Genome editing-mediated Utrophin upregulation in Duchenne Muscular Dystrophy stem cells


Scientific Other

Poster Number: 133


Kasturi Sengupta, PhD, Manoj Mishra, PhD, Emanuele Loro, PhD, Melissa Spencer, PhD, April Pyle, PhD, Tejvir Khurana, PhD


1. University of Pennsylvania, 2. University of Pennsylvania, 3. , 4. Molecular Biology Institute, Department of Neurology, Center for Duchenne Muscular Dystrophy at UCLA, 5. University of California, Los Angeles, 6. University of Pennsylvania

Utrophin upregulation is considered a promising therapeutic strategy for Duchenne Muscular Dystrophy (DMD). A number of miRNAs post-transcriptionally regulate utrophin expression by binding their cognate sites in the 3' UTR. Previously, we have shown that miRNA-mediated UTRN repression can be alleviated using miRNA let-7c site blocking oligonucleotides (SBOs) to achieve utrophin upregulation and functional improvement in mdx mice. Here, we designed and applied the CRISPR/Cas9-mediated genome editing to delete five miRNA binding sites (miR-150, miR-296-5p, miR-133b, let-7c and miR-196b) clustered in a 500 bp inhibitory miRNA target region (iMTR) within the UTRN 3' UTR, for achieving higher expression of endogenous utrophin protein. Successful deletion of the UTRN iMTR in DMD patient derived induced pluripotent stem cells (DMD-hiPSCs) resulted in ca. two-fold higher levels of utrophin protein expression. Differentiation of the UTRN edited DMD-hiPSCs (UTRNdeliMTR) by MyoD overexpression resulted in increased sarcolemmal alpha-sarcoglycan staining consistent with improved dystrophin glycoprotein complex (DGC) restoration. These results demonstrate that CRISPR/Cas9-based UTRN genome editing offers a novel strategy to "repress the repression" and achieve utrophin upregulation. In principle, this utrophin upregulation therapeutic strategy would be applicable to all DMD patients, irrespective of the dystrophin mutation status.
Funding: Orphan Disease Centre, Muscular Dystrophy Association (MDA)