Ontology highlight
ABSTRACT:
PROVIDER: PRJNA910947 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22677400_1.fastq.gz | Fastqsanger.gz | |||
| SRR22677400_2.fastq.gz | Fastqsanger.gz | |||
| SRR22677401_1.fastq.gz | Fastqsanger.gz | |||
| SRR22677401_2.fastq.gz | Fastqsanger.gz | |||
| SRR22677402_1.fastq.gz | Fastqsanger.gz |
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Project description:CRISPR/Cas9-engineered human T cells hold great promise in improving T cell effector functions for adoptive T cell therapies. To ensure safety during CRISPR/Cas9-editing optimized gRNAs, novel variants or prediction tools for indel outcomes have been developed. Still, several reports describe the occurrence of CRISPR-induced chromosomal aberrations, such as loss of large DNA fragments or even aneuploidy. So far, the measures to increase the safety of T cell products focused on the CRISPR Cas9 system. However, T cell-intrinsic features, such as their massive expansion after TCR stimulation, have not been fully taken into consideration. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of unwanted large deletions but with the downside of reduced knock-out efficiencies. An alternative strategy to reduce the risk of large deletions is the addition of the small molecule pifithrin-α after CRISPR/Cas9 editing. Pifithrin-α treatment results in smaller, more defined deletions and reduces the risk of CRISPR-induced chromosomal translocations and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells. Controlling T cell activation and the addition of pifithrin-α are easily accessible strategies for safer CRISPR/Cas9-engineering for adoptive T cell therapies.
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Project description:CRISPR/Cas9-engineered human T cells hold great promise in improving T cell effector functions for adoptive T cell therapies. To ensure safety during CRISPR/Cas9-editing optimized gRNAs, novel variants or prediction tools for indel outcomes have been developed. Still, several reports describe the occurrence of chromosomal truncations or loss of large DNA fragments of up to several kbs after CRISPR/Cas9 editing. So far, the measures to increase the safety of T cell products focused on the CRISPR Cas9 system. However, T cell-intrinsic features such as their massive expansion after TCR stimulation have not been taken into consideration. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with higher knock-out rates and larger deletions. Editing of non-activated T cells reduces the risk of unwanted large deletions but with the downside of reduced knock-out efficiencies. An alternative strategy to reduce the risk of large deletions is the addition of the small molecule pifithrin-α after CRISPR/Cas9 editing. Pifithrin-α treatment results in a smaller, more defined deletion pattern. PFT-a furthermore reduces the risk of unwanted chromosomal translocations and aneuploidy while maintaining the functionality of CRISPR-engineered T cells. Controlling T cell activation and the addition of pifithrin-α are easily accessible strategies for safer CRISPR/Cas9-engineering for adoptive T cell therapies.
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Project description:Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly dropped from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.
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