Gene Editing Technique Repairs Mutation Causing Duchenne Muscular Dystrophy

DMD is caused by mutations in the gene that encodes dystrophin, a protein crucial for maintaining muscle cell integrity and function, and the subsequent disruption of the dystrophin-associated protein complex (DAPC). The mutation occurs on the X-chromosome, and the disease effects about one of every 3,500 boys whose muscle function is so degraded that they die usually before reaching the age of 30.

CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with cas genes that code for proteins related to CRISPRs. Since 2013, the CRISPR/Cas system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 enzyme and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR/Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides (CRISPRs) that shepherd the Cas9 protein to the target gene on a DNA strand.

Investigators at the University of California, Los Angeles (USA) applied the CRISPR/Cas9 method to hiPSCs derived from DMD patients. They reported in the February 11, 2016, online edition of the journal Cell Stem Cell that they had successfully deleted a gene segment comprising up to 725 kilobases and rejoined the non-homologous ends to repair the DMD gene. This was the largest CRISPR/Cas9-mediated deletion shown to date in DMD.

Use of hiPSCs allowed evaluation of dystrophin in disease-relevant cell types. Cardiomyocytes and skeletal muscle myotubes derived from repaired hiPSC clonal lines had restored dystrophin protein. The internally deleted dystrophin was functional as demonstrated by improved membrane integrity and restoration of the dystrophin glycoprotein complex in vitro and in vivo.

"This work demonstrates the feasibility of using a single gene editing platform, plus the regenerative power of stem cells to correct genetic mutations and restore dystrophin production for 60% of Duchenne patients," said senior author Dr. April Pyle, associate professor of microbiology, immunology, and molecular genetics at the University of California, Los Angeles.

The investigators stressed that the CRISPR/Cas9 platform for Duchenne will probably require another 10 years of research before becoming available for clinical use. It is not yet available in clinical trials and has not been approved by the [US] Food and Drug Administration for use in humans.

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University of California, Los Angeles