Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice

Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4+ T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4+ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4+ T-cells ex vivo. Furthermore, levels of gene-marked CD4+ T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease.This work was supported by grants from the California Institute for Regenerative Medicine (CIRM grant DR1-01431 to ISYC), the National Institutes of Health (1RO1HL086409 and 3RO1HL086409-03S1 to DSA and 5T32AI060567), and the University of California Los Angeles AIDS Institute/Center for AIDS Research (5P30AI028697). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4(+) T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+) T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+) T-cells ex vivo. Furthermore, levels of gene-marked CD4(+) T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease

Protection from R5- and X4- tropic HIV-1-mediated CD4⁺ T-cell loss in hu-BLT mice by Dual sh1005/sh516.

<p>hu-BLT mice transplanted with Dual sh1005/sh516-transduced HSPC were challenged intravenously with either HIV-1_NFNSX or HIV-1_NL4-3 ∼12 weeks post-transplantation. Levels of marked CD4⁺ T-cells, shown as percentages within total CD3⁺ cells in peripheral blood (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053492#pone.0053492.s003" target="_blank">Table S2</a>), were normalized to that of the time point preceding decline of percentages of control-vector transduced cells to determine relative fold change over time. Values above and below 1.0 on the y-axis depict increases or decreases, respectively, in CD4⁺ T-cell levels. Closed circles: EGFP-marked. Open circles: mCherry-marked. Error bars: mean + SEM in n = 5 and n = 4 for HIV-1_NFNSX- and HIV-1_NL4-3- infected mice, respectively. Statistical significance calculated by two-way ANOVA/Bonferroni post-test. *  =  p<0.05, **  =  p<0.01, ***  =  p<0.001.</p

Vector stability and the effects of sh1005/sh516 co-expression on cell viability and HSPC differentiation potential.

<p>IL-2/PHA-stimulated PBMCs (4×10⁵) were transduced with lentiviral vectors at MOI 0.6–1.0. <b>A.</b> Representative data showing EGFP expression in vector-transduced PBMCs over time. <b>B.</b> Vector-transduced cells were subjected to CytoTox-Glo^TM Cytotoxicity Assay (Promega) four and six days post-transduction. Relative cytotoxicities were calculated by dividing the dead cell count by the total cell count and normalizing to that of mock-transduced cells. 1 μM Staurosporine (STS) and U6 promoter-driven sh1005 expression (U6-sh1005) served as positive controls. Error bars: mean + SD. <b>C.</b> OAS1 mRNA expression relative to β-actin was assessed by qRT-PCR analysis of total RNA isolated from vector-transduced PBMCs. PBMCs harvested two days post-electroporation with 500 pg/μL poly(I:C) served as positive control. Values were normalized to those of mock-transduced cells. Error bars: mean + SD. <b>D.</b> Cytokine-pre-stimulated mPB-CD34⁺ cells were transduced with lentiviral vectors at MOI 10. HSPC differentiation potential was assessed by counting colony forming units produced from mock- and vector- transduced mPB-CD34⁺ cells plated on semi-solid methylcellulose plates one day after transduction. CFU-GEMM: granulocyte/erythrocyte/macrophage/megakaryocyte colony forming units. C/BFU-E: erythroid colony/burst forming units. CFU-GM: granulocyte/monocyte colony forming units. Error bars: range of values between duplicate samples. Representative data of three independent experiments.</p

Down-regulation of CCR5 and inhibition of HIV-1 replication in MOLT4-CCR5 cells and PBMCs by Dual sh1005/sh516.

<p><b>A.</b> MOLT4/CCR5 (1×10⁵) cells were transduced with lentiviral vectors at MOI 0.5. EGFP and CCR5 expression was assessed three days post-transduction. CCR5 MFIs shown below plots as “MFI: MFI_EGFP−/MFI_EGFP+.” Representative data of three independent experiments. <b>B.</b> Sorted EGFP⁺ cells (2×10⁵) were infected with either HIV-1_NL4-3 at MOI 0.5 and HIV-1_{NFNSX SL9} at MOI 5.0. Levels of p24 antigen in culture supernatants were measured by ELISA four and seven days post-infection. Errors bars: mean + SD. <b>C.</b> IL-2/PHA stimulated PBMCs (4×10⁵) were transduced with lentiviral vectors at MOI 0.6–1.0. EGFP and CCR5 expression was measured at seven days post-transduction. CCR5 MFIs shown as in <b>A</b>. Representative data showing CCR5 expression in vector-transduced PBMCs. <b>D.</b> Sorted EGFP⁺ cells (5×10⁴) were infected with either HIV-1_{NFNSX SL9} at MOI 5.0, HIV-1_JR-CSF at MOI 1.0, or HIV-1_NL4-3 at MOI 0.1. p24 production was measured as in <b>B</b>.</p

Down-regulation of CCR5 in systemic lymphoid tissues and inhibition of HIV-1 replication <i>ex vivo</i> by Dual sh1005/sh516.

