A new gene editing technique can be used to correct mutations in muscle stem cells and pave the way for the first possible cell therapy for genetic muscle disorders. The ECRC team, led by Professor Simone Spuler, has published its results in the journal “JCI Insight”.
Muscle stem cells enable our muscles to build and regenerate for a lifetime through exercise. However, when certain muscle genes are mutated, the opposite occurs. In patients with muscular dystrophy, the skeletal muscle begins to weaken in childhood. Suddenly these children can no longer run, play the piano or climb stairs, and often by the age of 15 they are dependent on a wheelchair. There is currently no therapy for this condition.
Now we can access the gene mutations of these patients using CRISPR-Cas9 technology. We care for more than 2,000 patients in the Charité outpatient clinic for muscle disorders and quickly recognized the potential of the new technology. “
Professor Simone Spuler, Head of the Myology Laboratory at the Experimental and Clinical Research Center (ECRC)
The researchers immediately started working with some of the affected families and have now presented their results in the journal JCI Insight. In the families studied, the parents were healthy and had no idea that they had a mutated gene. The children all inherited one copy of the disease mutation from both parents.
Processed human muscle stem cells developed into muscle fibers in mice
The term “muscular dystrophy” refers to about 50 different diseases. “They all take the same course, but differ due to the mutation of different genes,” explains Spuler. “And even within the genes, different places can be mutated.” After a genome analysis of all patients, the researchers selected a family based on their particular form of the disease: Muscular dystrophy 2D / R3 of the limb girdle is relatively common, progresses quickly and has a suitable docking point for the “genetic scissors” close to the mutation on the DNA.
For the study, the researchers took a sample of muscle tissue from a ten-year-old patient, isolated the stem cells, multiplied them in vitro, and used base manipulation to replace a base pair at the mutated site. They then injected the processed muscle stem cells into mouse muscles, which can tolerate foreign human cells. These multiplied in rodents and mostly developed into muscle fibers. “This enabled us to show for the first time that it is possible to replace diseased muscle cells with healthy ones,” says Spuler. After further testing, the repaired stem cells are reintroduced to the patient.
Basic machining – a sophisticated technique
Basic editing is a newer and more sophisticated variant of the CRISPR-Cas9 gene editing tool. While in the “classic” method both DNA strands are cut with these molecular scissors, the Cas enzymes used for base processing only remove the residual glucose from a certain base and attach another, creating a different base at the target side? ˅. “This tool works more like a pair of tweezers than a pair of scissors and is perfect for targeting point mutations in a gene,” says Dr. Helena Escobar, molecular biologist on Spuler’s team. “It’s also a much safer method because unwanted changes are extremely rare. In the genetically repaired muscle stem cells, we haven’t seen any misoccounting in unintended regions of the genome.” Escobar is the lead author of the study and the one who developed the technique for the muscle cells.
Autologous cell therapy, in which a patient’s stem cells are removed, processed outside the body, and then re-injected into the muscle, means that people who are already in a wheelchair can no longer walk. “We cannot repair muscles that are already stunted and have been replaced by connective tissue,” emphasizes Spuler. And the number of cells that can be manipulated in vitro is also limited. However, the study provides the first evidence that a form of therapy is possible even for a group of previously incurable diseases, and it could be used to repair small muscle defects such as the one in the finger flexor.
One step closer to a cure
However, this is only the first step. “The next milestone will be to find a way to inject the basic editor directly into the patient. Once it’s in the body, it swims around for a short time, manipulating all the muscle stem cells, and then quickly collapsing again.” The team plans to begin its first experiments in a mouse model soon. If this works, newborns could be tested for gene mutations in the future, and curative therapy could be initiated at a time when comparatively few cells would need to be processed.
What exactly could in vivo therapy for muscular dystrophy look like? This is something that scientists have been testing for some time on animal models with viral vectors. However, Helena Escobar explains that the risk of miseditation and toxic effects is too high because these vectors remain in the body for too long. “An alternative would be mRNA molecules that contain the information that the publisher needs to synthesize the tools in vivo,” says the molecular biologist. “mRNA is broken down very quickly in the body, so that the therapeutic enzymes can only remain in an active state for a short time.” Therapy could likely be repeated if necessary. “We don’t yet know whether this has to be a therapy cycle that includes several applications.”
This therapeutic approach would mean that, in contrast to autologous cell therapy, not every patient would have to be treated individually. For any form of muscle therapy, one “tool” would be sufficient to heal the muscle atrophy before any major damage occurred. But there is still a long way to go.
Escobar, H., et al. (2021) Base editing repairs an SGCA mutation in human primary muscle stem cells. JCI Insight. doi.org/10.1172/jci.insight.145994.