PMC

LVsh5/C46 genomic DNA integration and LVsh5/C46-mediated shRNA expression in hematopoietic cells. CD4⁺ T lymphocytes and CD34⁺ HSPC were transduced with LVsh5/C46 at MOIs of 1 and 5, respectively. (a) Genomic DNA was isolated from the transduced cells, and C46 DNA copy number normalized to β-globin was determined by quantitative PCR. (b) RNA was extracted from the transduced cells, and CCR5 shRNA transcript level was determined by RT-qPCR using sh5 primers and was normalized to the expression of RNU38B microRNA. HSPC, hematopoietic stem/progenitor cell; MOI, multiplicity of infection; RT-qPCR, real-time quantitative PCR; shRNA, short hairpin RNA.

LVsh5/C46-modified CD34⁺ HSPC maintain their multilineage hematopoietic differentiation potential. CD34⁺ HSPC from three healthy donors were transduced with the indicated lentiviral vectors, and 2 weeks posttransduction, methylcellulose colony-forming unit (CFU) assays were performed. Colonies were enumerated for CFU erythrocyte (CFU-E); CFU granulocyte/monocyte (CFU-GM), and CFU granulocyte/erythrocyte/monocyte/megakaryocyte (CFU-GEMM) and compared to untransduced HSPC. (a) CD34⁺ cells were transduced with LVsh5/C46 vector at MOI of 10, and the relative colony counts were plotted against untransduced cells. (b–d) CD34⁺ cells were transduced with lentiviral vectors expressing sh5 alone, sh5 fused to enhanced green fluorescent protein reporter gene, C46 alone, or LVsh5/C46 (sh5/C46), and CFU assays were performed. Colonies from the lineages (b) CFU-GM, (c) CFU-E, and (e) CFU-GEMM were scored, and the relative colony counts were plotted against untransduced cells. HSPC, hematopoietic stem/progenitor cell; MOI, multiplicity of infection.

LVsh5/C46 inhibited R5-, X4-, and dual-tropic HIV-1 infection in Molt4/CCR5 cells. (a) Cells were transduced with LVsh5/C46 at MOI of 5, with transduction efficiency of ~80–100%, in three individual experiments. Cells were then challenged with the HIV-1 viral strains BaL (R5-tropic) and NL4-3 (X4-tropic), and viral replication was measured by p24 antigen. (b) LVsh5/C46-transduced Molt4/CCR5 cells were infected with increasing doses of SF2 virus (R5/X4) as indicated, and viral replication was determined by p24 protein levels. Results are from one representative experiment. (c) Molt4/CCR5 were transduced with the indicated lentiviral vectors (sh5, C46, or sh5/C46), followed by infection with BaL strain (R5-tropic) at MOI of 0.2, and p24 protein levels were assessed 7 and 10 days later. Results are from one representative experiment. (d) Cells were transduced with sh5, C46, or LVsh5/C46 (sh5/C46) lentiviral vectors at MOIs of 1 and 5 and were challenged with NL4-3 (X4-tropic) or BaL (R5-tropic) HIV-1 viruses. HIV-1 infection was measured by p24 protein levels on day 13 postinfection and was compared to untransduced cells. *P = 0.006 compared to untransduced cells. **P = 0.011 compared to untransduced cells. MOI, multiplicity of infection.

Introducing LVsh5/C46 vector into target cells. (a) Human CD4⁺ T lymphocytes were isolated from PBMCs by CD8 depletion followed by selection and expansion using CD3/CD28 beads. Cells were stained with CD4, CD3, and CD8 antibodies and analyzed by flow cytometry. Purified CD4⁺ T-lymphocyte cells are shown as CD4⁺/CD3⁺/CD8⁻ fraction in the lower right panel. (b) CD34⁺ HSPC were isolated from G-CSF mobilized peripheral blood, using CD34⁺ microbeads. Positive fraction was stained with CD34 antibody and analyzed by flow cytometry. (c, d) Purified cells were treated with LVsh5/C46 at the increasing doses (MOIs as indicated), in triplicate, and the percentage of (c) C46-expressing CD4⁺ cells and (d) CD34⁺ cells was determined by flow cytometry. (e) Purified CD4⁺ T lymphocytes and CD34⁺ HSPC cells were treated with GMP-grade LVsh5/C46 at MOI of 1 and 5, respectively, and LVsh5/C46 transduction was analyzed by flow cytometry, measuring the percentage of cells expressing cell-surface C46. G-CSF, granulocyte colony-stimulating factor; GMP, good manufacturing practice; HSPC, hematopoietic stem/progenitor cell; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell.

