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首页> 外文OA文献 >Lentiviral Vector Design for Optimal T Cell Receptor Gene Expression in the Transduction of Peripheral Blood Lymphocytes and Tumor-Infiltrating Lymphocytes
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Lentiviral Vector Design for Optimal T Cell Receptor Gene Expression in the Transduction of Peripheral Blood Lymphocytes and Tumor-Infiltrating Lymphocytes

机译：在外周血淋巴细胞和肿瘤浸润淋巴细胞转导中最佳T细胞受体基因表达的慢病毒载体设计

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Lentiviral vectors containing promoters of distinct origins, that is, strong viral promoters (cytomegalovirus [CMV] and murine stem cell virus [MSCV]), a cellular promoter (phosphoglycerate kinase [PGK]), and two composite promoters (CAG [a composite promoter sequence comprised of the CMV enhancer and portions of the chicken β-actin promoter and the rabbit β-globin gene] and SV40/CD43), were used to evaluate green fluorescent protein (GFP) reporter gene expression in human primary peripheral blood lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs). In PBLs, vectors containing the MSCV promoter were found to be optimal for expression in both minimally stimulated and highly activated lymphocytes. The stability of gene expression was monitored for up to 7 weeks in culture and the MSCV promoter-containing vector was found to be comparable to the cellular PGK promoter-containing vector. The MSCV promoter-containing lentiviral vector was also the most active in transduced TILs and these cells retained biological activity as measured by antimelanoma antigen reactivity. Using the knowledge gained in comparing individual promoters, a series of two-gene-containing lentiviral vectors was constructed in an attempt to produce the α and β chains of antitumor antigen T cell receptors (TCRs). Dual-promoter or internal ribosome entry site (IRES)-containing vector designs were evaluated and found to be unable to produce both chains of the TCR in amounts that led to significant biological activity. In contrast, if the α and β chains were linked by a 2A ribosomal skip peptide, both proper TCR chain pairing and biologically activity were observed. This paper emphasizes the need to optimize both promoter function and protein synthesis in constructs that require stoichiometric production of multiple protein subunits.
机译：慢病毒载体包含不同来源的启动子，即强病毒启动子（巨细胞病毒[CMV]和鼠类干细胞病毒[MSCV]），细胞启动子（磷酸甘油酸激酶[PGK]）和两个复合启动子（CAG [复合启动子]）包含CMV增强子和鸡β-肌动蛋白启动子和兔β-珠蛋白基因的一部分的序列]和SV40 / CD43），用于评估人原代外周血淋巴细胞（PBLs）中绿色荧光蛋白（GFP）报告基因的表达）和肿瘤浸润淋巴细胞（TIL）。在PBL中，发现含有MSCV启动子的载体对于在最小刺激和高度活化的淋巴细胞中表达都是最佳的。在培养中监测基因表达的稳定性长达7周，发现含MSCV启动子的载体与含细胞PGK启动子的载体相当。含有MSCV启动子的慢病毒载体在转导的TIL中也是最活跃的，这些细胞保留了按炭黑瘤抗原反应性测定的生物学活性。利用在比较各个启动子中获得的知识，构建了一系列包含两个基因的慢病毒载体，以尝试产生抗肿瘤抗原T细胞受体（TCR）的α和β链。评估包含双启动子或内部核糖体进入位点（IRES）的载体设计，发现无法产生导致显着生物学活性的TCR两条链。相反，如果α和β链通过2A核糖体跳跃肽连接，则可以同时观察到正确的TCR链配对和生物学活性。本文强调需要在需要化学计量产生多个蛋白质亚基的构建体中优化启动子功能和蛋白质合成。

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