title: Multiplexing seven miRNA-Based shRNAs to suppress HIV replication.
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.
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: Jang-Gi
authors.initials: JG
authors.lastname: Choi
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: Preeti
authors.initials: P
authors.lastname: Bharaj
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: Sojan
authors.initials: S
authors.lastname: Abraham
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: Hongming
authors.initials: H
authors.lastname: Ma
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: Guohua
authors.initials: G
authors.lastname: Yi
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: Chunting
authors.initials: C
authors.lastname: Ye
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Dang
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: N
authors.initials: N
authors.lastname: Manjunath
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: Haoquan
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Biomedical Sciences, Center of Excellence in Infectious Disease Research, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA.
authors.forename: Premlata
authors.initials: P
authors.lastname: Shankar
mesh_terms: D000818:Animals; D018791:CD4 Lymphocyte Count; D002460:Cell Line; D004195:Disease Models, Animal; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D015658:HIV Infections / Q000276:immunology / Q000821:virology*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000821:virology; D051379:Mice; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D019713:Receptors, CCR5 / Q000235:genetics; D016176:T-Lymphocyte Subsets / Q000378:metabolism / Q000821:virology; D014161:Transduction, Genetic; D014779:Virus Replication / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; D019713:Receptors, CCR5