Foamy Virus Vectors Expressing Anti-HIV Transgenes Efficiently Block HIV-1 Replication
Authors: Jason A Taylor, Lucia Vojtech, Ingrid Bahner, Donald B Kohn, Dorothee Von Laer, David W Russell, Robert E Richard
Journal: Molecular Therapy (2007)

Gene therapy has the potential to control human immunodeficiency virus (HIV) in patients who do not respond to traditional antiviral therapy. In this study, we tested foamy virus (FV) vectors expressing three anti-HIV transgenes, both individually and in a combination vector. The transgenes tested in this study are RevM10, a dominant negative version of the viral rev protein, Sh1, a short hairpin RNA directed against a conserved overlapping sequence of tat and rev, and membrane-associated C46 (maC46), a membrane-attached peptide that blocks HIV cell entry. FV vectors efficiently transduce hematopoietic stem cells and, unlike lentivirus (LV) vectors, do not share viral proteins with HIV. The titers of the FV vectors described in this study were not affected by anti-HIV transgenes. On a direct comparison of FV vectors expressing the individual transgenes, entry inhibition using the maC46 transgene was found to be the most effective at blocking HIV replication. A clinically relevant FV vector expressing three anti-HIV transgenes effectively blocked HIV infection in primary macrophages derived from transduced, peripheral blood CD34-selected cells and in a cell line used for propagating HIV, CEM×174. These results suggest that there are potential benefits of using FV vectors in HIV gene therapy. Gene therapy has the potential to control human immunodeficiency virus (HIV) in patients who do not respond to traditional antiviral therapy. In this study, we tested foamy virus (FV) vectors expressing three anti-HIV transgenes, both individually and in a combination vector. The transgenes tested in this study are RevM10, a dominant negative version of the viral rev protein, Sh1, a short hairpin RNA directed against a conserved overlapping sequence of tat and rev, and membrane-associated C46 (maC46), a membrane-attached peptide that blocks HIV cell entry. FV vectors efficiently transduce hematopoietic stem cells and, unlike lentivirus (LV) vectors, do not share viral proteins with HIV. The titers of the FV vectors described in this study were not affected by anti-HIV transgenes. On a direct comparison of FV vectors expressing the individual transgenes, entry inhibition using the maC46 transgene was found to be the most effective at blocking HIV replication. A clinically relevant FV vector expressing three anti-HIV transgenes effectively blocked HIV infection in primary macrophages derived from transduced, peripheral blood CD34-selected cells and in a cell line used for propagating HIV, CEM×174. These results suggest that there are potential benefits of using FV vectors in HIV gene therapy. IntroductionGene therapy has the potential to improve the outcome of treatment in patients with advanced human immunodeficiency virus (HIV) infection, and could supplement or potentially eliminate the need for complicated drug regimens.1Strayer DS Akkina R Bunnell BA Dropulic B Planelles V Pomerantz RJ et al.Current status of gene therapy strategies to treat HIV/AIDS.Mol Ther. 2005; 11: 823-842Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Several anti-HIV gene therapy trials targeting either lymphocytes2Morgan RA Walker R Gene therapy for AIDS using retroviral mediated gene transfer to deliver HIV-1 antisense TAR and transdominant Rev protein genes to syngeneic lymphocytes in HIV-1 infected identical twins.Hum Gene Ther. 1996; 7: 1281-1306Crossref PubMed Scopus (63) Google Scholar,3Ranga U Woffendin C Verma S Xu L June CH Bishop DK et al.Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals.Proc Natl Acad Sci USA. 1998; 95: 1201-1206Crossref PubMed Scopus (121) Google Scholar,4Levine BL Humeau LM Boyer J MacGregor RR Rebello T Lu X et al.Gene transfer in humans using a conditionally replicating lentiviral vector.Proc Natl Acad Sci USA. 2006; 103: 17372-17377Crossref PubMed Scopus (412) Google Scholar,5Lunzen J Glaunsinger T Stahmer I Baehr V Baum C Schilz A et al.Transfer of autologous gene-modified T Cells in HIV-infected patients with advanced immunodeficiency and drug-resistant virus.Mol Ther. 2007; 15: 1024-1033Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar or the hematopoietic stem cell6Kohn DB Bauer G Rice CR Rothschild JC Carbonaro DA Valdez P et al.A clinical trial of retroviral-mediated transfer of a rev-responsive element decoy gene into CD34+ cells from the bone marrow of human immunodeficiency virus-1-infected children.Blood. 1999; 94: 368-371PubMed Google Scholar,7Amado RG Mitsuyasu RT Rosenblatt JD Ngok FK Bakker A Cole S et al.Anti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study: myeloid and lymphoid reconstitution in human immunodeficiency virus type-1-infected patients.Hum Gene Ther. 2004; 15: 251-262Crossref PubMed Scopus (120) Google Scholar,8Podsakoff GM Engel BC Carbonaro DA Choi C Smogorzewska EM Bauer G et al.Selective survival of peripheral blood lymphocytes in children with HIV-1 following delivery of an anti-HIV gene to bone marrow CD34+ cells.Mol Ther. 2005; 12: 77-86Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar have been conducted, with only limited success.1Strayer DS Akkina R Bunnell BA Dropulic B Planelles V Pomerantz RJ et al.Current status of gene therapy strategies to treat HIV/AIDS.Mol Ther. 2005; 11: 823-842Abstract Full Text Full Text PDF PubMed Scopus (87) Google ScholarHematopoietic stem cell–directed anti-HIV gene therapy has the potential advantage that a small number of genetically modified stem cells can generate a renewable population of mature CD4+ T lymphocytes, dendritic cells, and monocytes protected from HIV infection. Lentivirus (LV) vectors have been demonstrated to efficiently transduce human hematopoietic stem cells9Miyoshi H Smith KA Mosier DE Verma IM Torbett BE Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors.Science. 1999; 283: 682-686Crossref PubMed Scopus (552) Google Scholar and have been tested in anti-HIV clinical trials.4Levine BL Humeau LM Boyer J MacGregor RR Rebello T Lu X et al.Gene transfer in humans using a conditionally replicating lentiviral vector.Proc Natl Acad Sci USA. 2006; 103: 17372-17377Crossref PubMed Scopus (412) Google Scholar However, LV vectors could potentially be mobilized by wild-type HIV, leading to uncontrolled vector production in vivo and increasing the possibility of recombination events between the vector and wild-type HIV.10Evans JT Garcia JV Lentivirus vector mobilization and spread by human immunodeficiency virus.Hum Gene Ther. 2000; 11: 2331-2339Crossref PubMed Scopus (44) Google Scholar,11Browning MT Schmidt RD Lew KA Rizvi TA Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions.J Virol. 2001; 75: 5129-5140Crossref PubMed Scopus (69) Google Scholar In addition, many anti-HIV gene products can interfere with LV vector production.12Banerjea A Li MJ Bauer G Remling L Lee NS Rossi J et al.Inhibition of HIV-1 by lentiviral vector-transduced siRNAs in T lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell-derived macrophages.Mol Ther. 2003; 8: 62-71Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar,13Mautino MR Morgan RA Inhibition of HIV-1 replication by novel lentiviral vectors expressing transdominant Rev and HIV-1 env antisense.Gene Ther. 2002; 9: 421-431Crossref PubMed Scopus (19) Google Scholar,14Li MJ Bauer G Michienzi A Yee JK Lee NS Kim J et al.Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs.Mol Ther. 2003; 8: 196-206Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar In particular, anti-REV transgenes such as the trans-dominant rev mutation, RevM10, and short hairpin RNAs targeting the tat/rev message, decrease LV vector titers. For these reasons, we are developing foamy virus (FV) vectors that express anti-HIV transgenes. FVs are found in a number of non-human primate species and have never been associated with disease.15Meiering CD Linial ML Historical perspective of foamy virus epidemiology and infection.Clin Microbiol Rev. 2001; 14: 165-176Crossref PubMed Scopus (144) Google Scholar,16Wolfe ND Switzer WM Carr JK Bhullar VB Shanmugam V Tamoufe U et al.Naturally acquired simian retrovirus infections in central African hunters.Lancet. 