Figure 1.

Expression of the dual-therapeutic anti–HIV-1 genes in T cell lines and in human primary cultures. (a) Schematic representation of LVsh5/C46 lentiviral vector. The CCR5 shRNA (1005)/FG12 vector (previously described)⁴¹ was digested by NdeI/XhoI to excise a fragment containing CCR5 shRNA (sh5) under the human H1 RNA polymerase III promoter. The insert was cloned into the same sites in an FG11F vector encoding membrane-anchored C46⁴³, under the Ubiquitin C promoter (UbC). Other components of the vector include 5′ and 3′ modified HIV-1 long terminal repeats (LTRs), a central polypurine tract (cPPT), and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). (b) PBMCs and Molt4/CCR5 cells were transduced with LVsh5/C46 at MOI of 1 and 2, respectively, in triplicate, and expression of sh5 and C46 was assessed by flow cytometry. CCR5 shRNA (sh5) expression was demonstrated by downregulation of CCR5 expression evidenced by CD195 staining. Cell-surface expression of C46 was detected by staining with 2F5 antibody. (c) The T cell line CEM.NKR.CCR5 was transduced with LVsh5/C46, and the expression of CCR5 and C46 was similarly assessed over 8 weeks in culture by flow cytometry. (d, e) Quantification of integrated LVsh5/C46 DNA and LVsh5/C46-mediated C46 mRNA synthesis in transduced PBMCs. Cells were treated with LVsh5/C46 at MOIs of 1, 5, and 10 (in duplicate), resulting in transduction efficiencies of 35, 62.5, and 74% (respectively) at 4 days postinfection as assessed by flow cytometry (data not shown). Genomic DNA and RNA were isolated at 8 days posttransduction. C46 DNA copy number per cell was determined by quantitative PCR and was normalized to β-globin (d). C46 RNA transcript levels were measured by RT-qPCR using C46 primers and were normalized to β2-microglobulin mRNA as a measure of relative C46 expression (e). PBMC, peripheral blood mononuclear cell; shRNA, short hairpin RNA.