This photo shows green dystrophin protein restored by dual-Cas3 in skeletal muscle derived from DMD patient iPSCs. Credit: Akitsu Hotta
Duchenne muscular dystrophy (DMD) is a muscle degeneration disorder caused by mutations affecting the dystrophin gene. On August 24 in the journal Stem Cell Reports, researchers show how a dual CRISPR RNA method restored dystrophin protein function in induced pluripotent stem cells derived from DMD patients.

The approach worked by removing large sections of the dystrophin gene, allowing the cells to skip faulty or misaligned sections of the genetic code. This yields truncated but still functional proteins for a wide variety of mutation patterns associated with DMD.

“Dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via multi-exon skipping induction,” says senior author Akitsu Hotta of Kyoto University. “We expect this study to enlighten 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, deleting a small section of the gene can only be used for a limited number of DMD patients. For example, the most common mono-exon skipping of exons 51, 53, and 45 can be applied to 13%, 8%, and 8% of DMD patients, respectively.

Multi-exon skipping (MES) has broad applicability to various DMD mutation patterns. By targeting the mutation hotspots in the dystrophin gene, MES from exon 45 to 55 was estimated to benefit more than 60% of DMD patients. Unfortunately, few techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases.

To overcome this hurdle, Hotta and his team used CRISPR-Cas3 to induce a deletion of up to 340 kilobases at the dystrophin exon 45-55 region in various DMD mutation patterns. Because it was rare to observe a deletion of more than a hundred kilobases using a single CRISPR RNA—which helps to locate the correct segment of DNA—the researchers used a pair of CRISPR RNAs inwardly sandwiching the target genomic region.

The authors note potential limitations of the dual CRISPR RNA system. First, there is variation in the deletion pattern, and the precise start and end points of the deletion cannot be fully controlled. This could be a drawback when a large but precise deletion is required. 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 might apply to future gene therapies once we’re able to deliver the dual-Cas3 components in vivo to skeletal muscle tissues safely and efficiently,” says Hotta. “The ability to induce several hundred kilobases of DNA deletion itself also has broad applicability for basic research when a large deletion is needed.”

Check out our AAV CDMO service to expedite your gene therapy research
About PackGene

PackGene is a CRO & CDMO technology company that specializes in packaging recombinant adeno-associated virus (rAAV) vectors. Since its establishment in 2014, PackGene has been a leader in the AAV vector CRO service field, providing tens of thousands of custom batches of AAV samples to customers in over 20 countries. PackGene offers a one-stop CMC solution for the early development, pre-clinical development, clinical trials, and drug approval of rAAV vector drugs for cell and gene therapy (CGT) companies that is fast, cost-effective, high-quality, and scalable. Additionally, the company provides compliant services for the GMP-scale production of AAVs and plasmids for pharmaceutical companies, utilizing five technology platforms, including the π-Alpha™ 293 cell AAV high-yield platform and the π-Omega™ plasmid high-yield platform. PackGene’s mission is to make gene therapy affordable and accelerate the launch of innovative gene drugs. The company aims to simplify the challenging aspects of gene therapy development and industrialization processes and provide stable, efficient, and economical rAAV Fast Services to accelerate gene and cell therapy development efforts from discovery phase to commercialization.

Related News

Scientists develop potential stealth cancer therapy

Credit: ACS Central Science (2024). DOI: 10.1021/acscentsci.4c00559Sneaking by cancer's defenses, by disguising tumor-fighting antibodies inside the molecules cancer uses to nourish tumor growth, is the basis of a novel therapy from Yale Cancer Center researchers at...

read more

Related Services

Plasmids GMP Services

Multiple scales & grade of solutions of various kind of plasmids suitable for multiple treatments in a fast and cost effective way.


AAV GMP Services

Ranging from small-scale AAV production, to large-scale AAV cGMP manufacturing for animal studies.


aav icon

Technology Platforms

PackGene’s proprietary π-Alpha™ 293 AAV High-yield Platform increases AAV production by 3 to 8 times that of traditional platforms.