CRISPR against myodystrophy
Adeno-associated viruses and CRISPR again coped with muscular dystrophy
Daria Spasskaya, N+1
American researchers who are developing approaches to correcting mutations in the dystrophin gene using CRISPR have described another effective system for resuming the synthesis of this protein in the muscles of animal models. To do this, scientists have developed a new mouse model of Duchenne myodystrophy. As reported in an article in Science Advances (Min et al., CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells), the expression of dystrophin in the muscles of experimental mice reached 90 percent of the norm.
Violation of dystrophin protein synthesis as a result of a congenital mutation leads to the development of Duchenne myodystrophy progressing with age, which leads to the death of the patient at the age of 20-30 years. Harmful mutations occur on average in one in 3600 boys.
The dystrophin gene has a complex structure and consists of many coding pieces – exons. Deletions in several of them lead to the appearance of a stop codon in the gene and the synthesis of a shortened non-functional protein. The researchers found out that it is possible to restore the synthesis of the working version of dystrophin by removing a piece with a mutation using the so-called "exon skipping" (exon skipping). Since the edges of the exons contain recognition sites for the Cas9 protein (PAM), it is quite convenient to cut out mutant exons using the CRISPR-Cas9 system.
Researchers from the University of Texas, led by Eric Olson, previously tested an approach with CRISPR to cut 51 exons in the dystrophin gene to eliminate the mutation that causes 13 percent of cases of the disease. In the new work, scientists focused on another type of mutation – deletion in the 44 exon of the gene. This mutation is responsible for 12 percent of Duchenne myodystrophy in humans.
In the previous case, scientists had the opportunity to test the system on beagle dogs, which have cases of myodystrophy due to a mutation common with humans. However, this time, to test the effectiveness of the removal of 44 exons, the authors of the work had to create a new model of myodystrophy and breed mice with the corresponding deletion in the genome. Dystrophin was not detected in the muscles of model animals.
The selection of the guide RNA for excision of 44 exons was carried out on stem cells taken from a patient with Duchenne myodystrophy. Heart muscle cells were then grown from the edited cells. Scientists have managed to find an effective guide RNA to a section of the gene that matches in a mouse and in a human. The guide RNA and the Cas9 protein gene were injected into mice as part of separate viral vectors. As before, the scientists used the adenoassociated serotype 9 virus (AAV9), which has the greatest affinity for muscles. In addition, CRISPR-Cas9 expression was limited by tissue-specific regulatory elements.
Viral particles were injected into the muscle or blood of animals. It turned out that with systemic delivery (via blood), the expression of the working form of dystrophin in the heart reaches 80 percent of the norm. When the concentration of the vector containing the guide RNA gene was increased by 10 times, the amount of dystrophin in the heart muscle was already 90 percent of the norm.
Absence of dystrophin (red) in human cardiomyocytes before and after therapy. A drawing from the UT Southwestern Discovery press release made while editing genetic defect behind Duchenne muscular dystrophy – VM.
From this experiment, the researchers made an important conclusion that the amount of guide RNA affects the expression of Cas9 and limits the effectiveness of editing.
For the only drug approved to date, the action of which is based on the same principle of exon skipping (eteplirsen), the excision efficiency is less than one percent.
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