1. ¹Division of Molecular Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
2. ²City of Hope Graduate School of Biological Sciences, City of Hope National Medical Center, Duarte, CA, USA
3. ³Department of Medical Microbiology, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands
4. ⁴Department of Computer and Information Science, Norwegian University of Science and Technology, Trondheim, Norway
5. ⁵Biotechnology Centre of Oslo, University of Oslo, Gaustadallé, Oslo, Norway

Correspondence: Dr JJ Rossi, Division of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010, USA. E-mail: jrossi@coh.org

⁶These authors contributed equally to this work.

Received 7 February 2008; Revised 15 August 2008; Accepted 16 August 2008; Published online 18 September 2008.

Abstract

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.