LVsh5/C46 vector does not induce deleterious effect on cells. PBMCs from three to five healthy donors were transduced with LVsh5/C46 (sh5/C46) at MOIs of 1 or 10 or and were compared to untransduced cells and to cells transduced with a control lentivirus vector encoding eGFP (enhanced green fluorescent protein) at MOI of 1. In different experiments, transduction efficiency was determined by flow cytometry to be 15–44% at an MOI of 1 and 36–64% at an MOI of 10. Cells were assessed for apoptosis, proliferation, and inflammation; results were normalized to untransduced cells; and Student’s t-test was performed. (a) Apoptosis was not induced in LVsh5/C46-transduced PBMCs. Caspase 3/7 activities were assessed 7 days posttransduction by luminescence assay (n = 3). Staurosporine was used as a positive control. (b) LVsh5/C46 did not alter viability or proliferative capacity of modified PBMCs. Cells were cultured for 7 days, and proliferation rate was assessed (n = 5). IL-2 was used as a positive control. (c–e) Transduced PBMCs were cultured for 8 days, and the level of the cytokines (c) interferon-γ (n = 3), (d) interleukin-6 (n = 4), and (e) TNF-α (n = 4) was determined. Cells treated with PHA were used as positive controls. *P < 0.05 compared to untransduced cells. **P < 0.01 compared to untransduced cells. MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor.

LVsh5/C46-modified PBMCs inhibit infection from laboratory and clinical strains of HIV-1. (a, b) PBMCs were transduced with the LVsh5/C46 (sh5/C46), C46 alone, sh5/GFP, or GFP vectors, at MOIs of 0.5, and 4 days later, transduction efficiency was determined by flow cytometry, (a) measuring CCR5 and C46 expression, or CCR5 and enhanced green fluorescent protein. (b) Cells were then infected with R5-tropic strain NFNSX or X4-tropic NL4-3, at MOIs of 0.02, and p24 protein levels was measured to assess HIV-1 replication. (c) PBMCs were transduced with LVsh5/C46 (sh5/C46) at MOI of five in four independent experiments, and 48 hours later were challenged with the HIV-1 laboratory strains BaL (R5-), SF162 (R5-), or Bru (X4-tropic) at MOIs of 0.14, 0.16, and 0.17, respectively. Four days postinfection, p24 levels were assessed and plotted relative to untransduced cells (this was done due to high variability between experiments and donor variability). Average p24 readings were: untransduced-BaL: 134,935 ng/ml (SE: 80,245)/transduced-BaL: 24,159 ng/ml (SE: 5,665). Untransduced-SF162: 72,153 ng/ml (SE: 30,041)/transduced-SF162: 20,121 ng/ml (SE: 6,181). Untransduced-Bru: 106,066 ng/ml (SE: 39,585)/transduced-Bru: 19,371 ng/ml (SE: 5,033). (d) PBMCs were transduced with LVsh5/C46 (sh5/C46) at MOI of 5, and 48 hours later were challenged with the HIV-1 clinical isolates 92US723 (R5/X4-tropic, Clade B) or 92UG021(X4-tropic, Clade D) in three independent experiments. Viral infection levels were determined relative to untransduced cells. Average p24 levels were: untransduced-92US723: 94,986 ng/ml (SE: 60,533)/transduced-92US723: 20,148 ng/ml (SE: 15,764). Untransduced-92UG021: 45,804 ng/ml (SE: 16,988)/transduced-92UG021: 15,536 ng/ml (SE: 9,401). GFP, green fluorescent protein; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell.

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) 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.