Engel Nils W, Steinfeld Israel, Ryan Daniel, Anupindi Kusala, Kim Samuel, Wellhausen Nils, Chen Linhui, Wilkins Katherine, Baker Daniel J, Rommel Philipp C, Jarocha Danuta, Gohil Mercy, Zhang Qian, Milone Michael C, Fraietta Joseph A, Davis Megan, Young Regina M, June Carl H
Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
Agilent Research Laboratories, Santa Clara, CA 95051.
Proc Natl Acad Sci U S A. 2025 May 20;122(20):e2427216122. doi: 10.1073/pnas.2427216122. Epub 2025 May 5.
Genome-editing technologies have enabled the clinical development of allogeneic cellular therapies, yet the optimal gene-editing modality for multiplex editing of therapeutic T cell product manufacturing remains elusive. In this study, we conducted a comprehensive comparison of CRISPR/Cas9 nuclease and adenine base editor (ABE) technologies in generating allogeneic chimeric antigen receptor (CAR) T cells, utilizing extensive in vitro and in vivo analyses. Both methods achieved high editing efficiencies across four target genes, critical for mitigating graft-versus-host disease and allograft rejection: or , , , and . Notably, ABE demonstrated higher manufacturing yields and distinct off-target profiles compared to Cas9, with translocations observed exclusively in Cas9-edited products. Functionally, ABE-edited CAR T cells exhibited superior in vitro effector functions under continuous antigen stimulation, including enhanced proliferative capacity and increased surface CAR expression. Transcriptomic analysis revealed that ABE editing resulted in reduced activation of p53 and DNA damage response pathways at baseline, along with sustained activation of metabolic pathways during antigen stress. Consistently, Assay for Transposase-Accessible Chromatin using sequencing data indicated that Cas9-edited, but not ABE-edited, CAR T cells showed enrichment of chromatin accessibility peaks associated with double-strand break repair and DNA damage response pathways. In a preclinical leukemia model, ABE-edited CAR T cells demonstrated improved tumor control and extended overall survival compared to their Cas9-edited counterparts. Collectively, these findings position ABE as superior to Cas9 nucleases for multiplex gene editing of therapeutic T cells.
基因组编辑技术推动了同种异体细胞疗法的临床发展，然而，用于治疗性T细胞产品制造的多重编辑的最佳基因编辑方式仍不明确。在本研究中，我们利用广泛的体外和体内分析，对CRISPR/Cas9核酸酶和腺嘌呤碱基编辑器（ABE）技术在生成同种异体嵌合抗原受体（CAR）T细胞方面进行了全面比较。两种方法在四个对减轻移植物抗宿主病和同种异体移植排斥至关重要的靶基因上均实现了高编辑效率：即 或 、 、 以及 。值得注意的是，与Cas9相比，ABE显示出更高的制造产量和独特的脱靶谱，易位仅在Cas9编辑的产品中观察到。在功能上，ABE编辑的CAR T细胞在持续抗原刺激下表现出卓越的体外效应功能，包括增强的增殖能力和增加的表面CAR表达。转录组分析表明，ABE编辑在基线时导致p53激活和DNA损伤反应途径减少，同时在抗原应激期间代谢途径持续激活。一致地，使用测序数据的转座酶可及染色质分析表明，Cas9编辑而非ABE编辑的CAR T细胞显示出与双链断裂修复和DNA损伤反应途径相关的染色质可及性峰的富集。在临床前白血病模型中，与Cas9编辑的CAR T细胞相比，ABE编辑的CAR T细胞显示出更好的肿瘤控制和更长的总生存期。总体而言，这些发现表明ABE在治疗性T细胞的多重基因编辑方面优于Cas9核酸酶。
