Park Tristen S, Abate-Daga Daniel, Zhang Ling, Zheng Zhili, Morgan Richard A
Surgery Branch, Center for Cancer Research, National Cancer Institute , Bethesda, Maryland.
Nucleic Acid Ther. 2014 Oct;24(5):356-63. doi: 10.1089/nat.2014.0486. Epub 2014 Jul 14.
To generate γ-retroviral vectors for stable conjoint expression of artificial microRNAs (amiR) and therapeutic genes in primary human lymphocytes, and to identify the design parameters that are key for successful vector generation. Gamma-retroviral vectors were designed to co-express both amiRs and a linked reporter gene, truncated CD34 (tCD34). Artificial miRs based on microRNAs miR-16, miR-142, miR-146b, miR-150, miR155, and miR-223 were inserted into sites within the intron of the vector and tested for tCD34 expression by flow cytometry (FACS). Different constructs were assembled with amiRs targeted to knockdown expression of suppressor of cytokine signaling 1 (SOCS1) or programmed cell death 1 (PDCD1, PD-1). Three of the six amiRs maintained tCD34 expression. Expansion of primary human T cells transduced with these amiR vectors, as well as transgene expression, were equivalent to control engineered T cells over a 40-day period. Knockdown of SOCS1 RNA and PD-1 expression by FACS was shown to vary between constructs, dependent on either the specific short interfering RNA sequence used in the amiR, or the microRNA backbone and location in the vector intron. Gamma-retroviral vectors that both efficiently knockdown endogenous gene expression and maintain linked transgene production can be produced, but empirical vector evaluations were best suited for optimal construct analysis.
为了生成γ-逆转录病毒载体，以便在原代人淋巴细胞中稳定联合表达人工微小RNA（amiR）和治疗性基因，并确定成功生成载体的关键设计参数。设计γ-逆转录病毒载体以共表达amiR和一个连接的报告基因，即截短的CD34（tCD34）。将基于微小RNA miR-16、miR-142、miR-146b、miR-150、miR155和miR-223的人工miR插入载体内含子内的位点，并通过流式细胞术（FACS）检测tCD34的表达。用靶向敲低细胞因子信号传导抑制因子1（SOCS1）或程序性细胞死亡1（PDCD1，PD-1）表达的amiR组装不同的构建体。六个amiR中的三个维持了tCD34的表达。用这些amiR载体转导的原代人T细胞的扩增以及转基因表达，在40天的时间内与对照工程化T细胞相当。通过FACS显示，SOCS1 RNA和PD-1表达的敲低在不同构建体之间有所不同，这取决于amiR中使用的特定短干扰RNA序列，或者微小RNA骨架和在载体内含子中的位置。可以生产出既能有效敲低内源性基因表达又能维持连接的转基因产生的γ-逆转录病毒载体，但经验性的载体评估最适合于优化构建体分析。
