Authors
Joseph G Skeate, Nicholas J Slipek, Walker S Lahr, Shambojit Roy, Bryce J Wick, Bibekananda Kar, Jae-Woong Chang, Erin M Stelljes, Alexandria K Gilkey, Prateek P Thenge, Miechaleen D Diers, Joshua B Krueger, Ethan M Niemeyer, Cara-Lin Lonetree, Mitchell G Kluesner, Jason B Bell, Kendell Clement, Paolo P Provenzano, Branden S Moriarity, Beau R Webber
Published in
Molecular therapy : the journal of the American Society of Gene Therapy. Jul 17, 2026. Epub Jul 17, 2026.
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 base editor 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/CD3ε/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.
PMID:
42470099
Bibliographic data and abstract were imported from PubMed on 18 Jul 2026.
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