Talk abstracts

Talk on Wednesday 04:45-05:00pm submitted by Tabatha Simmons

Intravenous delivery of a rAAV9 U7snRNA vector targeting exon 2 results in widespread dystrophin expression in the Dup2 DMD mouse model

Tabatha Simmons (The Center for Gene Therapy, Nationwide Childrens Hospital; The Department of Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus OH, USA), Nicolas Wein (The Center for Gene Therapy, Nationwide Childrens Hospital, Columbus OH, USA), Adeline Vulin-Chaffiol (The Center for Gene Therapy, Nationwide Childrens Hospital, Columbus OH, USA), Andrea Rutherford (The Center for Gene Therapy, Nationwide Childrens Hospital, Columbus OH, USA), Paul Janssen (Department of Physiology and Cell Biology, The Ohio State University, Columbus OH, USA), Kevin M Flanigan (The Center for Gene Therapy, Nationwide Childrens Hospital; Departments of Pediatrics and Neurology, The Ohio State University, Columbus OH, USA)

Abstract:
Accounting for around 6% of all cases of Duchenne muscular dystrophy, exon duplications provide an excellent avenue for new exon skipping therapies. We sought to test the efficacy of virally-mediated duplication skipping in the novel Dup2 mouse, modeling the most common single-exon duplication (exon 2) seen in DMD patients. A targeting construct was created containing four copies of a modified U7snRNA, each of which targets either the splice donor or acceptor sites of exon 2 (U7sn RNA-ACCA). Both tibilias anterior IM injections and tail vein IV injections were done in 8 week mice and then analyzed 4 weeks later at both the mRNA and protein level. RT-PCR reveals widespread exon 2 skipping, with the simultaneous presence of all 3 predicted transcripts – duplicated exon 2, wild-type, and deleted exon 2 – in variable proportions. Dystrophin expression and location was confirmed by immunoblot as well as by immunofluorescence. Treatment normalized hindlimb and forelimb grip strength and partially corrected extensor digitorum longus force deficits seen in untreated Dup2 mice. These results demonstrate the utility of the Dup2 mouse model as a tool for testing potential duplication exon-skipping strategies. They confirm that IV delivery of rAAV9.U7snRNA is able induce exon 2 skipping and to drive the production of a functional dystrophin protein, suggesting a promising strategy for future clinical development.

Keywords: AAV, muscle, therapy