# A singular base-editing platform for polyfunctional multiplex engineering of immune cells

Article · Skeate JG, Slipek NJ, Lahr WS, Roy S, Wick BJ, Kar B, Chang JW, Stelljes EM, Gilkey AK, Thenge PP, Diers MD, Krueger JB, Niemeyer EM, Lonetree CL, Kluesner MG, Bell JB, Clement K, Provenzano PP, Moriarity BS, Webber BR

- Archive id: doi:10.1016/j.ymthe.2026.07.033
- Published: 2026-07-18
- Venue: Molecular therapy : the journal of the American Society of Gene Therapy
- DOI: https://doi.org/10.1016/j.ymthe.2026.07.033
- PMID: 42470099
- Citations: 2
- Source URL: https://doi.org/10.1016/j.ymthe.2026.07.033
- Provenance: europepmc

## 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 knockin (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 single-guide RNA design strategy and a DNA template delivered by a recombinant adeno-associated virus, 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/CD3E/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.

_Abstract reproduced under CC-BY (article license as recorded by Europe PMC; abstract reproduced unmodified apart from markup removal)._

Topics: [CRISPR & Gene-Editing Therapies](https://space-kitty.com/topics/gene-editing-therapies)

---
Space Kitty Archive · https://space-kitty.com · API: https://space-kitty.com/api/v1 · MCP: https://space-kitty.com/mcp · Premium: $0.20/request via x402 (Base Sepolia (testnet)). Metadata: arXiv (CC0), OpenAlex (CC0), Europe PMC (CC BY/CC0 works), ClinicalTrials.gov.