29 | RNA-based combinatorial multi-target therapy for the treatment of Duchenne muscular dystrophy

Mathieu Horeau, C. Pezzini, M. Sandri | Department of Biomedical Sciences, University of Padova, Italy.

Duchenne muscular dystrophy (DMD) is a severe and fatal X-linked desease, caracterize by the lack of dystrophin leading to structural fragility of muscle fibers and promotes a pro-catabolic metabolic imbalance. To date, no curative treatment is available, partly due to the difficulty in restoring full dystrophin expression and developing non-immunogenic approaches. Reactivation of utrophin, a dystrophin’s paralogous protein naturally downregulated by microRNAs, has shown strong potential to compensate for dystrophin deficiency. The RNA_DMD project aims to address three major challenges that are 1) to reactivate utrophin expression to compensate for the loss of dystrophin, 2) to inhibit pro-catabolic pathways to promote muscle growth, 3) to develop non-immunogenic vectors for effective and tolerable combination therapy. We hypothesize that combination therapy could both restore muscle structural integrity and prevent or reverse muscle wasting and associated loss of function. To study aim 2, we used the bicistronic plasmid expressing green fluorescent protein (GFP) and bearing our shRNA against FOXO1/3 or MURF1caring by an adeno-associated virus with a muscle specific serotype (AAV-MYO). To date, we test the efficiency of our AAV-MYO-shRNA in wild-type (WT) mice by electroporation in tibialis anterior (TA) (1010 vg/muslce) and systemically (1012 vg/mouse). And then, we moved to 3-week-old D2-mdx mice (a severe model of DMD). Our preliminary data highlights in WT animals, that combined electroporation of AAV-MYOshMuRF1 and AAV-MYO-shFOXO1/3 in the tibialis anterior muscle leads to a reduction in the expression of MuRF1 (-24%; P=0.04), FOXO1 (-36%; P=0.002), and 3 (-39%; P=0.03) genes, associated with muscle hypertrophy described by a +30% increase in CSA (P=0.03) of TA. In 3-week-old D2-mdx mice separate intramuscular injection of AAV-MYO-shMuRF1 and AAVMYO- shFOXO1/3 led to a reduction after 4 weeks of MuRF1 transcript (-45%; P<0.001) and FOXO1 respectively (-27%; P=0.04).