Skeate Joseph G, Slipek Nicholas J, Lahr Walker S, Roy Shambojit, Wick Bryce J, Stelljes Erin M, Gilkey Alexandria K, Thenge Prateek P, Diers Miechaleen D, Kar Bibekananda, Krueger Joshua B, Niemeyer Ethan M, Lonetree Cara-Lin, Kluesner Mitchell G, Bell Jason B, Clement Kendell, Provenzano Paolo, Moriarity Branden S, Webber Beau R
Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
bioRxiv. 2025 Jul 16:2025.07.11.664404. doi: 10.1101/2025.07.11.664404.
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 (), 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.
目前设计抗原特异性受体的方法依赖于随机整合载体或双链断裂诱导的靶向整合，这两种方法都存在安全风险。为了实现一种用于多重基因敲除（KO）和基因敲入（KI）的一体化工具，我们扩大了胞嘧啶和腺嘌呤碱基编辑器（ABE）切口酶活性的应用，以刺激同源定向修复（HDR），并将临床相关的嵌合抗原受体（CAR）插入特定基因座。通过一种新颖的sgRNA设计策略和重组腺相关病毒（rAAV）递送的DNA模板，我们提高了ABE8e刺激的HDR在人T细胞中的效率。通过将靶向CD19、CD33或间皮素的CAR的KI与>95%的四重基因KO相结合，我们实现了具有增强功能的高活性即用型CAR T细胞产品的一步生成。重要的是，我们未检测到可检测到的易位或明显的脱靶编辑，并证明了其对多种癌细胞系和抑制性3D球体培养模型的疗效。这种用于T细胞同步工程重编程的迭代切口高效工程过程（INSERT）为先进的治疗性CAR T工程建立了一个安全、简化的平台。
