Restoration of Full-Length Dystrophin Expression in the Dup2 Mouse Induced by Systemic Delivery of a Peptide-Conjugated Morpholino Oligomer

Duchenne muscular dystrophy (DMD) is an x-linked recessive genetic disorder caused by mutations that disrupt the reading frame of the DMD gene and lead to lack of dystrophin protein. There are two approved gene corrective therapies that use antisense phosphorodiamidate morpholino oligomers (PMOs). These therapies alter splicing to exclude exon 51 or exon 53 from the mRNA, restoring translation of a truncated, yet functionally active protein. However, in the presence of single exon duplications, the antisense PMO therapies allow restoration of a wild-type (WT) DMD transcript and dystrophin protein. Here we report the application of cell-penetrating peptide-conjugated PMO (PPMO) to target the DMD exon 2 splice acceptor site in Dup2 mice. These mice carry a duplication of exon 2, which is the most common single duplication mutation in DMD patients, accounting for approximately 10% of all duplication mutations. In the presence of an exon 2 duplication, skipping can result in either of two therapeutic transcripts. Skipping a single copy results in a WT 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). The dose-escalation and time point studies were conducted in 12-week-old Dup2 mice which were intravenously injected with different doses of PPMO. Scrambled PPMO and saline-injected mice were used as controls. Dup2 mice (n=5-6) were sacrificed at 7 days post-injection in dose-escalation study and at 15-, 30- and 60 days post PPMO administration in time point study. Five muscles—tibialis anterior (TA), gastrocnemius (Gas), quadriceps (Quad), 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 in dose escalation study demonstrated a positive dose response; robust skipping of the duplicated exon 2 (both WT and Del2 transcripts) was seen at the higher does in all skeletal muscles: TA (40.8%), Gas (39.7%), Quad (53.5%), and Dia (53.1%), with minimal skipping observed in heart muscle (8%). The time-point study was performed using the highest dose from the escalation study and showed robust skipping of the duplicated exon 2 in most skeletal muscles at 15 days post-injection: TA (68.2%), Gas (45.1%), Quad (55.1%), Dia (62.4%) and heart (14.8%). The dystrophin expression was determined by western blot. The highest level of dystrophin was observed at 30 days post-injection with an average of 11% in heart, 40-52% in TA, Dia and Gas, and 71% in Quad. Dystrophin expression decreased in all skeletal muscles at 60 days post-injection with peak expression in Gas still showing an average of 19%. Future studies will evaluate longer term expression, with a goal to develop a meaningful therapy for patients with exon 2 duplications and potentially other 5’ DMD mutations.