Researchers at the Christiana Care Health System's Gene Editing Institute say they have developed a new system that allows them to not only repair damaged DNA within human cells, but also to determine when the DNA repair machinery has introduced unwanted genetic changes alongside, or instead of, the desired repair.

A team led by Eric Kmiec, Ph.D., director of the Gene Editing Institute at the Helen F. Graham Cancer & Research Institute at Christiana Care, published its findings using a modified version of the CRISPR/Cas9 gene-editing technique in PLOS ONE. The modified technique, called Excision and Corrective Therapy, or EXACT, uses an oligonucleotide to serve as both a bandage and a template during the repair of a genetic mutation.

“The advancement here is a new concept of using donor DNA as an oligonucleotide to act as a Band-Aid across a gap created by the CRISPR [ribonucleoprotein complex], and then allowing replication to fill in the gap, and then the oligonucleotide dissociates and on you go,” said Dr. Kmiec.

The published paper, titled “Insertional Mutagenesis by CRISPR/Cas9 Ribonuclear Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides,” describes how the EXACT CRISPR/Cas9 technique can be used to repair point mutations that can produce hereditary diseases in humans, such as sickle cell anemia or Gaucher'sdisease.

The present study follows an earlier report published in September in Scientific Reports in which Dr. Kmiec and his colleagues established that their technique functions using the “Band-Aid template” repair mechanism that they had predicted. In the PLOS ONE paper, Dr. Kmiec and his colleagues report using a single-stranded DNA template with a preassembled CRISPR/Cas9 ribonucleoprotein complex to fix a point mutation in human cells that have been engineered to express a fluorescent protein only if a single change in the DNA that encodes the fluorescent protein can be repaired.

The researchers note that their EXACT gene-editing approach does in fact result in a significant amount of point mutation repair, thereby producing cells that make functional fluorescent protein. More importantly, Dr. Kmiec and his team also characterize undesirable mutations that sometimes occur alongside of or instead of the desirable point mutation repair when using EXACT, referring to these undesirable side mutations as “collateral damage” or “on-site mutagenesis.”

“If you lose DNA, even one or two bases, even if you fix the point mutation the gene is disabled, because the gene can no longer code for the proper protein,” said Dr. Kmiec. “So even though you have successfully corrected the gene, the problem is that you’ve also introduced some sort of secondary mutation at the site, and that causes the gene to be completely nonfunctional.”

As reported by the team, on-site mutagenesis can occur even when repair of the point mutation has not taken place, meaning that CRISPR/Cas9 ribonucleoprotein complexes can produce additional indels at a target site without carrying out the function they were placed there to perform. In their PLOS ONE paper, the researchers map out exactly where and how indels occur during on-site mutagenesis, examining what happens to both copies of the DNA strand after the CRISPR/Cas9 ribonucleoprotein complex has done its work.

Overcoming the problem of on-site mutagenesis and the genetic scar tissue it leaves behind
will be necessary if CRISPR/Cas9-mediated gene therapy is to become useful in the clinical setting. As Dr. Kmiec and his colleagues suggest, solving this problem will not be easy, as the DNA-repair machinery that cells use to perform point mutation repairs is inherently error prone. Based on the greater mechanistic understanding provided by his recent studies, Dr. Kmiec says he remains optimistic that on-site mutagenesis is a problem that can be overcome.

“We are more optimistic now, seeing this data, that we will be able to fix point mutations efficiently, using this mechanism as opposed to other things that are now being reported in the literature,” Dr. Kmiec said. “It’s an advance that I think will give people hope that these kind of point mutations can be fixed if we use the proper tools to fix them.”

In order to take his CRISPR/Cas9 system into the clinical setting, Dr. Kmiec says it will be necessary to further stabilize the repair complex at the site of the mutation, which should cut down on the occurrence of on-site mutagenesis. He likens the repair process to the way in which a bandage can facilitate wound healing, noting that wounds “heal a lot faster if the bandage stays in place a lot longer. So the more times you wrap it with tape, or in this case, the more stable the binding, the more efficient the point mutation repair is going to be.”

The group also must figure out how to get the CRISPR/Cas9 machinery into the progenitor cells that give rise to mature, therapeutically relevant cells in the body. Dr. Kmiec says this is an active area of research for his laboratory. He hopes that CRISPR/Cas9 gene therapy with EXACT could be in human clinical trials at Christiana Care within 18 to 24 months.

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