Highly efficient gene knockout in tumor-infiltrating lymphocytes by adenine base editing

Area of Science:

• Immunology
• Gene Editing
• Cancer Therapy

Background:

• Immune checkpoint disruption in T cells can enhance tumor-infiltrating lymphocyte (TIL) therapy.
• CRISPR-Cas9 genome editing risks chromosomal aberrations due to double-strand DNA breaks.
• Cas9 nickase (nCas9) and base editing offer safer alternatives by inducing single-strand DNA breaks.

Purpose of the Study:

• To establish a Good Manufacturing Practice-compatible adenine base editing (ABE) procedure for engineering TILs.
• To evaluate the efficiency and safety of ABE for single and dual knockout of TIM3 and TIGIT in TILs.
• To assess the impact of ABE-mediated gene editing on TIL function and anti-tumor activity.

Main Methods:

• Developed a small-scale, GMP-compatible adenine base editing (ABE) protocol.
• Performed single and dual knockout of co-inhibitory receptors TIM3 and TIGIT in TILs using ABE.
• Assessed editing efficiency, insertion-deletion events, TIL expansion, phenotype, cytokine production, and killing capacity in vitro and in vivo.

Main Results:

• Achieved high knockout efficiency for TIM3 and TIGIT using ABE with minimal insertion-deletion events.
• ABE editing improved TIL expansion and function without adverse phenotypic changes.
• Edited TILs demonstrated enhanced cytokine production, serial killing of tumor cells, tumor spheroid infiltration, and in vivo tumor control.

Conclusions:

• Adenine base editing is a feasible and efficient method for multiplex editing of TILs.
• ABE offers a safer alternative to CRISPR-Cas9 for generating genetically engineered TILs for clinical applications.
• ABE-engineered TILs show promise for enhancing cancer immunotherapy efficacy.