SACF and GILA for in vitro transformation assessment of CRISPR/Cas9-edited cell therapy candidates: a multi-site study.
Tumorigenicity is a key safety concern for CRISPR/Cas9-based gene therapies, yet its assessment remains challenging due to the lack of relevant and sensitive in vivo models. This HESI Global multi-site study evaluated the Soft Agar Colony Formation (SACF) assay and the Growth in Low Attachment (GILA) assay for in vitro transformation testing of CRISPR/Cas9-edited cells as part of tumorigenicity risk assessment. Across four laboratories, MCF10A cells were spiked with varying concentrations of cells carrying a CRISPR/Cas9-mediated knockout of the known tumor suppressor PTPN12 to determine the assays' limit of detection (LOD), robustness and reproducibility. Both assays demonstrated a consistent LOD of 0.8% at most sites, with SACF showing a broader dynamic range and slightly stronger correlation across laboratories. GILA exhibited weaker correlation due to plateauing at high spike-in concentrations (>12.5%), and excluding these conditions improved inter-laboratory correlations. Additionally, the study identified key determinants of assay success, including the selection of appropriate cell lines and lots, and ensuring sufficient recovery time post-thaw. Together, these data establish multi-site reproducibility of SACF and GILA in the MCF10A-PTPN12 model evaluated here and support their use as animal-free, development-stage characterization tools contributing to tumorigenicity evidence generation.