143. Off-Target Analysis of a rAAV-U7snRNA Vector Used for the Treatment of Duchenne Patients By Exon Skipping

Abstract

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1 male in 5000 and caused by mutations in the gene encoding for the Dystrophin protein. So far, there are no strongly effective treatments but gene-based therapies are currently being developed. Among them, the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in shorter in-frame transcripts that are translated into functionally active Dystrophin. Exon skipping can be achieved using modified U7-small nuclear RNA (snRNA) in which a therapeutic antisense oligonucleotide (AO) is cloned. Such optimized U7snRNA, called U7.OPT, can be packaged in recombinant vectors derived from Adeno-Associated Virus (rAAV), allowing a long-lasting repair of the dystrophin messenger after one single administration.We recently published the high ability of a rAAV8.U7.OPT vector to transduce the skeletal muscle but also the liver and restore the open reading frame of the dystrophin transcript in GRMD dogs muscles injected in one forelimb. We are now moving forward to a phase I/II clinical trial in DMD patients treatable by skipping of the exon 53 of the dystrophin messenger.Because an abundant presence of the therapeutic U7.OPT RNA can in theory lead to partial hybridization to non-targeted messenger(s) with unwanted side effects, we looked for possible off-target events. Primary human myotubes and hepatocytes, untransduced or transduced with our clinical rAAV.U7.OPT vector, served as our in vitro model system. The skipping of exon 53 of the human dystrophin messenger upon vector transduction was validated. In silico analysis using the miRanda algorithm identified some gene candidates expressed in liver and muscle, i.e. messengers which could be the target of our dystrophin-specific AO. Three of them were analyzed by RT-PCR and RTqPCR and showed not detectable exon skipping events or differential expression. Other candidates are currently analyzed. Simultaneously, further investigation included a full transcriptomic analysis by RNA sequencing comparing the samples obtained from untransduced vs transduced primary cells. Preliminary results revealed differential gene expression patterns between the two types of samples. We are now exploring the respective impact of the rAAV transduction itself vs the AO effect per se to finally identity potential off-target events.The complete data will be presented and discussed in the context of our future clinical trial in DMD patients.This project is supported by AFM (Association Francaise contre les Myopathies) and ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Merieux and supported by research and innovation aid from the French public agency, BpiFrance.