CRISPR/methotrexate-integrated strategy for TCR-T cell engineering with reduced chromosome 14 loss.

Adoptive T cell therapy, particularly T cell receptor-engineered T (TCR-T) cell therapy, holds promise for cancer treatment in solid tumors and hematological malignancies. Conventional lentiviral TCR-T cell therapies face insertional mutagenesis risks, while CRISPR-mediated T cell receptor α constant (TRAC) locus targeting suffers from suboptimal knockin efficiency and chromosome 14 loss. To address these challenges, we introduced a non-viral strategy combining CRISPR-Cas9 electroporation with methotrexate (MTX) metabolic selection. Primary human T cells were engineered to integrate a CMV-pp65-specific TCR and an MTX-resistant dihydrofolate reductase (DHFR)-FS cassette into the TRAC locus via homology-directed repair (HDR). Systematic optimization of electroporation timing, buffer systems, and HDR enhancers achieved initial TCR integration efficiency of ∼20%. Subsequent 6-day MTX treatment enriched engineered cells to ∼70% purity while selectively depleting unedited and chromosomally aberrant clones. Fluorescence in situ hybridization revealed that MTX enrichment reduced CRISPR-associated chromosome 14 loss comparable to unedited T cells. Functionally, TRAC-TCR-T cells exhibited enhanced interferon (IFN)-γ/tumor necrosis factor alpha (TNF-α) secretion and reduced exhaustion markers versus lentiviral counterparts, while maintaining equivalent tumor clearance efficacy in vitro and in xenograft models. In conclusion, this integrated platform mitigates viral vector risks, alleviates concerns of CRISPR-associated genomic instability, and provides a good manufacturing practice (GMP)-compatible approach that may facilitate the development of safer adoptive TCR-T immunotherapies.