Most forms of muscular dystrophy are caused by a single faulty gene. However, the progressive muscle wasting has not yet been cured. Researchers have now laid the foundations for a possible therapeutic approach: With the help of mRNA, they have introduced the CRISPR/Cas9 gene scissors into human muscle stem cells. In this way, they were able to repair a defective gene. In cell culture, the repaired stem cells successfully formed muscle fibers. Clinical trials on patients are to follow.
A tiny change in the genome can have serious repercussions. In almost all of the around 50 known forms of muscular dystrophy, a single gene mutation means that certain proteins that are essential for muscle metabolism are missing. As a result, muscle tissue breaks down over time, leading to muscle weakness and eventually the inability to move, swallow, or grasp objects on their own. Existing drugs can delay the progression of the disease and increase the life expectancy of those affected. Muscular dystrophy is not yet curable.
Gene repair in muscle stem cells
For a cure it would be necessary to repair the defective gene. A team led by Christian Stadelmann from the Experimental and Clinical Research Center (ECRC) of the Max Delbrück Center for Molecular Medicine (MDC) and the Charité – Universitätsmedizin Berlin has now researched a possibility for such a gene therapy. “For years we have been pursuing the idea of taking muscle stem cells from sick people, repairing the modified genes using CRISPR/Cas9 gene scissors and injecting the treated cells back into the muscles so that they multiply there and form new muscle tissue,” explains Stadelmann’s colleague Helena Escobar.
For the current study, the researchers took muscle stem cells from donors of different ages and sexes. The researchers used mRNA to smuggle the construction manual for the CRISPR/Cas9 gene scissors into the cells. In earlier experiments with mice, they used so-called plasmids instead of this messenger RNA – circular, double-stranded DNA molecules from bacteria. In contrast to single-stranded mRNA, however, there is a risk with plasmids that they will unintentionally integrate into the human genome and lead to unwanted effects. “We would therefore not have been able to treat patients like this,” says Escobar.
mRNA as a transporter
The mRNA, on the other hand, is broken down in the cell after a short time. “Thus, mRNA-mediated delivery provides a platform to prompt the cell to produce gene-modifying enzymes for therapeutic genome-editing applications in a time-limited manner and without the risk of integration into the genome,” the researchers write. In doing so, they rely on a principle similar to that used in the mRNA vaccination against the corona virus. “In the vaccines, the mRNA molecules contain the genetic information for the construction of the spike protein of the virus, with which the pathogen penetrates human cells,” explains Stadelmann. “For our purposes, we use mRNA molecules that contain the instructions for building the gene scissors.” Unlike vaccines, which cannot change human DNA, the gene scissors can repair defective genes in a targeted manner.
So that the mRNA could get into the cells, the researchers temporarily made the cell membranes of the removed muscle stem cells permeable to larger molecules. To do this, they used a process called electroporation, in which they applied an electrical field to the cells and thus temporarily changed the membrane properties. They used a trick to check whether the mRNA was actually absorbed: “Using mRNA, which contained the genetic information for a green fluorescent dye, we were initially able to demonstrate that almost all stem cells absorb the mRNA molecules,” reports Stadelmann.
Clinical studies planned
In order to prove that it is actually possible to edit the cells’ genome with this method, the researchers used gene scissors to change the gene for a surface molecule of the muscle stem cells. This molecule has no effect on the functionality of the cells, but can be easily detected. In fact, they succeeded in making targeted changes in the gene that became visible through a change in the expression of the surface molecule. Tests with another molecular genetic tool, which, unlike the CRISPR/Cas9 gene scissors, does not cut the DNA but instead exchanges a base with pinpoint accuracy, were also successful. In cell culture, the genetically engineered muscle stem cells were able to fuse with each other and form young muscle fibers, just like healthy muscle cells.
“We are now planning to start a first clinical study with five to seven patients suffering from muscular dystrophy towards the end of the year,” says Stadelmann’s colleague Simone Coiler. Muscle stem cells are to be removed from the patients, repaired and then transplanted back into the muscle – similar to what the researchers have already tested on mice. First of all, this method is only suitable for repairing small muscles. “Sick people who are in a wheelchair will not just get up and start walking after our therapy,” says Coiler. But for many of those affected, it is already a big step forward when a small muscle that is important for gripping or swallowing, for example, works better again.
In the long term, the researchers also want to develop methods in which the molecular tools can be introduced not only into isolated muscle stem cells, but directly into the muscle. In this case it might also be possible to repair larger muscles like those needed for standing and walking.
Source: Christian Stadelmann (Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin) et al., Molecular Therapy Nucleic Acids, doi: 10.1016/j.omtn.2022.02.016