Gene from the bench
CRISPR cured mice of muscular dystrophy without genome editing
Daria Spasskaya, N+1
The use of an artificial gene activator based on the catalytically inactive dCas9 protein made it possible to effectively increase the expression of the laminin gene in the muscles of mice with type 1A muscular dystrophy. This method not only prevented muscle degradation in young animals, but also improved the condition of already sick mice. Article by Kemaladewi et al. A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene is published in Nature.
One of the varieties of MDC1A muscular dystrophy (type 1A congenital muscular dystrophy) develops as a result of a mutation in the Lama2 gene. The gene encodes the a-chain of the laminin protein, which is necessary for the interaction of muscle fibers with Schwann cells. These auxiliary cells of the nervous tissue are involved in the formation of the electrically insulating myelin sheath of the nerve. As a result of a violation of the interaction, skeletal muscle degeneration and violation of its innervation occur.
There is no cure for this disease, however, scientists have previously shown that the lack of Lama2 can be compensated by additional expression of the Lama1 gene, which encodes a different type of laminin a-chain. The introduction of Lama1 into the muscles, however, is complicated by the huge size of the gene, which does not allow it to be delivered to the body in a standard way (as part of an adenovirus).
The staff of the research center at the Children's Hospital in Toronto (Canada), together with American colleagues, successfully demonstrated on a mouse model of the disease that the expression of their own Lama1 can be increased using an artificial transactivator based on the CRISPR-dCas9 system. As a result of a mutation in the catalytic domain, dCas9 is not able to cut DNA, but is able to bind to it in the place indicated by a short RNA guide (guide RNA). If a repressor or activator domain is attached to such a protein, dCas9 can be turned into a transcription factor to control gene expression.
A scheme of genetic constructs for creating an artificial gene activator based on dCas9. Figures from the article by Kemaladewi et al.
In this paper, scientists have chosen a "small" Cas9 from Staphylococcus aureus and stitched it with the viral transactivator domain VP64. The artificial activator was first tested on mouse fibroblasts and the three most effective RNA guides to the regulatory part of the Lama1 gene were selected. For the treatment of animals, the system was packaged in adenovirus type AAV9, which has a high affinity for muscle tissue.
An initial test in mice showed that all three RNA guides are needed at once for effective induction of Lama1 expression in muscles. A preventive experiment showed that the introduction of an adenovirus with an activator and guides into the blood of newborn mice prevents degeneration of skeletal muscles and the development of seizures with age (by the seventh week).
In addition, the authors also showed the therapeutic potential of the genetic construct in already sick mice — the administration of a high dose of adenovirus to three-week-old animals (there were 7-9 animals in groups) with signs of paralysis not only stopped the progression of the disease, but also improved the mobility of animals by the sixth week.
The results of one of the functional mobility tests (vertical activity) in mice from the control group (red dots) and mice that were injected with both constructs (activator and guides) as part of the adenovirus (black dots). By the seventh week, the animals have improved. On the right, a section of skeletal muscle in a mouse with dystrophy (-) and after treatment (+).
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