Sangamo Announces Nature Biotechnology Publication of New Strategies for Optimizing the Specificity of Gene Editing Nucleases
“When attempting to improve the specificity of genome editing tools, on-target editing efficiency is often sacrificed,” said
In order to adjust the binding affinity of the zinc finger recognition domain, the authors substituted a discrete, positively charged residue in the zinc finger framework to eliminate a nonspecific contact with the negatively charged phosphate backbone of the DNA. By varying the number of fingers bearing this substitution, the authors showed in cellular studies that they could effectively tune ZFN affinity into an optimally specific range, with no loss of on-target efficiency.
In a related series of cellular studies, the authors screened single-amino acid substitutions in the Fok1 nuclease domain in order to identify those able to improve specificity by slowing down catalysis. The goal of the study was to observe whether mutations would provide more time for the ZFNs to selectively dissociate from off-target sites prior to a cleavage event, which would improve global specificity. These studies yielded single-residue substitutions that could increase specificity by more than 1000-fold.
In a final study detailed in the manuscript, Sangamo scientists applied these two strategies in a therapeutically relevant setting by designing ZFNs that targeted the endogenous TCR-alpha gene in T-cells. Treatment of these T-cells with optimized ZFNs resulted in a greater than 98% on-target knockout efficiency of the TCR-alpha gene with undetectable off-target activity at a median assay background level of 0.01%. Sangamo believes these engineered improvements to the specificity of its ZFN genome editing platform have the potential to enable the routine generation of designed nucleases capable of high efficiency editing with minimal or no detectable off-target activity.
These results add to Sangamo’s body of research demonstrating the high degree of precision, efficiency, and specificity of ZFNs for genome editing. In
This press release contains forward-looking statements based on Sangamo's current expectations. These forward-looking statements include, without limitation, statements relating to the ability to engineer the ZFN structure to enable on-target modification, the effects of these strategies on Sangamo’s genome editing tools, the safety attributes associated with these strategies and their ability to help ensure the safety of Sangamo’s gene editing tools in the clinic, whether or not these effects may be replicated in the clinic, the potential for these engineered improvements to improve specificity on designed nucleases, and other statements that are not historical fact. These statements are not guarantees of future performance and are subject to certain risks, uncertainties and assumptions that are difficult to predict. Factors that could cause actual results to differ include, but are not limited to, risks and uncertainties related to: early preclinical data, including the risk that the early preclinical data may not warrant regulatory approvals to conduct any human clinical trials, and may not be representative of the results of any such human clinical trials; whether Sangamo’s gene editing tools and strategies will produce any beneficial therapeutic effect in humans; Sangamo's ability to develop commercially viable products; and the potential for technological developments by Sangamo's competitors that will obviate Sangamo's ZFN technology. These risks and uncertainties are described more fully in Sangamo's Quarterly Report on Form 10-Q for the quarter ended
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