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Restore a functional dystrophin gene using the genome editing tool CRISPR-case.9 corrected a common mutation behind Duchenne Muscular Dystrophy (DMD), removal of exon 44, in mice and human cells, a study shows.
The same research laboratory had already proved that the technique made it possible to correct a simple and effective way a range of mutations causing DMD. New the results represent another important step towards a possible clinical application of CRISPR-Cas9 in therapy for DMD, the researchers say.
The studyCRISPR-Cas9 corrects the deletion mutations of exon 44 of Duchenne muscular dystrophy in mice and human cells"Was published in the newspaper Progress of science.
The most common types of mutations that cause DMD are those that result in the removal of exon 44 from the dystrophin (DMD) gene.
An exon is a part of the gene that gives production instructions, or codes, for a piece of the protein that corresponds to that gene. In most mutations causing DMD, a deletion in an exon at the level of DMD gene alters the way the gene is read by the cellular machinery, generating a defective dystrophin protein.
Building on the recent positive results obtained using CRISPR approaches for DMD, Professor Eric Olson's laboratory at University of Southwestern Medical Center and his colleagues have developed a similar strategy to correct the deletion mutations of the exon 44 underlying the disease.
As this type of mutation can be corrected in about 12% of patients by making small changes to the neighboring virusxons, researchers employee CRISPR-case.9 genetically modify the exons surrounding exon 44 and repair the DMD gene.
CRISPR-Cas9 is a newly discovered system that can be used to cut or add small pieces of DNA to a specific location in the genome, a process known as gene editing.
The CRISPR-Cas9 system is highly valued by the scientific community as it is faster and more accurate than other existing gene editing tools.
For this to work, two key components are needed: a guide RNA and a DNA-cutting enzyme called Cas9.
The guide RNA is a small piece of RNA (a genetic messenger copied from DNA) that contains a short sequence capable of binding to the target region of the genome. It also binds to the enzyme Cas9, guiding it to the targeted location.
Once in this specific region, the Cas9 enzyme cuts the DNA. Then, the cell's own DNA repair mechanisms allow the addition or deletion of pieces of genetic material or the replacement of segments with a custom DNA sequence.
In this case, the researchers designed several guide RNAs to direct Cas9 to exons 43 and 45, and to modify the modifications, which flank the defective exon 44.
Using a viral vector commonly used in gene therapy, called AAV9, the researchers introduced the genetic material coding for Cas9 and guide the RNAs inside the cells.
The technique has proved effective in correcting the DMD dystrophin restoration gene and protein in cardiac muscle cells (cardiomyocytes) derived from stem cells of DMD patients and bearing the deletion of exon 44.
To show that this type of treatment could be administered to a living organism, researchers genetically modified mice to give them a deletion of exon 44 similar to that present in patients. These mice with CRISPR-Cas9 by injections into the muscle of the leg.
The results showed that three weeks after the injections, the DMD the gene was corrected and the production of dystrophin protein was restored both in the heart and skeletal muscles, including the muscles of the forelimb and hindlimb and in the thorax (diaphragm) of mice.
In addition, the study also tested different assays and guide RNA sequences to optimize the gene correction protocol. These tests showed that the proportion of Cas9 to guide the RNA strongly influences the efficiency of the method.
The same laboratory had already shown that this tool and other CRISPR tools could correct the genetic defects that caused DMD. Given their promising efforts, the team received a grant from Parent Project Dystrophy to continue their studies and identify potential risks associated with the method in a clinical setting.
The project leader, Olsen, with support from Cure Duchenne, has also launched a company to develop CRISPR / Cas9 therapy for DMD.
The murine deletion model of exon 44 developed for this study, combined with optimized delivery-optimized RNA and AAV vectors, "should facilitate progression to the long-term correction of dystrophin mutations in mice, a prelude to a possible clinical translation, "says the report.
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