Researchers have demonstrated the CRISPR-Cas9 gene editing tool can be used to treat muscular dystrophies. Muscular dystrophies are inherited conditions that cause muscles to weaken, causing a progressive loss of muscle function, which can result in the weakening of the heart or muscles required for breathing. Muscular dystrophies are caused by mutations in the genes responsible for maintaining the structure and function of the muscles and are usually the result of a single mutation in a gene. There are approximately 50 different disorders that fall under the umbrella term of muscular dystrophies, and while the mutations that cause those disorders are different, the symptoms and results are similar.
A team of researchers at the Experimental and Clinical Research Center (ECRC) of the Max Delbrück Center for Molecular Medicine and Charité, Germany have been exploring the use of the CRISPR gene editing tool for correcting the mutations that cause muscular dystrophies. “We have for several years been pursuing the idea of taking muscle stem cells from diseased patients, using CRISPR-Cas9 to correct the faulty genes, and then injecting the treated cells back into the muscles so that they can proliferate and form new muscle tissue,” said Dr. Helena Escobar, corresponding author of the study.
The challenges faced by the researchers were the absence of a method to isolate and propagate muscle stem cells, and their susceptibility to extensive ex vivo manipulations. The researchers had previously had some success using plasmids to introduce genetic instructions for the gene editor into stem cells, which worked in mice with muscle atrophy; however, the technique could not be used for treating patients due to the risk that plasmids could unintentionally integrate into human DNA, which could cause undesirable effects that would be difficult to assess.
The researchers have now developed a new technique that involves harvesting muscle stem cells from diseased patients and using mRNA-based delivery of SpCas9 and an adenine base editor. The mRNA would normally be too large to get inside the stem cells, so the researchers used a process called electroporation, which makes the cell membranes temporarily more permeable to larger molecules. The researchers found that this process allowed them to get the mRNA into almost all stem cells. The researchers used CRISPR-Cas9 to genetically alter the stem cells and showed the genetically altered muscle stem cells were just as healthy and capable as unaltered muscle stem cells, and could fuse with each other to form young muscle fibers.
The researchers say they achieved >90% efficient genome editing in human muscle stem cells from many donors, regardless of age and gender, and without any enrichment step. Professor Simone Spuler, head of the Myology Lab at the ECRC, said they are now planning a clinical trial toward the end of the year on 5-7 patients with muscular dystrophies.
You can read more about the study in the paper – mRNA-mediated delivery of gene editing tools to human primary muscle stem cells – which was recently published in Molecular Therapy Nucleic Acids. DOI: 10.1016/j.omtn.2022.02.016