Westhaus Adrian, Barba-Sarasua Elena, Chen Yuyan, Hsu Kenneth, Scott Suzanne, Knight Maddison, Haase Florencia, Mesa Mora Santiago, Houghton Benjamin C, Roca-Pinilla Ramon, Kalajdzic Predrag, O'Neill Geraldine, Thrasher Adrian J, Santilli Giorgia, Lisowski Leszek
Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia; Infection, Immunity and Inflammation Research and Teaching Department, Great Ormond Street Institute of Child Health, University College, London, UK.
Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia.
Mol Ther. 2025 Jun 4;33(6):2801-2818. doi: 10.1016/j.ymthe.2024.12.012. Epub 2024 Dec 12.
Chimeric antigen receptor (CAR) T cell (CAR-T) therapies present options for patients diagnosed with certain leukemias. Recent advances of the technology included a method to integrate the CAR into the T cell receptor alpha constant (TRAC) locus to take advantage of the endogenous promoter and regulatory elements for CAR expression. This method used adeno-associated viral (AAV) vectors based on AAV6 to deliver the donor template encoding the CAR construct. Since the original publication, improvements have been made to this targeted CAR integration technique; however, none of those techniques focused on improving the AAV vector used to deliver the therapeutic cargo. The herein presented study developed a novel AAV capsid directed evolution platform that allows for specifically selecting for novel AAV capsid variants that enable more efficient targeted gene editing-mediated CAR construct integration into the TRAC locus in primary T cells. Using this new platform, we selected several novel AAVs that enable more efficient editing in T cells than AAV6. Two novel capsids, AAV-T1 and AAV-T2, were able to mediate 5-fold improvement for on-target knockin, which resulted in 5-fold reduction of the vector dose to produce highly cytolytic T cells against a brain tumor cell line.
嵌合抗原受体（CAR）T细胞（CAR-T）疗法为被诊断患有某些白血病的患者提供了治疗选择。该技术的最新进展包括一种将CAR整合到T细胞受体α恒定区（TRAC）基因座的方法，以利用内源性启动子和调控元件来表达CAR。这种方法使用基于AAV6的腺相关病毒（AAV）载体来递送编码CAR构建体的供体模板。自最初发表以来，这种靶向CAR整合技术已有改进；然而，这些技术都没有专注于改进用于递送治疗性物质的AAV载体。本文介绍的研究开发了一种新型AAV衣壳定向进化平台，该平台能够特异性地筛选新型AAV衣壳变体，从而更有效地将靶向基因编辑介导的CAR构建体整合到原代T细胞的TRAC基因座中。利用这个新平台，我们筛选出了几种新型AAV，它们在T细胞中的编辑效率比AAV6更高。两种新型衣壳AAV-T1和AAV-T2能够使靶向敲入提高5倍，这使得产生针对脑肿瘤细胞系的高细胞毒性T细胞所需的载体剂量减少了5倍。