<p>Tissues were isolated from a Dual sh1005/sh516-transduced HSPC-transplanted mouse at eleven weeks post-transplantation. <b>A.</b> Representative data showing CCR5 expression in EGFP- and mCherry- marked human CD4⁺CD3⁺CD45⁺CD19⁻ T-cells within a gated lymphocyte population. <b>B.</b> Splenocytes were depleted of human CD8⁺ cells and murine CD45⁺ cells and then stimulated with PHA/IL-2 for two days. Five days later, EGFP⁺ and mCherry⁺ cells were then sorted by FACS at >97% purities. Sorted cells (5×10⁴) were then infected with either HIV-1_NL4-3 at MOI 0.5, HIV-1_{NFNSX SL9} at MOI 5.0, or HIV-1_JR-CSF at MOI 1.0 for four hours. Cells were then washed five times before culturing. HIV-1 replication was monitored by p24 ELISA analysis of culture supernatants at four and seven days post-infection. Error bars: mean + SD. Representative data of three independent experiments.</p

Reconstitution of Dual sh1005/sh516-transduced HSPCs in humanized BLT mice.

<p><b>A.</b> Schematic of generating vector-transduced HSPC-transplanted hu-BLT mouse. NSG mouse is treated with Busulfan 24 hours pre-transplantation. CD34⁺ and CD34⁻ cells are isolated from human fetal liver (FL). CD34⁺ cells are transduced with either therapeutic (EGFP-marked) or control (mCherry-marked) vectors. Therapeutic vector- and control vector- transduced CD34⁺ cells are mixed at a 50 50 ratio. The cell mixture is then 1) combined with CD34⁻ cells, solidified with Matrigel, and implanted under the kidney capsule with a human fetal thymus segment (FT) and also 2) intravenously injected. <b>B.</b> EGFP and mCherry reporter gene expression was monitored in human CD45⁺, CD3⁺CD45⁺, and CD19⁺CD45⁺ cells within a gated lymphocyte population in peripheral blood at twelve weeks post-transplantation. CD4/CD8 ratios were analyzed in mCherry- and EGFP- marked CD3⁺CD45⁺ cells. Data were generated from n = 13 mice for both Mono sh1005- and Dual sh1005/sh516- HSPC-transplanted animals from an aggregate of three donors. Error bars: mean + standard error of mean (SEM) in n = 13 per group.</p

Concurrent down-regulation of CCR5 and HIV-1 via sh1005/sh516 co-expression.

<p><b>A.</b> Schematics of lentiviral vectors. Asterisks depict sites of vector LTR mutagenesis in sh516-expressing vectors. <b>B.</b> Down-regulation of CCR5 and HIV-1 reporter gene expression. CEM-NKR.CCR5 cells (1×10⁵) were transduced with mCherry-marked lentiviral vectors at MOI 0.5 then seven days later infected with NL4-3 EGFP R-E-at MOI 0.8. EGFP (top panel) and CCR5 (bottom panel) expression was analyzed at three days post-infection. Fold inhibition of gene expression, shown in the top right corner of each histogram, was calculated as MFI in untransduced (mCherry⁻) cells divided by MFI in transduced (mCherry⁺) cells. LTRm: Vector LTR-modified.</p

Selection of a single potent anti-HIV-1 shRNA.

<p><b>A.</b> Schematic of candidate shRNA screening. McIntyre <i>et al</i>. ranked HIV-1_NL4-3-derived target sequences by percent conservation of published and proprietary HIV-1 sequence databases then ranked the top ninety-six 19-mer target sequences based on suppression, specificity, and percent conservation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053492#pone.0053492-McIntyre1" target="_blank">[40]</a>. The resultant top eight shRNAs were selected for validation in our lentiviral vector based setting. <b>B.</b> Candidate shRNA targeted genes and sequences. <b>C.</b> HEK-293T cells (7×10⁴) were simultaneously transduced with lentiviral vectors bearing shRNAs at MOI 0.3 and infected with NL4-3.Luc.R-E- at MOI 0.3. Two days post-challenge, cells were harvested and luciferase activities were measured. Luciferase units were normalized by transduction efficiency and protein concentration and made relative to that of the No shRNA control. Relative luciferase units at or below the dashed line (shLuc positive control) were considered sufficient knockdown of luciferase expression. Error bars: mean + standard deviation (SD). <b>D.</b> Representative EGFP/HSA expression data of virus-challenged vector-transduced HEK-293T cells. HEK-293T cells (7×10⁴) were transduced with lentiviral vectors bearing anti-HIV-1 shRNAs at MOI 0.3 and then challenged two days post-transduction with NL4-3.HSA.R+.E- at MOI 0.5. Two days post-challenge, cells were harvested and expression of HSA at the cell surface and EGFP was analyzed by flow cytometry. HSA mean fluorescent intensity (MFI) information is shown below plots as “MFI: MFI_EGFP-HSA+/MFI_EGFP+HSA+ (MFI_EGFP-HSA+ ÷ MFI_EGFP+HSA+ normalized to that of No shRNA cells)”.</p