MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy
MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy
复制标题
DOI：
10.1038/gt.2011.148
复制
发表时间：
2012-09-01
期刊：
影响因子：
5.1
通讯作者：
Moritz, T.
中科院分区：
文献类型：
--
作者：
Lachmann, N.;Jagielska, J.;Moritz, T.
内源 microRNA (miRNA) 表达可用于细胞类型特异性转基因表达，因为将 miRNA 靶序列添加到转基因 cDNA 中可以在表达相应 miRNA 的细胞中特异性地下调转基因。在这里，我们研究了 miRNA-150 靶序列特异性抑制淋巴细胞基因表达的潜力，从而防止转基因诱导的淋巴毒性。通过定量逆转录酶 PCR 证实了分化的 B 细胞和 T 细胞中 miRNA-150 的丰富表达。使用单顺反子和双顺反子慢病毒载体研究miRNA-150靶序列对淋巴造血系统转基因表达的影响。体外研究证明可有效下调小鼠 B220(+) B 和 CD3(+) T 细胞中的转基因表达，这一概念在小鼠移植模型中得到了进一步验证。同样，在 B220(+) B 和 CD4(+) 或 CD8(+) T 细胞中观察到转基因活性显着受到抑制，而 CD11b(+) 骨髓细胞、lin(-) 和 lin(-)/Sca1(+) 祖细胞或 lin(-)/Sca1(+)/c-kit(+) 干细胞中的表达几乎不受影响。没有发现 miRNA-150 靶向转导的淋巴造血细胞的毒性。因此，我们的结果证明了 miRNA-150 靶向特异性抑制淋巴细胞转基因表达的适用性，并进一步支持基因治疗方法中 miRNA 靶向细胞类型特异性转基因表达的概念。
Endogenous microRNA (miRNA) expression can be exploited for cell type-specific transgene expression as the addition of miRNA target sequences to transgenic cDNA allows for transgene downregulation specifically in cells expressing the respective miRNAs. Here, we have investigated the potential of miRNA-150 target sequences to specifically suppress gene expression in lymphocytes and thereby prevent transgene-induced lymphotoxicity. Abundance of miRNA-150 expression specifically in differentiated B and T cells was confirmed by quantitative reverse transcriptase PCR. Mono-and bicistronic lentiviral vectors were used to investigate the effect of miRNA-150 target sequences on transgene expression in the lymphohematopoietic system. After in vitro studies demonstrated effective downregulation of transgene expression in murine B220(+) B and CD3(+) T cells, the concept was further verified in a murine transplant model. Again, marked suppression of transgene activity was observed in B220(+) B and CD4(+) or CD8(+) T cells whereas expression in CD11b(+) myeloid cells, lin(-) and lin(-)/Sca1(+) progenitors, or lin(-)/Sca1(+)/c-kit(+) stem cells remained almost unaffected. No toxicity of miRNA-150 targeting in transduced lymphohematopoietic cells was noted. Thus, our results demonstrate the suitability of miRNA-150 targeting to specifically suppress transgene expression in lymphocytes and further support the concept of miRNA targeting for cell type-specific transgene expression in gene therapy approaches.