肿瘤细胞治疗研究
英文原题：Platinum TALEN-mediated nonviral gene editing facilitates clinical-scale production of cancer antigen-reactive T cells.
Platinum TALEN-mediated nonviral gene editing facilitates clinical-scale production of cancer antigen-reactive T cells.
Platinum TALEN介导的非病毒基因组编辑促进了内源性TCR被所需TCR的替换，并可应用于治疗性T细胞产品的临床生产。
近年来，靶向基因组编辑已成为设计型细胞产品临床开发的下一代工具。尽管成簇规律间隔短回文重复序列-Cas9（CRISPR-Cas9）是治疗性基因组编辑中最常采用的核酸酶，但其广泛应用仍受限于潜在的脱靶效应和高额专利许可费。铂转录激活样效应物核酸酶（TALEN）是一种经修饰的 TALEN，其携带非重复可变双残基（non-RVD）变异，比缺乏 non-RVD 变异的传统 TALEN 具有更高效率。
在本研究中，我们利用靶向T细胞受体（TCR）基因位点的Platinum TALEN和单链DNA同源定向修复（HDR）模板，旨在制备经TCR替换的人类T细胞，这些细胞被重编程以识别癌症抗原并杀死癌细胞。
该系统可重复地从约50 mL外周血中在临床规模上制备TCR工程化T细胞。所得基因组编辑T细胞在CD4+和CD8+组分中均保留naïve/naïve样和记忆表型细胞，并在体外对癌细胞系表现出高效的细胞溶解活性。
BACKGROUND AIMS: Recently, target-genome editing has emerged as a next-generation tool for the clinical development of designed cellular products. Although Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) is the most frequently adopted nuclease for therapeutic genome editing, its widespread use is still hampered by potential off-target effects and high patent royalties. Platinum transcription activator-like effector nuclease (TALEN) is a modified TALEN that harbors non-repeat-variable di-residue (non-RVD) variations and confers higher efficiency than conventional TALENs lacking non-RVD variations. METHODS: In this study, using Platinum TALEN targeting T-cell receptor (TCR) gene loci and a single-stranded DNA homology-directed repair (HDR) template, we aimed to produce TCR-replaced human T cells reprogrammed to recognize a cancer antigen and kill cancer cells. RESULTS: This system can reproducibly produce TCR-engineered T cells from ∼50 mL of peripheral blood on a clinical scale. The resulting genome-edited T cells retain naïve/naïve-like and memory phenotype cells in both CD4+ and CD8+ fractions and exhibit efficient cytolytic activity against cancer cell lines in vitro. CONCLUSION: In conclusion, Platinum TALEN-mediated nonviral genome editing facilitates the replacement of the endogenous TCR with a desired TCR and can be applied to the clinical manufacturing of therapeutic T-cell products.
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