NEW YORK (GenomeWeb) – Using the CRISPR/Cas9 genome editing system, researchers from the University of Texas Southwestern Medical Center were able to prevent Duchenne muscular dystrophy in a mouse model of the disease.
As the researchers reported in the online early edition of Science today, they were able to edit germline mutations in the dystrophin gene to produce mosaic mice containing both corrected and uncorrected versions of the gene. Still, the researchers were able to prevent the disease phenotype in the mice.
"With the anticipated technological advances that will facilitate genome editing of postnatal somatic cells, this strategy may one day allow correction of disease-causing mutations in the muscle tissue of patients with DMD," Eric Olson, a professor at UT-Southwestern, and his colleagues said in their paper.
DMD is an X-linked condition that affects about one in 3,500 boys. It is marked by progressive muscle weakness and is often fatal by the age of 25 years due to breathing difficulties and cardiomyopathy.
For this study, the researchers aimed to correct the nonsense mutation in exon 23 of the Dmd gene that the mdx mouse model of Duchenne muscle dystrophy harbors.
Olson and his colleagues designed a single-guide RNA targeting that exon and a single-stranded oligodeoxynucleotide to be used as a template for the homology-directed repair that can occur after Cas9 creates a double-stranded break at the targeted site. Non-homologous end joining, the researchers noted, could also correct the point mutation.
After optimizing their method, the researchers applied it to the mdx mice. Using RFLP analysis, the researchers identified 11 mice with corrected genes, seven that were corrected through homology-directed repair and four by non-homologous end joining.
Sequencing of the mice revealed that CRISPR/Cas9–mediated germline editing led to genetically mosaic corrected mice, which had between 2 percent and 100 percent correction of the Dmd gene. Mosaicism, they noted, occurs if editing takes place after the zygote stage and repair happens in only a subset of embryonic cells.
The researchers noted that the mice developed into adults and exhibited no signs of tumors or other abnormal phenotypes.
Additionally, they searched the genomes of the mice for evidence of off-target genome editing. They particularly focused on 32 sites in the mouse genome with similarity to the site they targeted, but found no indications of off-site editing as compared to controls.
Olson and his colleagues also tested various muscle tissues from the mice, finding that mice with more than 40 percent of mdx alleles corrected by homology-directed repair or with 83 percent corrected through in-frame non-homologous end joining did not exhibit the dystrophic muscle phenotype and expressed dystrophin in their myofibers.
Correction of only 17 percent of mutant Dmd alleles, the researchers noted, was enough to enable dystrophin expression in a majority of myofibers, with the expression level rivaling that of wild-type mice. This, the researchers said, suggest that the corrected skeletal muscle cells confer a selective advantage.
Over time, the researchers added, skeletal muscle, though not cardiac muscle, increasingly contained more corrected myofibers, indicating progressive rescue with age. This, they said, may occur as corrected satellite cells — muscle stem cells — are recruited into damaged myofibers.
This suggests that direct CRISPR/Cas9 editing of satellite cells in vivo could be a way to promote muscle repair in DMD, they said.
Still, they noted that genome editing isn't ready for the clinic as delivery method issues and safety concerns need to be addressed, though the approach remains promising.
"Despite the challenges listed above, with rapid technological advances of gene delivery systems and improvements to the CRISPR/Cas9 editing system, the approach we describe could ultimately offer therapeutic benefit to DMD and other human genetic diseases in the future," Olson and his colleagues said.