Through a year-long study of mice, researchers from Duke University have demonstrated that treatment with CRISPR genome editing technology can safely and steadily correct a genetic disorder called Duchenne muscular dystrophy (DMD). The study was published online in the February 21 issue of Nature Medicine.

In 2016, Charles Gersbach, associate professor of biomedical engineering at the Rooney family in Duke, published a method for successfully using CRISPR to treat animal models of hereditary diseases, and its strategy could be translated into human therapy. Since then, many other cases have been researched, and several more genome editing therapies for human diseases are currently in clinical trials.

Gersbach's latest research focuses on the mouse model of DMD, which is caused by the body's inability to produce dystrophin, a long protein chain that binds the interior of the muscle fiber to the surrounding support structure. Anti-dystrophin is encoded by a gene containing 79 protein coding regions and is called an exon. If one or more exons are disrupted or deleted by a genetic mutation, the strand will not be constructed, causing the muscles to slowly tear and deteriorate. Most patients are in a wheelchair when they are 10 years old and cannot live to their 20s or 30s.

Since 2009, Gersbach has been working on potential genetic therapies for DMD. His lab was one of the first labs to focus on CRISPR / Cas9, a modified version of a bacterial defense system that can locate and slice invaded DNA. His method is to use CRISPR / Cas9 to cut the dystrophin exon around the gene mutation, allowing the body's natural DNA repair system to re-sew the remaining genes together to create a shortened but functional dystrophin gene.

“People generally believe that genetic editing can lead to permanent genetic changes,” Gersbach said. “However, it is important to explore the theoretical possibilities that may undermine the effects of gene editing.” Therefore, the purpose of this new study is to explore factors that may alter the long-term effects of CRISPR/Cas9-based gene editing.

Christopher Nelson, a postdoctoral researcher at Gersbach Laboratories who led the work, intravenously administered a single dose of CRISPR therapy to adult and newborn mice bearing the defective dystrophin gene. In the following year, the researchers measured how many muscle cells were successfully edited and what types of genetic alterations were made, as well as any immune responses to the bacterial CRISPR protein Cas9.

Other studies have reported that the immune system of mouse can respond to Cas9, which may interfere with the benefits of CRISPR therapy. A few research groups have also reported that some people have pre-existing immunity to Cas9 protein, probably because of previous exposure to bacterial hosts. "The good news is that although we observed antibodies and T cells responding to Cas9, none of them seemed to produce any toxicity in these mice," the authors said. "This response did not prevent the therapy from successfully editing dystrophin genes and the ability to produce long-term protein expression."

However, the authors acknowledge that the function of the immune system of mouse is usually completely different from that of the human. DMD screening among newborns is not currently widely available; most Duchenne diagnoses occur in children between the ages of three and five. To address this challenge, Gersbach said that inhibiting immune system activity during treatment may be a viable approach. In addition, researchers are investigating potential strategies that limit the expression or delivery of Cas9 to muscle cells in a short period of time, which may reduce immune testing.

The author has previously investigated the off-target editing potential of CRISPR/Cas9, inadvertently modified other sites in the genome, and reported minimal activity at possible off-target sites. However, other recent studies have reported that CRISPR can sometimes perform gene editing at the correct site, but not in the expected manner. For example, some studies have shown that CRISPR can cut genetic fragments that are much larger than expected, or that DNA fragments can be inserted into the cleavage site. These types of edits were not previously reported in the genome editing study because the method used only detected the expected edits.

To comprehensively map all the editors that occurred in the dystrophin gene, the authors performed DNA sequencing. Surprisingly, in addition to the expected removal of target exons, many types of editing have taken place.

Depending on the type of tissue and the dose used for CRISPR, up to half of the target edits result in these alternative sequence changes. Although this result is surprising, unexpected sequence changes do not appear to affect the safety or efficacy of the CRISPR/Cas9 gene editing method for DMD.

“Because the dystrophin gene is already flawed, in these cases, these editorial situations are not necessarily of concern. However, any unexpected results may deprive you of the efficiency of the gene editing you are trying to achieve, which supports the importance of designing in objectively identifying and mitigating alternative editing methods in future research."

Previous studies have shown that some types of editing may occur, but this is one of the first comprehensive measurements of these events in animal models using treatment-related methods. Looking ahead, this phenomenon needs to be carefully monitored and better understood.

Author's Bio: 

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