2004; 363: 932-937Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar,17Switzer WM Bhullar V Shanmugam V Cong ME Parekh B Lerche NW et al.Frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates.J Virol. 2004; 78: 2780-2789Crossref PubMed Scopus (182) Google Scholar FV vectors have transduction rates of hematopoietic stem cells comparable to those of LV vectors and have been tested in mice, dogs, and human CD34+ cells using the xenogeneic transplanted NOD-SCID mouse.18Vassilopoulos G Trobridge G Josephson NC Russell DW Gene transfer into murine hematopoietic stem cells with helper-free foamy virus vectors.Blood. 2001; 98: 604-609Crossref PubMed Scopus (71) Google Scholar,19Kiem HP Allen J Trobridge G Olson E Keyser K Peterson L et al.Foamy-virus-mediated gene transfer to canine repopulating cells.Blood. 2007; 109: 65-70Crossref PubMed Scopus (45) Google Scholar,20Trobridge GD Russell DW Helper-free foamy virus vectors.Hum Gene Ther. 1998; 9: 2517-2525Crossref PubMed Google Scholar,21Leurs C Jansen M Pollok KE Heinkelein M Schmidt M Wissler M et al.Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells.Hum Gene Ther. 2003; 14: 509-519Crossref PubMed Scopus (106) Google Scholar,22Josephson NC Vassilopoulos G Trobridge GD Priestley GV Wood BL Papayannopoulou T et al.Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors.Proc Natl Acad Sci USA. 2002; 99: 8295-8300Crossref PubMed Scopus (81) Google Scholar There is little risk of recombination between FV vectors and HIV, and transgenes that inhibit the HIV lifecycle should have no effect on FV vector titers.23Park J Nadeau P Zucali JR Johnson CM Mergia A Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs.Virology. 2005; 343: 275-282Crossref PubMed Scopus (18) Google ScholarWe have tested three promising anti-HIV gene therapy strategies in an FV vector system. The most extensively tested strategy in clinical trials uses a trans-dominant rev, RevM10, which inhibits the nuclear translocation of partially spliced and unspliced HIV transcripts.24Malim MH Freimuth WW Liu J Boyle TJ Lyerly HK Cullen BR et al.Stable expression of transdominant Rev protein in human T cells inhibits human immunodeficiency virus replication.J Exp Med. 1992; 176: 1197-1201Crossref PubMed Scopus (164) Google Scholar The second strategy uses RNA interference by creating a short hairpin RNA directed against an early, multiply spliced RNA encodingTat and Rev.25Lee NS Dohjima T Bauer G Li H Li MJ Ehsani A et al.Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.Nat Biotechnol. 2002; 20: 500-505Crossref PubMed Scopus (137) Google Scholar The third strategy is based on membrane-associated C46 (maC46), a gp41-derived, 46-amino-acid peptide that inhibits HIV cell entry.26Egelhofer M Brandenburg G Martinius H Schult-Dietrich P Melikyan G Kunert R et al.Inhibition of human immunodeficiency virus type 1 entry in cells expressing gp41-derived peptides.J Virol. 2004; 78: 568-575Crossref PubMed Scopus (135) Google ScholarIn this study, we compared FV vectors containing the individual transgenes and a vector containing all three transgenes. FV vectors containing anti-HIV transgenes, including the vector with all three transgenes, ΔΦSh1maC46Rev, could be produced at titers comparable to vectors containing only the green fluorescent protein (GFP) reporter gene. Transduction of human macrophages differentiated from transduced peripheral blood CD34+ cells or CEMx174 cells resulted in a significant decrease in HIV-1 replication.ResultsAnti-HIV FV vectors can be produced without adverse effects on titerWe engineered FV vectors that express Sh1, RevM10, maC46, or all three transgenes (Figure 1). Some of the proposed studies required flow sorting of cells transduced with the anti-HIV vector. Cells expressing maC46 can be identified using the human monoclonal antibody 2F5. Versions of the FV vectors that did not contain maC46 were constructed to express GFP. An