505. Treatment of DMD 5’ Mutations Through Two Different exon2 Skipping Strategies: Intramuscular Delivery of rAAV9.snRNA Mediated Skipping and Antisense Morpholino Oligomers

Abstract

To date, exon-skipping therapies for Duchenne muscular dystrophy (DMD) patients have focused on patients with out-of-frame exon deletions, in whom treatment results in larger but in-frame internal deletions which lead to translation of an internally truncated but partially functional dystrophin protein. We are developing exon-skipping therapies for exon duplication mutations, accounting for around 6% of all mutations, with the intent to induce production of wild-type transcripts and protein. As a model of the most common single exon duplication, we have developed a new mouse with a duplication of exon 2 (the Dup2 mouse) that largely recapitulates the findings in the standard mdx mouse. Using this mouse, we are testing both virally (AAV) mediated skipping induced by a modified U7snRNA (rAAV9.U7.ACCA), and antisense oligomer-induced skipping. Intramuscular injections of the tibialias anterior (TA) (N=6 muscles each) were performed rAAV9.U7.ACCA at 6 doses between 1×1010 and 1×1012 total vector genomes. Intramuscular (TA) injections of an exon 2-directed antisense peptide-morpholino conjugate (PPMO) were performed at doses of either 10 or 20 ug total PPMO (N=3 each). For each study, mice were sacrificed 1 month post injection and muscle analysis of DMD mRNA and dystrophin protein expression. Treatment with either modality results in significant skipping of the duplicated exon 2, and expression of a functional dystrophin isoform at levels of up to 30% of normal with AAV-mediated skipping. Physiology has been assessed in the AAV-treated mice, in which correction of TA absolute force deficits in comparison to the background Bl6 strain are seen, along with a partial yet significant correction of eccentric contraction injury in comparison to untreated Dup2 mice. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Over-skipping – to the exclusion of exon 2 entirely – results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin that allows for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, consistent with the observation that patients expressing it have minimally symptomatic (or even asymptomatic) Becker muscular dystrophy (BMD), and suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5’ end of the gene.