Single CRISPR treatment provides long-term benefits in mice

Researchers at Duke University have shown that a single systemic treatment using genome editing technology CRISPR could safely and stably fix a genetic disorder – Duchenne muscular dystrophy – for longer periods of time. One year in mice, despite the observed immune responses and alternative results of gene editing.

The study appears online February 18 in the newspaper Nature Medicine.

In 2016, Charles Gersbach, a biomedical associate professor of the Rooney family at Duke, published one of the first successful uses of CRISPR to treat an animal model of genetic disease with a strategy likely to be translated into human therapy. . Since then, many other examples have been published and several genome editing therapies targeting human diseases are currently undergoing clinical trials and others are underway.

Gersbach's latest research focuses on a mouse model of DMD, caused by the body's inability to produce dystrophin, a long protein chain that binds inside a muscle fiber to its muscle structure. surrounding support.

Dystrophin is encoded by a gene containing 79 protein coding regions, called exons. If one or more exons are disrupted or removed by an inherited mutation, the chain is not built, which causes the muscle to slowly shred and deteriorate. Most patients are wheelchair users at the age of 10 and do not live beyond the age of 20 or in their early thirties.

Gersbach has been working on potential genetic treatments for Duchenne since 2009. His laboratory was one of the first to focus on CRISPR / Cas9, a modified version of a bacterial defense system that targets and separates the brain. DNA invading viruses. His approach uses CRISPR / Cas9 to extract dystrophin exons around the genetic mutation, letting the body's natural DNA repair system gather the remaining gene to create an abbreviated – but functional – version of the dystrophin gene.

"As we continue to work to develop CRISPR-based genetic therapies, it is essential to test our hypotheses and rigorously evaluate all aspects of this approach," Gersbach said. "The purpose of our experiments was to test some ideas discussed in the field, which will help us understand the potential of CRISPR for the treatment of genetic diseases in general and Duchenne muscular dystrophy in particular. long-term durability of the response to potential immune responses against the bacterial protein Cas9 ".

The first eight-week study showed that functional dystrophin was restored and muscle strength increased. However, he did not explore the long-term sustainability of the treatment.

"It is widely accepted that gene editing leads to permanent gene correction," Gersbach said. "However, it is important to explore the theoretical possibilities that could undermine the effects of gene editing, such as the loss of treated cells or an immune response."

The purpose of this new study was to explore the factors that may alter the long-term effects of CRISPR / Cas9-based gene editing.

Christopher Nelson, the Gersbach Postdoctoral Fellow who led the work, administered a single dose of intravenous CRISPR treatment to adult and neonatal mice harboring a defective dystrophin gene. Over the next year, the researchers measured how many muscle cells had been successfully edited and what types of genetic alterations had been made, as well as the generation of any immune responses against the CRISPR bacterial protein, Cas9, which acts as a "chisel". it makes cuts in the genome.

Other studies have shown that the immune system of the mouse can generate a response to Cas9, likely to interfere with the benefits of CRISPR therapies. Several groups also reported that some people already had immunity to Cas9 protein, probably due to previous exposure to the bacterial host.

"The good news is that even though we observed both antibody and T-cell responses to Cas9, neither of these seemed to cause any toxicity in these mice," said Nelson. "The response also did not prevent the therapy from effectively altering the dystrophin gene and producing long-term protein expression."

The results also suggested approaches to address potential challenges, if any. For example, the researchers observed that when two-day-old mice with no fully developed immune system were treated intravenously, no immune response was detected. The CRISPR genome edition has remained stable and, in some cases, even strengthened over the course of a year. One could imagine administering infants' therapy as a method of circumventing or modulating an undesirable immune response.

Gersbach and Nelson, however, recognize that the immune system of the mouse often functions very differently from the human immune system. And screening for DMD in newborns is not yet widespread; most Duchenne diagnoses occur when children are three to five years old. To meet this challenge, Gersbach said suppressing the immune system during treatment may be a viable approach.

Researchers are also investigating potential strategies to limit the expression or delivery of Cas9 to muscle cells for short periods of time, which may reduce immune detection.

"We were pleased to find that all mice were doing well one year after treatment, but our results show that it is necessary to put more emphasis on the immune response as we progress toward larger animal models, "Nelson said.

Nelson and Gersbach have already investigated the potential of non-targeted CRISPR / Cas9 editing to unintentionally alter other genome sites and report minimal activity on possibly untargeted sites. However, other recent studies have indicated that CRISPR can sometimes perform genetic modifications on the correct site, but not in the intended manner. For example, some studies have shown that CRISPR can cut much larger genetic sections than expected or that DNA fragments can be integrated at the site of the cut. These types of modifications have not heretofore been reported in genome editing studies because the methods used only detected the desired editing.

To exhaustively map all the changes that occur in the dystrophin gene, Nelson used a DNA sequencing approach that agnostically signals any type of modification. Surprisingly, many types of modifications were made in addition to the planned elimination of the targeted exon, including a high level of DNA sequence insertion from the viral vector coding for the CRISPR / Cas9 system.

Depending on the tissue type and dosage of CRISPR administered, up to half of the changes on the target resulted in these alternative sequence changes. Although this result was surprising, unexpected sequence changes do not appear to affect the safety or efficacy of this CRISPR / Cas9 gene editing approach for DMD .

"None of these changes would necessarily be of concern in this case because the dystrophin gene is already defective," said Nelson. "That being said, any unplanned results could potentially hinder the effectiveness of the gene editing that you are trying to achieve, highlighting the importance of designing ways to improve the quality of life. identify and objectively mitigate alternative modifications in future studies. "

"Previous studies have suggested that some of these other types of modifications could take place," said Gersbach. "But this is one of the first comprehensive measurements of these events in an animal model using a therapeutically relevant approach.In the future, this phenomenon needs to be closely monitored and better understood. the potential of genome editing in the treatment of diseases. "

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