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.

中文翻译：

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定制衣壳定向进化技术以改进 AAV 介导的 CAR-T 生成


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 常数 （TRAC） 基因座的方法，以利用 CAR 表达的内源性启动子和调节元件。该方法使用基于 AAV6 的腺相关病毒 （AAV） 载体来递送编码 CAR 构建体的供体模板。自最初发布以来，这种有针对性的 CAR 整合技术已经进行了改进;然而，这些技术都没有专注于改进用于递送治疗货物的 AAV 载体。本文提出的研究开发了一种新型 AAV 衣壳定向进化平台，该平台允许专门选择新型 AAV 衣壳变体，从而能够更高效地靶向基因编辑介导的 CAR 构建体整合到原代 T 细胞中的 TRAC 基因座中。使用这个新平台，我们选择了几种新型 AAV，它们能够在 T 细胞中比 AAV6 更高效地进行编辑。两种新型衣壳 AAV-T1 和 AAV-T2 能够介导靶向敲入的 5 倍改善，这导致载体剂量减少 5 倍，以产生针对脑肿瘤细胞系的高溶细胞性 T 细胞。