Aagaard L A, Zhang J, von Eije K J, Li H, Saetrom P, Amarzguioui M, Rossi J J
Division of Molecular Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA.
Gene Ther. 2008 Dec;15(23):1536-49. doi: 10.1038/gt.2008.147. Epub 2008 Sep 18.
Many microRNAs (miRNAs) are encoded within the introns of RNA Pol II transcripts, often as polycistronic precursors. Here, we demonstrate the optimization of an intron encoding three endogenous miRNAs for the ectopic expression of heterologous anti-HIV-1 small interfering RNAs (siRNAs) processed from a single RNA polymerase II primary miRNA. Our expression system, designated as MCM7, is engineered from the intron-embedded, tri-cistronic miR-106b cluster that endogenously expresses miR-106b, miR-93 and miR-25. Manipulation of the miR-106b cluster demonstrated a strict requirement for maintenance of the native flanking primary miRNA (pri-miRNA) sequences and key structural features of the native miRNAs for efficient siRNA processing. As a model for testing the efficacy of this approach, we have replaced the three endogenous miRNAs with siRNAs targeting the tat and rev transcripts of human immunodeficiency virus type 1 (HIV-1). This study has enabled us to establish guidelines for optimal processing of the engineered miRNA mimics into functional siRNAs. In addition, we demonstrate that the incorporation of a small nucleolar RNA TAR chimeric decoy (snoRNA) inserted within the MCM7 intron resulted in a substantial enhancement of HIV suppression in long-term acute infectious HIV-1 challenges.
许多微小RNA（miRNA）由RNA聚合酶II转录本的内含子编码，通常作为多顺反子前体。在这里，我们展示了一个编码三种内源性miRNA的内含子的优化，用于从单个RNA聚合酶II初级miRNA加工而来的异源抗HIV-1小干扰RNA（siRNA）的异位表达。我们的表达系统称为MCM7，是由内含子嵌入的三顺反子miR-106b簇改造而来，该簇内源性表达miR-106b、miR-93和miR-25。对miR-106b簇的操作表明，对于高效的siRNA加工，严格需要维持天然侧翼初级miRNA（pri-miRNA）序列和天然miRNA的关键结构特征。作为测试该方法有效性的模型，我们用靶向人类免疫缺陷病毒1型（HIV-1）tat和rev转录本的siRNA取代了三种内源性miRNA。这项研究使我们能够建立将工程化miRNA模拟物加工成功能性siRNA的最佳指南。此外，我们证明，在MCM7内含子中插入一个小核仁RNA TAR嵌合诱饵（snoRNA），在长期急性感染性HIV-1挑战中，可显著增强对HIV的抑制作用。
