Assessing a single-cell multi-omic analytic platform to characterize ex vivo-engineered T cell therapy products

Genetically engineered CD8+ T cells are being explored for the treatment of various cancers. Analytical characterization represents a major challenge in the development of genetically engineered cell therapies, especially assessing the potential off-target editing and product heterogeneity. As conventional sequencing techniques only provide information at the bulk level, they are unable to detect off-target CRISPR translocation or editing events occurring in minor cell subpopulations. Here we report the analytical development of a single-cell multi-omic DNA and Protein assay to characterize genetically engineered cell products for safety and genotoxicity assessment. We were able to quantify on-target edits, off-target events, and potential translocations at the targeting loci with per-cell granularity, providing important characterization data of the final cell product. Conclusion: Single cell multi-omics approach provides the resolution required to understand the composition of cellular products and identify critical quality attributes (CQAs). Single-cell, amplicon-based DNA and protein sequencing were performed on edited T cells from four different donors (edited CGT products) upon simultaneous knocking out of the three loci: TRAC, TRBC1 and TRBC2.