Cell and Gene Therapy Laboratory, Center for Basic Research II, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
Gene Ther. 2010 Mar;17(3):380-8. doi: 10.1038/gt.2009.147. Epub 2009 Nov 12.
Several gene therapy applications require the transfer and simultaneous expression of multiple genes in the same cell. In this study, we analyzed the potential for coordinated expression of an endogenous bidirectional promoter located on chromosome X, which controls the expression of the heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2) and alpha-galactosidase (GLA) genes. The promoter was cloned in both transcriptional orientations in a foamy virus (FV) vector backbone, whereas the enhanced green fluorescent protein (EGFP) and low-affinity nerve growth factor receptor (DeltaLNGFR) reporter genes were cloned in the 5'-3' and 3'-5' transcriptional orientations, respectively. In all the cell lines tested, both vectors showed high levels of transgene coexpression that reached 76% of total positive cells (range from 76 to 18%). Comparison of EGFP and DeltaNGFR levels revealed that the side of the promoter that drives the expression of the HNRNPH2 gene in the genome was stronger and in accordance to its in situ activity. When tested with CD34(+) cells, transgene coexpression reached 35.3% of all positive cells in progenitor assays and 16.8% of all positive cells after transplantation in NOD/severe combined immunodeficient mice. In summary, we show that the endogenous promoter used in this study holds bidirectional activity in the context of FV vectors and can be used in gene therapy applications requiring synchronized expression of two genes.
几种基因治疗应用需要在同一细胞中转导和同时表达多个基因。在这项研究中，我们分析了位于 X 染色体上的内源性双向启动子控制异质核核糖核蛋白 H2（HNRNPH2）和α-半乳糖苷酶（GLA）基因表达的协同表达潜力。该启动子在泡沫病毒（FV）载体骨架中以两种转录方向被克隆，而增强型绿色荧光蛋白（EGFP）和低亲和力神经生长因子受体（DeltaLNGFR）报告基因分别以 5'-3'和 3'-5'转录方向被克隆。在所有测试的细胞系中，两种载体均显示出高水平的转基因共表达，达到总阳性细胞的 76%（范围从 76%到 18%）。比较 EGFP 和 DeltaNGFR 水平表明，驱动基因组中 HNRNPH2 基因表达的启动子一侧更强，并与其原位活性一致。在对 CD34(+)细胞进行测试时，在祖细胞测定中转基因共表达达到所有阳性细胞的 35.3%，在 NOD/严重联合免疫缺陷小鼠移植后达到所有阳性细胞的 16.8%。总之，我们表明，本研究中使用的内源性启动子在 FV 载体的背景下具有双向活性，可用于需要两个基因同步表达的基因治疗应用。
