Brown Brian D, Gentner Bernhard, Cantore Alessio, Colleoni Silvia, Amendola Mario, Zingale Anna, Baccarini Alessia, Lazzari Giovanna, Galli Cesare, Naldini Luigi
San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, via Olgettina, 58, 20132 Milan, Italy.
Nat Biotechnol. 2007 Dec;25(12):1457-67. doi: 10.1038/nbt1372. Epub 2007 Nov 16.
We have shown previously that transgene expression can be suppressed in hematopoietic cells using vectors that are responsive to microRNA (miRNA) regulation. Here we investigate the potential of this approach for more sophisticated control of transgene expression. Analysis of the relationship between miRNA expression levels and target mRNA suppression suggested that suppression depends on a threshold miRNA concentration. Using this information, we generated vectors that rapidly adjust transgene expression in response to changes in miRNA expression. These vectors sharply segregated transgene expression between closely related states of therapeutically relevant cells, including dendritic cells, hematopoietic and embryonic stem cells, and their progeny, allowing positive/negative selection according to the cells' differentiation state. Moreover, two miRNA target sites were combined to restrict transgene expression to a specific cell type in the liver. Notably, the vectors did not detectably perturb endogenous miRNA expression or regulation of natural targets. The properties of miRNA-regulated vectors should allow for safer and more effective therapeutic applications.
我们之前已经表明，使用对微小RNA（miRNA）调控有响应的载体，可以在造血细胞中抑制转基因表达。在此，我们研究这种方法对转基因表达进行更精细控制的潜力。对miRNA表达水平与靶mRNA抑制之间关系的分析表明，抑制取决于miRNA的阈值浓度。利用这一信息，我们构建了能够响应miRNA表达变化而快速调节转基因表达的载体。这些载体在包括树突状细胞、造血干细胞和胚胎干细胞及其后代等治疗相关细胞的密切相关状态之间，能够显著区分转基因表达，从而可根据细胞的分化状态进行正/负选择。此外，两个miRNA靶位点相结合，可将转基因表达限制在肝脏中的特定细胞类型。值得注意的是，这些载体未检测到对内源性miRNA表达或天然靶标的调控产生明显干扰。miRNA调控载体的特性应有助于实现更安全、更有效的治疗应用。
