PMO-based miRNA site blocking oligo (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne Muscular Dystrophy (DMD)

Non-Gene Targeting Therapies
Kasturi Sengupta, PhD, Emanuele Loro, PhD, Tejvir Khurana, PhD
1. University of Pennsylvania, 3. University of Pennsylvania

Upregulation of utrophin, a dystrophin related protein, is a promising therapeutic approach for the treatment of Duchenne Muscular Dystrophy (DMD). Utrophin expression is repressed at the post-transcriptional level by a group of miRNAs targeting the utrophin (UTRN) mRNA 3’UTR. Among them, miRNA let-7c is highly conserved and expressed in skeletal muscle tissues. Here we have designed five phosphorodiamidate morpholino oligomer (PMO)-based site blocking oligos (let-7c PMO SBOs) complementary to the miRNA let-7c binding site in UTRN 3’UTR, with the goal of inhibiting miRNA let-7c interaction with UTRN mRNA and thus upregulating utrophin. To test the efficacy of different SBO's we used our previously described mouse muscle reporter cell line (C2C12 UTRN 5’luc3’) in which the luciferase gene is flanked with UTRN 5’- and 3’-UTR sequences. The five different let-7c PMO SBOs (S24, S28, S31, S32 and S56) were evaluated in this reporter cell line, for the higher luciferase expression as a readout for their site blocking efficiency. The let-7c SBO PMOs were further tested in C2C12 myoblast cell line for alleviation of miRNA let-7c repression and higher expression of endogenous utrophin protein. Among the five let-7c PMO SBOs, the S56 PMO resulted in ca. two-fold higher expression of utrophin protein in vitro. The S56 SBO was used for preclinical studies by treating one-month old mdx mice at 80 mg/kg for five weeks. Treatment resulted in two-fold higher utrophin protein expression in skeletal muscles and improvement in dystrophic pathophysiology. In summary, we showed that PMO-based miRNA let-7c site blocking has potential therapeutic applicability for upregulating utrophin expression as a therapeutic approach for DMD.

Funding: Muscular Dystrophy Association (MDA)