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

Gene Therapy volume 15, pages 1536–1549 (2008)Cite this article

An Erratum to this article was published on 18 November 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.

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Acknowledgements

This work was funded by a fellowship to LAA from the Alfred Benzon Foundation, a post-doctoral fellowship to MA from the Norwegian Research Council and NIH AI42552. AI29329 and HL07470 grants to JJR. KJE is supported by ZonMW through a VICI grant. We thank A Ehsani for his suggestions about the positions for inserting the Tar decoy and members of the Rossi lab for their support.

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  1. J Zhang and K J von Eije: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Molecular Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA

    L A Aagaard, J Zhang, K J von Eije, H Li, P Sætrom, M Amarzguioui & J J Rossi

  2. City of Hope Graduate School of Biological Sciences, City of Hope National Medical Center, Duarte, CA, USA

    J Zhang & J J Rossi

  3. Department of Medical Microbiology, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands

    K J von Eije

  4. Department of Computer and Information Science, Norwegian University of Science and Technology, Trondheim, Norway

    P Sætrom

  5. Biotechnology Centre of Oslo, University of Oslo, Gaustadallé, Oslo, Norway

    M Amarzguioui

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  1. L A Aagaard
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Correspondence to J J Rossi.

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Aagaard, L., Zhang, J., von Eije, K. et al. Engineering and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs. Gene Ther 15, 1536–1549 (2008). https://doi.org/10.1038/gt.2008.147

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