Gene editing corrects a muscular dystrophy in the laboratory and opens the door to its repair

2023-08-24 18:00:03

Gene editing has successfully corrected Duchenne muscular dystrophy (DMD) in the laboratory. The results, which are published in the journal «Stem Cell Reports», open the door to its repair, a muscle wasting disorder caused by mutations affecting the dystrophin gene.

The researchers of the Kyoto University (Japan) show how dual gene editing – CRISPR RNA -, the famous molecular scissors, restored the function of the dystrophin protein in induced pluripotent stem cells derived from patients with Duchenne muscular dystrophy.

The therapy removed large sections of the dystrophin gene, allowing the cells to skip faulty or misaligned sections of the genetic code. This generates truncated but functional proteins for a wide variety of disease-associated mutation patterns.

“Dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore the dystrophin protein by inducing multi-exon skipping,” explains lead author Akitsu Hotta. «We hope this study will shed light on new ways to treat DMD patients. and other genetic disorders that require extensive deletions.”

Due to significant variations in the mutation patterns affecting the dystrophin gene, deletion of a small section of the gene can only be used in a limited number of DMD patients. For example, the most common monoexon skipping of exons 51, 53, and 45 may apply to 13%, 8%, and 8% of DMD patients, respectively.

Multiple exon skipping (MES) has broad applicability to various DMD mutation patterns. By targeting mutation hotspots in the dystrophin gene, exon 45 to 55 MES was estimated to benefit more than 60% of DMD patients.

few techniques

Unfortunately, few techniques are available to induce a large deletion covering target exons spread over several hundred kilobases.

To overcome this obstacle, Hotta and his team used CRISPR-Cas3 to induce a deletion of up to 340 kilobases in the region of dystrophin exon 45-55 in various DMD mutation patterns.

Because it was rare to see a deletion of more than 100 kilobases using a single CRISPR RNA, which helps locate the correct segment of DNA, the researchers used a pair of CRISPR RNAs interspersing the target genomic region.

The researchers acknowledge possible limitations of the dual CRISPR RNA system. First, there is variation in the removal pattern and the precise start and end points of removal cannot be fully controlled. «This could be inconvenient when a large but precise removal is required.», they explain.

Second, the study did not demonstrate the functionality of the recovered dystrophin protein. Third, other methods should be developed to improve the overall genome editing efficiency of the Cas3 system.

“Our dual-Cas3 system could be applied to future gene therapies once we are able to deliver the dual-Cas3 components in vivo to skeletal muscle tissues safely and efficiently,” Hotta said. The ability to induce deletion of several hundred kilobases of DNA also has wide applicability for basic research when a large deletion is needed.”