Related Experiment Video
Updated: May 21, 2025
Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation
Published on: February 28, 2025
DNA-dependent protein kinase inhibitors PI-103 and samotolisib augment CRISPR/Cas9 knock-in efficiency in human T
Emina Džafo1, Morteza Hafezi1, Greta Maria Paola Giordano Attianese1
1Ludwig Institute for Cancer Research, Lausanne Branch, Department of Oncology, University of Lausanne (UNIL), and Lausanne University Hospital (CHUV), Lausanne, Switzerland.
Abstract:
The adoptive transfer of autologous peripheral blood T cells gene-modified to express preselected, tumor antigen-specific T-cell receptors (TCRs) is a promising treatment for solid cancers. While gene-transfer by viral transduction is highly efficient, the insertional site is not targeted and persistence of the T cells is oftentimes limited. In contrast, site-specific integration of the TCR into the TCR α chain (TRAC) locus by CRISPR/Cas9 has been shown to enable more consistent and physiologic levels of exogenous TCR expression coupled with superior persistence and tumor control in preclinical studies. Here, we sought to improve the efficiency of CRISPR/Cas9 mediated TCR knock-in (KI) into the TRAC locus of primary human T cells. In addition to the previously reported DNA-dependent protein kinase (DNA-PK) inhibitor M3814, we demonstrated that PI-103 and samotolisib markedly increase KI efficiency in a process that is good manufacturing process (GMP)-compatible. Importantly, samotolisib enabled the generation of a potent T-cell product, having no negative impact on T-cell viability, phenotype, expansion, effector function, and tumor control. Overall, we conclude that our GMP-compatible CRISPR/Cas9 protocol comprising samotolisib to augment TCR KI efficiency is suitable for the generation of genetically modified T cells for clinical use.
Insights
Improving CRISPR/Cas9 gene editing for cancer therapy, this study enhances T-cell receptor (TCR) knock-in efficiency using specific inhibitors. This leads to better T-cell persistence and tumor control for potential clinical applications.
Area of Science:
- Immunology
- Gene Therapy
- Oncology
Background:
- Adoptive T-cell therapy shows promise for solid cancers.
- Viral transduction for T-cell receptor (TCR) gene transfer has limitations in targeting and cell persistence.
- CRISPR/Cas9 mediated site-specific integration into the TCR α chain (TRAC) locus offers improved T-cell persistence and tumor control.
Purpose of the Study:
- To enhance the efficiency of CRISPR/Cas9 mediated TCR knock-in (KI) into the TRAC locus of primary human T cells.
- To identify novel compounds that improve KI efficiency in a Good Manufacturing Process (GMP)-compatible manner.
- To evaluate the impact of enhanced KI on T-cell product potency and functionality.
Main Methods:
- Utilized CRISPR/Cas9 gene editing for TCR KI into the TRAC locus of human T cells.
- Investigated the efficacy of DNA-dependent protein kinase (DNA-PK) inhibitors, including M3814, PI-103, and samotolisib, in enhancing KI efficiency.
- Assessed T-cell viability, phenotype, expansion, effector function, and tumor control following gene modification with samotolisib.
Main Results:
- PI-103 and samotolisib significantly increased CRISPR/Cas9 mediated TCR KI efficiency.
- Samotolisib demonstrated GMP-compatibility and enhanced KI efficiency without compromising T-cell viability, phenotype, expansion, or effector function.
- The developed protocol, using samotolisib, resulted in potent T-cell products with effective tumor control in preclinical models.
Conclusions:
- A GMP-compatible CRISPR/Cas9 protocol was established to improve TCR KI efficiency in human T cells.
- Samotolisib is identified as a potent enhancer of TCR KI, suitable for clinical T-cell manufacturing.
- This optimized approach facilitates the generation of genetically modified T cells for enhanced cancer immunotherapy.