internal ribosomal entry signal was used to express GFP in vectors that had a transgene transcribed by RNA polymerase II, while a cytomegalovirus (CMV) promoter expressing GFP was included in the RNA polymerase III-dependent, Sh1-expressing vector. A vector that expresses all three genes was constructed, in which the murine stem-cell virus (MSCV) long terminal repeat promoter directs the expression of maC46 and the phosphoglycerate kinase promoter directs the expression of RevM10. The two genes share a common termination signal in the FV deleted 3′ long terminal repeat. As shown in Figure 1, all the FV vectors can be produced as concentrated stock with titers of over 107/ml on HT1080 cells. The anti-HIV genes did not interfere with vector production, as determined by comparing against titers obtained with a similar vector plasmid expressing GFP (pΔΦMscvF).Because the Sh1 and RevM10 gene products interfere with Rev and Tat, two HIV proteins necessary for LV vector production, we also measured the effect of the plasmids, pΔΦU6Sh1, pΔΦMscvRev, and pΔΦSh1maC46Rev, on LV vector production. FV plasmids were cotransfected with a 3 plasmid, second-generation LV vector system using 293 cells.27Naldini L Blomer U Gallay P Ory D Mulligan R Gage FH et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Scopus (3974) Google Scholar LV vector titers of the supernatants were determined by staining HT1080 cells for the expression of the LV transgene, β-galactosidase (Figure 2). The inclusion of the FV plasmid expressing the cell-entry inhibitor maC46 alone did not affect LV vector production; this was as expected, becausethe vector does not use the HIV envelope. The inclusion of pΔΦMscvRev had a modest (twofold) but significant (P < 0.006) effect on LV vector titer. When pΔΦU6Sh1 was included in the transfection, LV vector titer decreased >200-fold. Cotransfection of the combination construct, pΔΦSh1maC46Rev, decreased LV vector titer >1,500-fold and showed a significant difference from the results obtained with the use of pΔΦU6Sh1 (P < 0.0002). Combining the Sh1 and RevM10 transgenes has previously been demonstrated to improve the anti-HIV effect and limit the development of resistant HIV strains.28Unwalla HJ Li HT Bahner I Li MJ Kohn D Rossi JJ Novel Pol II fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin RNA and protein.J Virol. 2006; 80: 1863-1873Crossref PubMed Scopus (47) Google ScholarFigure 2Lentivirus vector inhibition assay. Foamy virus plasmids were cotransfected with a lentivirus (LV) vector plasmid system in which the LV vector expresses β-galactosidase. The supernatant from each transfection was titered on HT1080 cells. Three independent transfections were performed for each condition, and the mean titer + SD is represented.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FV vectors containing maC46 block entry of HIVWe used an entry inhibitor assay to test the maC46 transgene in our constructs. TZM-bl cells are a HeLa-derived cell line constitutively expressing CD4 and CCR5. The β-galactosidase gene is under the control of the HIV-1 long terminal repeat, and is expressed upon HIV entry and Tat expression. TZM-bl cells were transduced with either ΔΦMscvmaC46 or ΔΦSh1maC46Rev vector stocks at a multiplicity of infection (MOI) of 3 or 9 and infected with HIVJR-FL at an MOI of 0.025. Both ΔΦMscvmaC46 and ΔΦSh1maC46Rev inhibited HIV cell entry, as measured by the decrease in β-galactosidase-positive cell foci (Figure 3). A dose-dependent effect was observed, with a decrease in foci when the MOI was increased from 3 to 9 (P < 0.01 for ΔΦMscvmaC46 and P < 0.05 for ΔΦSh1maC46Rev). This result indicates that the maC46 transgene is expressed at an effective level in both constructs when target cells are transduced at MOIs appropriate for a clinical trial.Figure 3Human immunodeficiency virus (HIV) cell-entry inhibition assay. β-galactosidase expression in TZM-bl cells is HIV dependent. TZM-bl cells transduced by the indicated foamy virus vector were infected in triplicate with HIVJR-FL at a multiplicity of infection (MOI) of 0.025, and stained for β-galactosidase expression 2 days later. The average number of foci for three wells (+SD) is represented.