CRISPR-Cas9-mediated ON-target genotoxicity – CRISPR-genotox
CRISPR-Cas9 gene therapy: importance of assessing potential genotoxic risks to mitigate them
Numerous clinical trials using CRISPR technology for gene therapy are underway in many medical fields (cancer, rare genetic diseases, infectious diseases, etc.). The first drug has been granted marketing authorization in France. These new tools modify the genome of cells by creating a double-strand break in the DNA. It is important to assess the potential risks associated with this manipulation.
The objective of this work is to assess genotoxicity at the targeted locus in human cells of clinical interest. It could induce genomic instability that is harmful to the cell. To this end, innovative, sensitive methods are being developed at the single-cell level for high sensitivity. The first objective is to detect, quantify, and describe potential chromosomal rearrangements. The second objective is to understand the mechanisms involved in order to understand why only a small proportion of cells exhibit chromosomal rearrangements. The final objective is to prevent the occurrence of these unwanted events.
The work carried out has enabled a better description and understanding of the heterogenous genotoxicity induced by CRISPR-Cas9 nuclease at the targeted locus.
These studies, through the development of highly sensitive methods, allow for a better detection and description the risks associated with the use of these innovative technologies (cytometry and scSNP-DNAseq).
Here, by understanding of the mechanisms involved in the occurrence of adverse effects, we proposed methods to monitor genotoxicity and preventive solutions that can be used in the laboratory and in gene therapy.
The work carried out has enabled a better description and understanding of genotoxicity induced by CRISPR-Cas9 nuclease at the targeted locus.
These studies, through the development of highly sensitive methods, allow (i) a better understanding of the risks associated with the use of these innovative technologies, and (ii) an understanding of the mechanisms involved in the occurrence of adverse effects in order to propose reliable detection methods and prevention solutions that can be used in the laboratory and in gene therapy.
In particular, the work has highlighted the role of p53 and the cell cycle in the occurrence of adverse events. These results have led to the proposal of a protocol using palbociclib to reduce the genotoxicity of CRISPR-Cas9 nuclease and to propose a high-performance genotoxicity analysis method (scSNP-DNAseq).
Our work has led to the development of:
- an editing protocol that drastically reduces the genotoxicity of the nuclease (palbociclib)
- a sensitive and innovative method for analyzing genotoxicity using DNA sequencing at the individual cell level
A national FHU Redgene network has been created to monitor the genotoxicity of the drug Casgevy using CRISPR technology in patients with hemoglobinopathies. We are participating in this network by proposing scSNP-DNAseq technology for longitudinal monitoring of the genome stability of genetically modified cells.
CRISPR-Cas9 nuclease is a very promising technology for gene therapy. The first clinical trials have started. However, genome editing, to be safe, must be precise and reliable. Genotoxicity at the targeted locus (ON-target) is little studied. Unexpectedly, we observed megabase-scale terminal chromosomal deletions, following the use of Cas9 nuclease to edit the genome at the UROS (Chr10) and the globin (Chr11) loci in cell lines. These results raise a potential new worrisome safety issue for CRISPR use in clinic. Indeed, theses undesired outcomes can lead to the loss of many genes. This nuclease side-effect was recently confirmed in human embryos. Its prevalence is unknown in human non-embryonic primary cells. This project will evaluate whether Cas9-mediated large genome modifications occur in primary cells currently involved in CRISPR-Cas9 gene therapies, measure their functional impact, and reveal the molecular mechanism(s) to find solutions to secure CRISPR-Cas9 technology.
Project coordination
Aurélie BEDEL (INSERM U1035 BIOTHÉRAPIES DES MALADIES GÉNÉTIQUES ET CANCERS)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partnership
U1035 BMGIC INSERM U1035 BIOTHÉRAPIES DES MALADIES GÉNÉTIQUES ET CANCERS
Help of the ANR 322,313 euros
Beginning and duration of the scientific project:
September 2021
- 36 Months
