Trials

Abstract

Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene mutations that disrupt the mRNA reading frame. In some cases, forced exclusion of a single exon can restore the reading frame, given rise to a shorter, but still functional dystrophin protein (so called quasi-dystrophin). One potential treatment of the disorder has utilized antisense oligoribonucleotide (AO) to induce removal of disease-associated exons during pre-mRNA processing. Indeed, this approach has been successfully used in DMD cells in vitro with antisense sequences against splice junctions of exon 51. Skipping of this exon would theoretically restore a functional quasi- dystrophin in a significant subset of DMD patients with Δ45-50,Δ47-50,Δ48-50, Δ49-50, Δ50 and Δ52 genotypes. However, since the AO are not self-renewed, they can not achieve long term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV and lentiviral vectors.We have designed AAV and lentiviral vectors harboring chimeric U7 snRNA carrying antisense sequences against exon 51 of the human dystrophin gene (U7-ex51). Lentiviral vectors expressing this U7-ex51 were tested on human myoblasts, whereas AAV vectors were injected in the transgenic hDMD mice (carrying the human dystrophin gene).We confirmed the skipping of the exon 51 in vitro in human myoblasts after transduction with the lentiviral vector encoding U7-ex51 by RT-PCR. We also detected the skipping of the exon 51 after intramuscular injection of an AAV-U7ex51 vector in the transgenic hDMD mouse.We have also tested the efficacy of these vectors to restore dystrophin expression in myoblasts from patients with Δ49-50 and Δ52 deletions.In this study, we provide evidence that efficient skipping of exon 51 can be achieved in human cells and also in vivo after intramuscular injection in a transgenic hDMD mice through U7snRNA shuttle.These results offer very promising tools for clinical treatment of DMD. Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene mutations that disrupt the mRNA reading frame. In some cases, forced exclusion of a single exon can restore the reading frame, given rise to a shorter, but still functional dystrophin protein (so called quasi-dystrophin). One potential treatment of the disorder has utilized antisense oligoribonucleotide (AO) to induce removal of disease-associated exons during pre-mRNA processing. Indeed, this approach has been successfully used in DMD cells in vitro with antisense sequences against splice junctions of exon 51. Skipping of this exon would theoretically restore a functional quasi- dystrophin in a significant subset of DMD patients with Δ45-50,Δ47-50,Δ48-50, Δ49-50, Δ50 and Δ52 genotypes. However, since the AO are not self-renewed, they can not achieve long term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV and lentiviral vectors. We have designed AAV and lentiviral vectors harboring chimeric U7 snRNA carrying antisense sequences against exon 51 of the human dystrophin gene (U7-ex51). Lentiviral vectors expressing this U7-ex51 were tested on human myoblasts, whereas AAV vectors were injected in the transgenic hDMD mice (carrying the human dystrophin gene). We confirmed the skipping of the exon 51 in vitro in human myoblasts after transduction with the lentiviral vector encoding U7-ex51 by RT-PCR. We also detected the skipping of the exon 51 after intramuscular injection of an AAV-U7ex51 vector in the transgenic hDMD mouse. We have also tested the efficacy of these vectors to restore dystrophin expression in myoblasts from patients with Δ49-50 and Δ52 deletions. In this study, we provide evidence that efficient skipping of exon 51 can be achieved in human cells and also in vivo after intramuscular injection in a transgenic hDMD mice through U7snRNA shuttle. These results offer very promising tools for clinical treatment of DMD.