Studies by researchers in the U.S. and Canada suggest that small differences in the genetic codes of individuals could have a significant impact on the efficacy, side effects and safety of treatments based on CRISPR/Cas9 gene editing. The teams at Harvard Medical School, Boston Children’s Hospital, and the Université de Montréal say their findings suggest that a one size fits all approach to therapeutic gene editing may not be equally effective for all patients, and that the editing technologies may need to be adjusted to each patient’s genome sequence to maximize efficacy and also prevent potentially harmful off-target effects.

“Humans vary in their DNA sequences, and what is taken as the 'normal' DNA sequence for reference cannot account for all these differences,” says Stuart Orkin, M.D., of Dana-Farber Boston Children's Cancer and Blood Disorders Center. “We recommend that common variation be taken into account in designing targeting systems for therapeutic editing, to maximize efficacy and minimize potential safety concerns.”

The researchers report their findings in the Proceedings of the National Academy of Sciences (PNAS) in a paper entitled, “Human Genetic Variation Alters CRISPR-Cas9 On- and Off-Targeting Specificity at Therapeutically Implicated Loci.”

The first trials evaluating CRISPR/Cas9 gene editing in a clinical setting are already ongoing, and additional trials are in the planning. “The development of successful therapies not only requires treatment efficacy but also requires that patient safety remain paramount,” the authors point out. “This requires assessing for toxicity related to reagent delivery, to the genome-editing reagents themselves, and to off-target effects.”

Most research that has been carried out to help understand potential on- and off-target effects of genome editing technologies, and to aid in the design of effective and safe guide RNAs (gRNAs), has been based on either a reference genome or the genomes of cell lines. As an alternative approach, Harvard Medical School’s Matthew Canver led a study to evaluate the potential effects of interindividual genetic variation on CRISPR/Cas9 gene editing. They analyzed 7444 previously published human whole-genome sequences, alongside a list of about 3000 different gRNAs that might be used to target genes within 30 different therapeutically relevant locations on the genome.

Their results indicated that about half of the gRNAs might be affected by small genetic variations, single-nucleotide polymorphisms, or insertions/deletions including at their target sites. “If there are genetic differences at the site that CRISPR reagents are targeting for therapy, you are at risk for decreased efficacy or treatment failure,” Canver explains. “A difference in just a single base pair can cause a decrease in binding efficiency due to a mismatch with the gRNA. Overall, this can cause a reduction in treatment efficacy.”  

In a few instances genetic variations might even cause the gRNA to target the CRISPR reagents to the wrong site on the genome, the findings suggested. “In rare cases, there was the potential to create very potent 'off-target' sites—where CRISPR reagents could bind and cut where they're not intended to. If an off-target effect happens to be in, say, a tumor suppressor gene, that would be a big concern.”

Although the research looked specifically at CRISPR/Cas9 gene editing, the team says its findings could have implications for the design of other gene-editing technologies, such as zinc-finger nucleases and TAL (transcription activator-like) effector nucleases (TALENs). “The unifying theme is that all these technologies rely on identifying stretches of DNA bases very specifically,” says Canver. “So, a variant that affects the target sequence could reduce gRNA binding. Variants can also lead to binding at new sites that could potentially cause harm. As these gene-editing therapies continue to develop and start to approach the clinic, it's important to make sure each therapy is going to be tailored to the patient that's going to be treated.”

In their paper, the researchers advocate carrying out preclinical studies to consider genetic variation at the gDNA design stage and then validate gRNAs for clinical translation. The overall aim is to increase the likelihood of offering “safe, effective, and personalized therapeutic options for all patients regardless of genotype.” They further suggest that it may be prudent to carry out whole-genome sequencing (WGS) on each patient before they undergo treatment. “WGS data would allow for in silico on- and off-target analysis, which may identify patients predisposed to treatment failure and/or adverse outcomes before therapy initiation.…It may be advisable for safety considerations to exclude gRNAs with predicted off-target sites within or near important genes such as tumor suppressors….”



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