The CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) method for genome editing is a powerful, new medical technology with many applications in biomedical research, including the potential to treat human genetic diseases, such as Duchenne Muscular Dystrophy (DMD). CRISPR/Cas9 allows researchers to edit parts of the genome by removing, adding, or changing sections of the DNA sequence.

How does CRISPR/Cas9 work?

The CRISPR method is based on bacteria’s natural system to protect itself from viral infections. When bacteria detect the presence of virus DNA, the CRISPR system captures a piece of the virus’ DNA and slides it into a section of its own DNA, keeping a bit of it behind to help recognize and defend against the virus the next time it attacks. This is done by producing two types of short RNA, one of which contains a sequence that matches that of the invading virus. These two RNAs form a complex with a protein called Cas9. Cas9 is a nuclease — a type of enzyme that can cut DNA. When the matching sequence known as a guide RNA finds its target within the viral genome, the Cas9 cuts the target DNA thus disabling the virus.

CRISPR/Cas9 as a Potential DMD Treatment

DMD affects 1 out of 5000 male births and is caused by mutations in the dystrophin gene. Three different laboratories reported using the CRISPR/Cas9 method for targeting the point mutation in exon 23 of the mdx mouse that creates a stop codon and serves as a model of DMD. Each of the studies demonstrated efficacy using a two-vector system of AAV-CRISPR rather than single vectors for the guide RNA and the Cas9, with all three laboratories agreeing on restoration of dystrophin-positive fibers. Moreover, they were able to demonstrate functional recovery in the CRISPR/Cas9-treated mice, including more grip strength, improved force generation, resistance against eccentric contraction and reduced blood levels of creatine kinase. At the cell level, there was reversal of muscle necrosis, fewer infiltrating inflammatory cells and decreased fibrosis. Probably the most interesting points were that dystrophin expression was seen in vascular smooth muscle, which is an important source of oxygenation during muscle activity, and that cardiomyocytes demonstrated the restoration of dystrophin.

Parent Project Muscular Dystrophy (PPMD) has just awarded a $2.2 million grant to Jerry Mendell, MD, PhD; co-PI Louise Rodino-Klapac, PhD, and Nationwide Children’s Hospital, which will support exploring gene therapy as DMD potential treatment. Later in January, PPMD will be launching a Gene Transfer Initiative that will consist in a deeper exploration of CRISPR/Cas9 method as a potential DMD therapeutic approach.

Application of CRISPR/Cas9 technology is also being researched for the treatment of Cystic Fibrosis, Hemophilia, and cancer.

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