P.141PPMO-mediated skipping therapy of duplicated exon 2 in the DMD gene

Abstract

Duchenne muscular dystrophy (DMD) is caused by fatal mutations that disrupt the reading frame of the DMD gene, leading to lack of dystrophin protein. The only approved gene corrective therapy is an antisense phosphorodiamidate morpholino oligomer (PMO) that alters splicing to exclude exon 51 from the mRNA, restoring translation of a shortened but functionally active protein. Use of antisense PMO skipping in the presence of single exon duplications, in contrast, may restore a wild-type DMD transcript and dystrophin protein. Here we report the application of cell-penetrating peptide-conjugated PMO, or PPMO, to target the DMD exon 2 splice acceptor site in Dup2 mice, which carry a duplication of exon 2 (the most common single duplication mutation in DMD patients, accounting for around 10% of all duplication mutations). In the presence of an exon 2 duplication, skipping can result in two therapeutic transcripts. Skipping of a single copy of exon 2 results in a wild-type transcript, whereas skipping both copies results in a transcript lacking exon 2 entirely (Del2), but from which functionally active dystrophin protein can be expressed via utilization of an exon 5 internal ribosome entry site (IRES). A single intravenous injection of PPMO was delivered to 12 week old mice at three dose levels; saline injection and scrambled PPMO were used as controls. All mice (n=5-6 each) were sacrificed one week post-injection. Five muscles–tibialis anterior (TA), gastrocnemius (Gas), quadriceps (Qua), diaphragm (Dia) and heart were harvested for analysis of DMD mRNA transcripts by RT-PCR, and for dystrophin protein expression by western blot and immunofluorescence staining. The single injection resulted in a clear dose response of exon skipping, maximal at the highest dose tested; robust skipping of the duplicated exon 2 (both WT and Del2 transcripts) was seen in skeletal muscles, including TA (40.8%), Gas (39.7%), Qua (53.5%), and Dia (53.1%), with minimal skipping observed in heart muscle (8%). Dystrophin protein restoration was seen at levels of up to 13% in Dia and up to 15% in Gas, and at lower levels in other tissues, likely reflecting the single injection and short time period allowed for expression. We are evaluating longer term expression, with a goal of developing a meaningful therapy for patients with exon 2 duplications and potentially other 5′ DMD mutations.