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

Clinical significance of methicillin-resistant coagulase-negative staphylococci obtained from sterile specimens

Distinguishing true coagulase-negative staphylococci bacteremia from contamination remains a challenge. We conducted a retrospective analysis of 183 patients with methicillin-resistant coagulase-negative staphylococci (MR-CoNS)-positive and methicillin-resistant Staphylococcus aureus-positive cultures obtained from sterile sites such as blood, synovial fluid, ascitic fluid, and cerebrospinal fluid. Of the 209 MR-CoNS isolates, 83 (39.7%) were considered infection associated, and 126 (60.3%) were considered contamination. MR-CoNS isolates cultured from synovial fluid were more likely to be infection associated (. P= 0.009). The median interval from insertion of a central venous catheter to onset of infection tended to be longer in MR-CoNS infection cases than in methicillin-resistant S. aureus infection cases (41 days versus 14 days, P= 0.055). In conclusion, our results suggest that the proportion of cases of true MR-CoNS infection may be higher than previously reporte

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

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

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

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