Myorediting
CRISPR will make the hearts of patients with Duchenne muscular dystrophy beat
Daria Spasskaya, N+1
The researchers edited heart muscle cells with mutations leading to the development of Duchenne myodystrophy. Using the CRISPR-Cas9 system, mutant sections of the gene were "ejected" from mRNA, and a beating heart muscle was grown from cells with "corrected" DNA. The study is published in the journal Science Advances (Long et al., Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing).
The DMD gene is the largest in the human genome and encodes the dystrophin protein, a structural protein that ensures the connection of muscle fibers with the surrounding matrix. Disruption of dystrophin synthesis due to stop mutations in the gene leads to the development of progressive muscular dystrophy with age - a disease known as Duchenne myodystrophy. In addition to the fact that patients eventually lose the ability to walk, they also develop cardiomyopathy – degradation of the heart muscle, which eventually leads to the death of a person aged 20-30 years.
The dystrophin gene has a complex structure and contains many non-coding regions that separate the "significant" regions of the gene – exons. During mRNA splicing, non-coding regions are cut out, and exons are "glued" together. The gene and its product are very large (the gene contains 79 exons), and the researchers found that removing one or more exons leads to the formation of a shortened, but still functional protein. Thus, the exon that the stop mutation got into can be thrown away, and this will partially restore muscle function. This approach to the therapy of myodystrophy, which is called "exon skipping", is one of the most promising to date and is undergoing clinical trials on patients, and one of the drugs has been approved by the FDA.
Researchers from the University of Texas Southwestern Medical Center (USA) used the CRISPR-Cas9 system to implement the "exon pass". The convenience of using this system in this case is determined by the fact that the sequences indicating to the splicing proteins where to cut the mRNA contain PAM motifs for Cas9, which determine the nuclease targets. A cut in this sequence for a specific exon and subsequent repair by the mechanism of non-homologous end joining, by which double-stranded breaks in DNA are "healed" by default, will lead to the fact that the exon will not be recognized by splicing proteins and will not be included in mature mRNA.
Sequences for mRNA splicing and for recognition by Cas9 protein (Chengzu Long et al / Science Advances 2018).
There are about three thousand mutations in the DMD gene that determine the development of Duchenne myodystrophy, which are grouped in the "hot spots" of mutagenesis, concentrated mainly in 12 exons. The scientists selected guide RNAs for making incisions using Cas9 in all 12 exons, and thus created a universal set for "skipping exons" suitable for most patients. CRISPR has already been used to edit the dystrophin gene, but only one exon was targeted in preliminary trials.
Since cardiomyopathy does not develop in model mice with myodystrophy, the technology was tested on human cardiomyocytes (heart muscle cells), which were obtained from stem cells with various impaired dystrophin functions. It turned out that "skipping exons" using CRISPR-Cas effectively restores the expression of dystrophin in cell culture.
Cardiomyocyte culture without editing (left) and with reduced dystrophin (right)
To test the functionality of such tissue, the researchers grew an artificial heart muscle in a cup by mixing cardiomyocytes with connective tissue cells. Compared to the muscle grown from the original mutant cells, the "edited" muscle normally contracted. Scientists have found that to support contractile function, it is enough that 30-50 percent of the edited cells are in the muscle.
The authors of the work called their technology mioredaktion. Theoretically, it is suitable for delivering an editing system to the heart muscle of patients using viruses (a standard delivery tool in gene therapy), however, the authors consider it rather as a technology for editing patient cells ex vivo with subsequent implantation into the heart muscle. The technology of editing cells using CRISPR-Cas9 outside the patient's body has already been approved in China and the USA for the treatment of cancer.
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