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Protection of macrophages derived from transduced, CD34-selected hematopoietic progenitorsIn order to test FV anti-HIV vectors in peripheral blood CD34-selected cells, a method was developed to differentiate these cells into macrophages which could then be tested for protection from HIV. Frozen human CD34-selected cells were thawed and transduced overnight with the FV vectors at an MOI of 3, resulting in gene transfer rates of between 15 and 30%. Vectors that did not express maC46 were engineered to express GFP so that fluorescence-activated cell sorting (FACS) could be performed (Figure 1). Plasmids containing the GFP-expressing vectors for RevM10 and Sh1 (pΔΦMscvRevIF and pΔΦU6Sh1CF) showed LV vector titer inhibition rates similar to those for pΔΦMscvRev and pΔΦU6Sh1, respectively, when using the same method as shown in Figure 2 (data not shown). The cells were flow sorted on day 4 for GFP or maC46 expression and then expanded for 34 additional days in conditions that supported macrophage differentiation. The cells in culture were demonstrated to be macrophages based on lipid uptake and the surface expression of CD14, HLA-DR, and CD168 (Supplementary Figure S1). The adherent cells were re-plated in at least three wells and infected the following day with HIVJR-FL at an MOI of 0.25. Media were exchanged every 4 days and p24 levels in the culture supernatant were followed over time. The results are shown in Figure 4a. Cells transduced with ΔΦMscvmaC46, ΔΦU6Sh1CF, or ΔΦSh1maC46Rev had minimal-to-undetectable p24 production throughout the 24-day HIV infection. In contrast, non-transduced cells or cells transduced with ΔΦMscvF or ΔΦMscvRevIF had p24 levels that increased throughout the experiment. The levels of p24 from cells transduced with ΔΦMscvmaC46, ΔΦU6Sh1CF, and ΔΦSh1maC46Rev are all significantly different from those of the non-transduced controls at day 24 (P = 0.04, 0.04, and 0.02, respectively). Figure 4b shows a compilation of data points from three independent experiments for p24 production at days 14 or 16. There is a dramatic difference in HIV production as measured by p24 levels of the macrophages transduced with ΔΦMscvmaC46, ΔΦU6Sh1CF, and ΔΦSh1maC46Rev as compared to non-transduced macrophages or macrophages transduced with ΔΦMscvF or ΔΦMscvRevIF.Figure 4Production of p24 after human immunodeficiency virus infection of macrophages differentiated from transduced human CD34+ cells. (a) CD34+ cells were transduced overnight with foamy virus (FV) vectors, sorted at day 4 and differentiated for 34 additional days. Adherent cells were re-plated and challenged with HIVJR-FL at a multiplicity of infection of 0.25. HIV infection of transduced cells was performed in triplicate (ΔΦMscvmaC46 and ΔΦU6Sh1CF) or quadruplicate (non-transduced control, ΔΦMscvRevIF, ΔΦMscvF, and ΔΦSh1maC46Rev). (b) Compilation of three independent experiments showing p24 production on days 14–16 after HIV infection. All data points are shown. The horizontal line represents the average p24 for each condition. Green fluorescent protein (GFP) includes both ΔΦMscvF (eight data points) transduced cells, and cells transduced with an FV vector that expresses GFP from the phosphoglycerate kinase promoter (three data points). NT, non transduced; MF, ΔΦMscvF, GFP, ΔΦMscvF and ΔΦPgkF; RevIF, ΔΦMscvRevIF; Sh1CF, ΔΦU6Sh1CF; M46, ΔΦMscvmaC46; SMR, ΔΦSh1maC46Rev.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Selection and protection of transduced CEM×174 cells following HIV infectionIn order to further compare the FV vectors, we tested their ability to block HIV-1 replication in CEM×174 cells. The CEM×174 cell line expresses both CD4 and CXCR4,29Salter RD Howell DN Cresswell P Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids.Immunogenetics. 