肿瘤细胞治疗研究
英文原题：An Engineered Adeno-Associated Virus Variant Enables Efficient Gene Editing in Human T Cells.
同时，使用Tot3在人原代T细胞中实现了程序性细胞死亡蛋白1（PD-1）敲除和CAR过表达，敲除和敲入效率分别高达70%和55%。
CAR-T（CAR-T）细胞通过基因编辑系统与重组腺相关病毒（rAAV）共同构建，为治疗白血病提供了一种有前景的策略。rAAV作为同源定向修复的安全有效供体模板，因为它可以避免整合到宿主基因组中。然而，只有少数AAV血清型能够在低感染复数（MOI）下高效转导人原代T细胞，同时具有高包装效率。为了解决这个问题，从AAV2肽库中衍生的变体在Jurkat细胞中进行了筛选，随后在原代T细胞中进行了验证。经过三轮选择后，发现了一个位于VR-VIII区域之外的高排名序列NNSKLTV，并将其命名为Tot3。Tot3表现出与AAV2相似的转导效率，但MOI低27倍。此外，Tot3表现出更高的包装效率和降低的热稳定性。同时，使用Tot3在人原代T细胞中实现了程序性细胞死亡蛋白1（PD-1）敲除和CAR过表达，敲除和敲入效率分别高达70%和55%。这些CAR-T细胞在弥漫性B细胞淋巴瘤小鼠模型中表现出显著增强的抗肿瘤活性和延长的生存时间。
Chimeric antigen receptor T (CAR-T) cells, created by gene editing systems along with recombinant adeno-associated virus (rAAV), provide a promising strategy for treating leukemia. rAAVs serve as a safe and effective donor template for homology-directed repair because they can avoid integrating into the host genome. However, only a few AAV serotypes can efficiently transduce human primary T cells at low multiplicities of infection (MOIs) with high packaging efficiency. To address this problem, variants derived from an AAV2 peptide library were screened in Jurkat cells and later validated in primary T cells. A high-ranking sequence identified outside the VR-VIII region, NNSKLTV, was discovered after three rounds of selection and was named Tot3. Tot3 demonstrated transduction efficiency similar to AAV2, but at a 27-fold lower MOI. In addition, Tot3 exhibited greater packaging efficiency and reduced thermal stability. Simultaneously, programmed cell death protein 1 (PD-1) knockout and CAR overexpression were achieved in human primary T cells using Tot3, with knockout and knock-in efficiencies reaching up to 70% and 55%, respectively. These CAR-T cells demonstrated significantly enhanced antitumor activity and increased survival times in a mouse model of diffuse B cell lymphoma.
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