Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs

被引：74

作者：

Aagaard L.A. ^{[1
]}

Zhang J. ^{[1
,2
]}

von Eije K.J. ^{[1
,3
]}

Li H. ^{[1
]}

Sætrom P. ^{[1
,4
]}

Amarzguioui M. ^{[1
,5
]}

Rossi J.J. ^{[1
,2
]}

机构：

[1] Division of Molecular Biology, Beckman Research Institute of City of Hope, Duarte, CA

[2] City of Hope Graduate School of Biological Sciences, City of Hope National Medical Center, Duarte, CA

[3] Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam

[4] Department of Computer and Information Science, Norwegian University of Science and Technology, Trondheim

[5] Biotechnology Centre of Oslo, University of Oslo, Gaustadallé, Oslo

来源：

Gene Therapy | 2008年 / 15卷 / 23期

基金：

美国国家卫生研究院;

关键词：

RNAi; HIV-1; intron; MCM7; microRNAs; siRNAs; polycistron; TAR; nucleolar;

D O I：

10.1038/gt.2008.147

中图分类号：

学科分类号：

摘要：

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.

引用

页码：1536 / 1549

页数：13

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