Promoter choice: Who should drive the CAR in T cells?
Promoter choice: Who should drive the CAR in T cells?
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DOI：
10.1371/journal.pone.0232915
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发表时间：
2020-07-24
期刊：
影响因子：
3.7
通讯作者：
McLellan, Alexander D.
中科院分区：
文献类型：
--
作者：
Rad, Ali Hosseini S. M.;Poudel, Aarati;McLellan, Alexander D.
嵌合抗原受体（CAR）T细胞疗法是B细胞恶性肿瘤的有效治疗，具有治疗其他血液系统癌症和实体瘤的新兴潜力。CAR盒内启动子的强度将改变T细胞的细胞表面上的CAR-多肽水平-影响T细胞中活化、存活和记忆细胞形成的动力学。除了CAR之外，启动子可用于驱动其他感兴趣的基因以增强CAR T细胞功能。从单个RNA转录物表达多个基因可以通过经由核糖体跳跃位点连接基因来有效地实现。然而，启动子可能在其转录较长RNA的能力上不同，或者可能干扰慢病毒的产生或转导频率。在这项研究中，我们比较了强的良好表征的启动子CMV，EF-1，hPGK和RPBSA驱动编码三种产物的单个RNA的功能表达的能力：GFP，CAR，加上额外的细胞存活基因Mcl-1。尽管四种启动子产生类似的高慢病毒滴度，但EF-1给出了原代T细胞的最佳转导功效。在启动子驱动编码GFP、CAR和Mcl-1的长RNA表达的能力方面发现了主要差异，突出了启动子选择作为需要表达长且复杂的mRNA的基因治疗应用的重要考虑因素。
Chimeric antigen receptor (CAR) T cell therapy is an effective treatment for B cell malignancies, with emerging potential for the treatment of other hematologic cancers and solid tumors. The strength of the promoter within the CAR cassette will alter CAR-polypeptide levels on the cell surface of the T cell-impacting on the kinetics of activation, survival and memory cell formation in T cells. In addition to the CAR, promoters can be used to drive other genes of interest to enhance CAR T cell function. Expressing multiple genes from a single RNA transcript can be effectively achieved by linking the genes via a ribosomal skip site. However, promoters may differ in their ability to transcribe longer RNAs, or could interfere with lentiviral production, or transduction frequencies. In this study we compared the ability of the strong well-characterized promoters CMV, EF-1, hPGK and RPBSA to drive functional expression of a single RNA encoding three products: GFP, CAR, plus an additional cell-survival gene, Mcl-1. Although the four promoters produced similarly high lentiviral titres, EF-1 gave the best transduction efficacy of primary T cells. Major differences were found in the ability of the promoters to drive expression of long RNA encoding GFP, CAR and Mcl-1, highlighting promoter choice as an important consideration for gene therapy applications requiring the expression of long and complex mRNA.