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AssigneesInstitut National de la Sante et de la Recherche Medicale, Chu De Bordeaux, Universite De Bordeaux
InventorsFrançois MOREAU-GAUDRY, Aurélie BEDEL, Sabrina FAYET
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The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has impelled gene editing into clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and sensitive detection methods. Here, the inventors developed two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). They revealed rare complex chromosomal rearrangements caused by Cas9-nuclease and showed that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppressed the appearance of LOH without compro-2024/235844 mising editing. These data were confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk and design safer protocols.
A method of preventing ON-target genotoxicity induced by a nuclease in a population of cells comprising the steps of i) inducing G0/G1 phase cell cycle arrest in the population of cells by contacting the cells with an agent that selectively induces G1 phase cell cycle arrest and ii) editing the population of cells induced in step i) with the nuclease.
A method of altering a target sequence of a nucleic acid molecule in a population of cells for which a G0/G1 phase cell cycle arrest is induced comprising contacting the target sequence of the nucleic acid molecule with (a) a nuclease and (b) one or more guide RNA(s).
A method of treating or preventing a disorder associated with increased expression of a target polynucleotide sequence in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a nuclease in combination with a therapeutically effective amount of an agent that induces G0/G1 cell cycle arrest.
Dependencies are inferred from the claim text.