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

The disruption of immune checkpoints in T cells is a promising tool for improving the efficacy of tumor-infiltrating lymphocyte (TIL) therapy. While CRISPR-Cas9 genome-editing is efficient, Cas9 nucleases induce double-strand DNA breaks and risks improper translocations, inversions, and chromosomal deletions in engineered T cells. Cas9 nickase (nCas9) used in base-editing cuts only a single strand of DNA, reducing genetic aberrations in modified cells. Here, we established a small-scale, good manufacturing practice-compatible adenine base editing (ABE) procedure for both single and dual knockout of co-inhibitory receptors TIM3 and TIGIT in TILs. ABE-mediated conversion of A·T to G·C pairs in TIM3 and TIGIT specific splice-sites led to high knockout efficiency, with negligible insertion-deletion events post editing. Using melanoma and ovarian TILs, we show that target-specific editing by ABE of TIM3 and TIGIT improved (1) TIL fold-expansion during the rapid expansion protocol without adversely impacting phenotype, (2) cytokine production, and (3) serial killing upon co-culture with autologous patient-derived tumor cells <i>in vitro.</i> Moreover, dual edited TILs were able to infiltrate tumor spheroids <i>in vitro</i> and control patient-derived tumors <i>in vivo</i>. Taken together, we show the feasibility of ABE multiplex editing as a promising tool for engineering TILs for clinical applications.