1985; 21: 235-246Crossref PubMed Scopus (509) Google Scholar and is used as a T cell model for HIV infection.30Velpandi A Nagashunmugam T Murthy S Cartas M Monken C Srinivasan A Generation of hybrid human immunodeficiency virus utilizing the cotransfection method and analysis of cellular tropism.J Virol. 1991; 65: 4847-4852Crossref PubMed Google Scholar,31Piller SC Dubay JW Derdeyn CA Hunter E Mutational analysis of conserved domains within the cytoplasmic tail of gp41 from human immunodeficiency virus type 1: effects on glycoprotein incorporation and infectivity.J Virol. 2000; 74: 11717-11723Crossref PubMed Scopus (61) Google Scholar In order to minimize the number of cells with multiple integration events, CEM×174 cells were transduced with each of the anti-HIV FV vectors shown in Figure 5a at an MOI of 0.5. As measured by Southern blot analysis, gene transfer was between 25 and 50% when this MOI was used (data not shown). Unselected transduced cells were infected with HIVNL4-3 (MOI of 0.001). By day 8 there was a rapid rise in p24 in all of the samples because of infection of the non-transduced cells (data not shown). By day 16 of the infection there were differences in total cell number and the amount of p24 production between the various transduced cell populations (Figure 5b). The total number of cells in the control, non-transduced culture decreased 11-fold. Cells transduced with ΔΦMscvRev were not protected from HIV infection, as determined on the basis of this assay which showed that both the decrease in cell number and the p24 production levels were similar to those of non-transduced cells.When compared with non-transduced cells, ΔΦU6Sh1 transduced cells had 30% more cells that survived (P < 0.02), with a non-significant decrease in p24 production per cell. Cells transduced with ΔΦMscvmaC46 were protected from HIV with 5.2 times more surviving cells (P < 0.00004) and a 3.1-fold decrease in p24 produced per cell (P < 0.05), relative to non-transduced cells. Cells transduced with the combination vector ΔΦSh1maC46Rev had a lower p24 production per cell (P < 0.005) and greater cell survival (P < 0.03) as compared to cells transduced with ΔΦMscvmaC46.Figure 5Human immunodeficiency virus (HIV) infection of unsorted transduced CEM×174 cells. (a) Schematic of vectors used for transducing CEM×174 cells in this assay. For the Southern blot analysis, hybridization with a single probe was used to identify each of the four vector inserts by differential fragment size using HindIII (H) and PstI (P) endonuclease double digestion. Predicted fragment sizes are as follows: ΔΦU6Sh1—2,014 base pair (bp), ΔΦMscvRev—2277 bp, ΔΦMscvmaC46—2472 bp, and ΔΦSh1maC46Rev—2814 bp. (b) p24 and cell count at day 16 after HIV infection of CEM×174 cells transduced with foamy virus (FV) vectors at a multiplicity of infection of 0.5. Infections were performed in triplicate, and the mean values + SD are represented. (c) Genomic Southern blot analysis. CEM×174 cultures transduced with each of the four FV vectors were pooled and subjected to HIV infection in duplicate cultures. DNA was extracted from cells at days 4, 8, and 16 and probed with the DNA fragment indicated in a. Subsequently, the membrane was re-probed with a Col1A1 probe as a DNA loading control. (d) The average of the signals from the two samples on the genomic Southern blot analysis in c was quantified and adjusted using the DNA loading controls for determining the average copy number of each of the four vectors over time. NT = non-transduced; Rev = ΔΦMscvRev; Sh1 = ΔΦU6Sh1; C46 = ΔΦMscvmaC46; SMR = ΔΦSh1maC46Rev.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to directly compare the effectiveness of the vectors, an in vitro competition assay was developed. Cells transduced by the different vectors were pooled and provirus copy numbers were tracked over time following HIV infection. CEM×174 cells were transduced with each of the four vectors (Figure 5a) at an MOI of 0.5, mixed together in equal ratios, and infected with HIVNL4-3. All four populations of unsorted transduced cells had similar growth and cell numbers prior to pooling (data not shown). Cells were removed for genomic DNA extraction at regular intervals. The relative