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Background TK-6302 consists of autologous enriched and expanded CD4 and CD8 T cells which are genome edited using CRISPR technology to express a PRAME-targeting TCR, a costimulatory single-chain CD8 co-receptor and a FAS switch receptor (FAS-SwR). The endogenous TCR alpha and TCR beta genes are knocked out using two single-stranded guide RNAs (sgRNA). A multicistronic transgene cassette encoding for the TCR, the CD8 coreceptor, and the FAS-SwR is knocked in at the TCR alpha constant (TRAC) locus. Successful knock-out (KO) and knock-in (KI) will result in full replacement of the endogenous TCR with exogenous PRAME-TCR, preventing formation of mixed TCR alpha/beta dimers and enhancing PRAME-TCR expression by eliminating the competition for assembly with CD3 molecules to be expressed at the cell surface.Methods On-target editing was measured by FACS, dPCR and AMP-Sequencing. Potential off-target effects of sgRNAs were nominated using ONE-seq and DEUX-seq, followed by confirmation using rhAMP-sequencing. Chromosomal integrity was assessed by SAFER detect, directional Genome Hybridization (dGH), G-banding, and dPCR. Correct knock-in of the HDR template was analysed by targeted locus amplification (TLA). Single cell amplicon sequencing (Tapestri) was used to characterize the genome editing events at cellular level. Studies were conducted with TK-6302 produced from at-scale manufacturing runs with healthy donor material representing the intended clinical product.Results TK-6302 contained 39-48% TCR negative T cells (knock out) and 24-42% PRAME-TCR positive cells (Knock-in) measured by FACS. Off-target nomination and confirmation process resulted in the identification of one single off-target site with a very low editing frequency. Consistent with previous reports, anticipated chromosomal translocations between on-target sites were either undetectable or occurred at very low frequencies and did not confer a growth advantage. TLA demonstrated the specific integration in the TRAC locus (chromosome #14). Tapestri and dGH single cell data revealed expected integration patterns and confirmed the single off-target hit. Data collection for chromosomal integrity using full scale runs using healthy donors is ongoing and data will be presented.Conclusions Comprehensive preclinical evaluation supports the safety of the genome editing process used to engineer TK-6302, with no concerning off-target activity or evidence of genomic instability observed. These findings provide a robust foundation for the clinical development of TK-6302 across PRAME-expressing solid tumor indications.