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bioRxiv · 10.1101/2025.07.11.664404

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.
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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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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.. 2025-07-16. A Singular Base Editing Platform for Polyfunctional Multiplex Engineering of Immune Cells. https://doi.org/10.1101/2025.07.11.664404

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