Multiplexing seven miRNA-Based shRNAs to suppress HIV replication

Jang Gi Choi, Preeti Bharaj, Sojan Abraham, Hongming Ma, Guohua Yi, Chunting Ye, Ying Dang, N. Manjunath, Haoquan Wu, Premlata Shankar

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here, we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. We inserted multiple shRNAs in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singly expressed counterpart. We used this system to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc -/- mice (Hu-PBL model) efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 infection.

Original languageEnglish (US)
Pages (from-to)310-320
Number of pages11
JournalMolecular Therapy
Volume23
Issue number2
DOIs
StatePublished - Feb 3 2015

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Pharmacology
  • Drug Discovery

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  • Cite this

    Choi, J. G., Bharaj, P., Abraham, S., Ma, H., Yi, G., Ye, C., Dang, Y., Manjunath, N., Wu, H., & Shankar, P. (2015). Multiplexing seven miRNA-Based shRNAs to suppress HIV replication. Molecular Therapy, 23(2), 310-320. https://doi.org/10.1038/mt.2014.205