论文部分内容阅读
抗肿瘤T细胞受体(T cell receptor,TCR)基因治疗在临床上已获得了巨大进展,但仍然存在一些技术瓶颈。例如,内外源性TCR链随机组合形成自身反应性TCR分子、外源基因随机插入导致抑癌基因灭活等。为解决这些问题,作者提出建立低/单拷贝TCR基因置换技术:即结合逆转录病毒和重组酶介导的盒式交换(recombinase mediated cassette exchange,RMCE)技术,实现TCR基因定点置换以构建TCR稳定表达系统。首先,通过逆转录病毒转导在16.113和Jurkat76细胞引入了含有lox P和FRT位点的EGFP(enhanced green fl uorescent protein)基因;然后,利用RMCE方法将EGFP置换成MAGE-A1的TCRαβ基因,置换效率高达5%。在Jurkat76细胞表面检测到了TCR和CD3分子,并能与MAGE-A1特异性HLA-A2多聚体结合。预计利用这一TCR基因置换系统结合干细胞技术可快速产生抗原特异性的T细胞,为TCR-T细胞治疗的安全应用提供一个新策略。
There have been many clinical advances in the gene therapy of T cell receptor (TCR), but there are still some technical bottlenecks. For example, internal and external TCR chains are randomly combined to form autoreactive TCR molecules, and random insertion of exogenous genes leads to inactivation of tumor suppressor genes. To address these issues, the authors propose the establishment of a low / single copy TCR gene replacement technique that combines site-specific transposition of TCR genes with TCR to stabilize TCR by combining retroviral and recombinase mediated cassette recombination mediated cassette exchange (RMCE) Expression system. First, an EGFP (enhanced green florescent protein) gene containing loxP and FRT sites was introduced into 16.113 and Jurkat76 cells by retroviral transduction; then, the EGFP was replaced with MCE-A1’s TCRαβ gene by the RMCE method, Efficiency up to 5%. TCR and CD3 molecules were detected on the surface of Jurkat76 cells and bound to MAGE-A1-specific HLA-A2 multimers. This TCR gene replacement system combined with stem cell technology is expected to rapidly generate antigen-specific T cells, providing a new strategy for the safe use of TCR-T cell therapy.