A Singular Base Editing Platform for Polyfunctional Multiplex Engineering of Immune Cells
Skeate, J. G.; Slipek, N. J.; Lahr, W. S.; Roy, S.; Wick, B. J.; Stelljes, E. M.; Gilkey, A. K.; Thenge, P. P.; Diers, M. D.; Kar, B.; Krueger, J. B.; Niemeyer, E. M.; Lonetree, C.-l.; Kluesner, M. G.; Bell, J. B.; Clement, K.; Provenzano, P.; Moriarity, B. S.; Webber, B. R.
Show abstract
Current methods to engineer antigen-specific receptors rely on randomly integrating vectors or double-strand break induced targeted integration, both of which pose safety risks. To implement an all-in-one tool for multiplex knockout (KO) and knock in (KI), we expand the use of cytosine and adenine base editor (ABE) nickase activity to stimulate homology-directed repair (HDR) and insert clinically relevant chimeric antigen receptors (CARs) into specific loci. Through a novel sgRNA design strategy and a recombinant adeno-associated virus (rAAV) delivered DNA template, we enhanced the efficiency of ABE8e-stimulated HDR in human T cells. By combining KI of CD19-, CD33-, or mesothelin-targeting CARs with >95% quadplex gene KO (B2M/CD3{varepsilon}/PDCD1/CISH), we achieve single-step generation of highly functional off-the-shelf CAR T cell products with enhanced function. Importantly, we found no detectable translocations or significant off-target edits and demonstrated efficacy against multiple cancer lines, and a suppressive 3D spheroid culture model. This efficient engineering process of Iterative Nicking for Synchronous Engineered Reprogramming of T cells (INSERT) establishes a safe, simplified platform for advanced therapeutic CAR T engineering.
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