Genome Editing-Mediated Utrophin Upregulation in Duchenne Muscular Dystrophy Stem Cells

Kasturi Sengupta; Manoj K Mishra; Emanuele Loro; Melissa J Spencer; April D Pyle; Tejvir S Khurana

doi:10.1016/j.omtn.2020.08.031

Genome Editing-Mediated Utrophin Upregulation in Duchenne Muscular Dystrophy Stem Cells

Mol Ther Nucleic Acids. 2020 Aug 29:22:500-509. doi: 10.1016/j.omtn.2020.08.031. eCollection 2020 Dec 4.

Authors

Kasturi Sengupta¹, Manoj K Mishra¹, Emanuele Loro¹, Melissa J Spencer^{2

3}, April D Pyle^{2

4}, Tejvir S Khurana¹

Affiliations

¹ Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
² Molecular Biology Institute, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.
³ Department of Neurology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁴ Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, 90095, USA.

Abstract

Utrophin upregulation is considered a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). A number of microRNAs (miRNAs) post-transcriptionally regulate utrophin expression by binding their cognate sites in the 3' UTR. Previously we have shown that miRNA: 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 used CRISPR/Cas9-mediated genome editing to delete five miRNA binding sites (miR-150, miR-296-5p, miR-133b, let-7c, miR-196b) clustered in a 500 bp inhibitory miRNA target region (IMTR) within the UTRN 3' UTR, for achieving higher expression of endogenous utrophin. Deleting the UTRN IMTR in DMD patient-derived human induced pluripotent stem cells (DMD-hiPSCs) resulted in ca. 2-fold higher levels of utrophin protein. Differentiation of the UTRN edited DMD-hiPSCs (UTRNΔIMTR) by MyoD overexpression resulted in increased sarcolemmal α-sarcoglycan staining consistent with improved dystrophin glycoprotein complex (DGC) restoration. These results demonstrate that CRISPR/Cas9-based UTRN genome editing offers a novel utrophin upregulation therapeutic strategy applicable to all DMD patients, irrespective of the dystrophin mutation status.

Keywords: CRISPR-Cas9; DMD-hiPSC; Duchenne muscular dystrophy; IMTR; UTRNΔIMTR; gene therapy; stem cells; utrophin.

Abstract

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