copy number was determined using Southern blot analysis. Using a single probe, the genomic DNA was cut with two restriction enzymes that resulted in a distinct fragment size for each vector (Figure 5a and c), and quantified by Phosphorimager analysis after correcting for DNA loading (Figure 5d). During HIV infection the overall copy number of cells transduced by ΔΦMscvmaC46 and ΔΦSh1maC46Rev increased, while the copy number of cells transduced by ΔΦU6Sh1 and ΔΦMscvRev decreased. The low hybridization signal on day 8 was secondary to the presence of infected dying cells, resulting in a high level of degraded DNA in the samples (data not shown). This demonstrates that CEMx174 cells transduced with ΔΦMscvmaC46 and ΔΦSh1maC46Rev have a selective advantage over cells transduced with either ΔΦU6Sh1 or ΔΦMscvRev during HIV infection (Figure 5c and d). The selected, ΔΦSh1maC46Rev-transduced cells were protected with a lower provirus copy number relative to selected ΔΦMscvmaC46 transduced cells.DiscussionThis study demonstrates that FV vectors are well-suited for anti-HIV gene therapy, on the basis of the following findings: (i) FV vector titers are not affected by anti-HIV transgenes; (ii) transgene expression of maC46 or sh1 effectively blocks HIV replication in macrophages; (iii) expression of maC46 is necessary to effectively block HIV infection and confer a selective advantage in a cell line (CEM×174 cells) used for propagating HIV; and (iv) an FV vector expressing three anti-HIV transgenes effectively blocks HIV infection in CEM×174 cells and in macrophages.One of the major drawbacks of using LV vectors in HIV gene therapy is that LV vector production requires functional HIV proteins. Two of the anti-HIV transgenes that we studied had a significant adverse effect on LV vector titer (Figure 2). This result has been previously observed when anti-HIV transgenes are expressed in an LV vector, and this is a major hurdle in developing a clinical trial.12Banerjea A Li MJ Bauer G Remling L Lee NS Rossi J et al.Inhibition of HIV-1 by lentiviral vector-transduced siRNAs in T lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell-derived macrophages.Mol Ther. 2003; 8: 62-71Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar,13Mautino MR Morgan RA Inhibition of HIV-1 replication by novel lentiviral vectors expressing transdominant Rev and HIV-1 env antisense.Gene Ther. 2002; 9: 421-431Crossref PubMed Scopus (19) Google Scholar,14Li MJ Bauer G Michienzi A Yee JK Lee NS Kim J et al.Inhibition of HIV-1 infection by lentiviral vectors expressing Pol III-promoted anti-HIV RNAs.Mol Ther. 2003; 8: 196-206Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar In contrast, FV vector stocks expressing the RevM10 and Sh1 can be produced at sufficient titer to pursue a clinical study.As part of these studies, we directly compared three well studied anti-HIV transgenes in FV vectors. Although RevM10 is the most tested of the three strategies, it was found to be the least effective in our studies, showing a modest effect only in the LV cotransfection assay (Figure 2), and was similar to controls in the other assays (Figures 4 and 5). When expressed as a single transgene in an FV vector, RevM10 had less of an anti-HIV effect than has been reported using other vector systems.32Bauer G Valdez P Kearns K Bahner I Wen SF Zaia JA et al.Inhibition of human immunodeficiency virus-1 (HIV-1) replication after transduction of granulocyte colony-stimulating factor-mobilized CD34+ cells from HIV-1-infected donors using retroviral vectors containing anti-HIV-1 genes.Blood. 1997; 89: 2259-2267PubMed Google Scholar,33Bonyhadi ML Moss K Voytovich A Auten J Kalfoglou C Plavec I et al.RevM10-expressing T cells derived in vivo from transduced human hematopoietic stem-progenitor cells inhibit human immunodeficiency virus replication.J Virol. 1997; 71: 4707-4716PubMed Google Scholar,34Bahner I Sumiyoshi T Kagoda M Swartout R Peterson D Pepper K et al.Lentiviral vector transduction of a dominant-negative rev gene into human CD34+ hematopoietic progenitor cells potently inhibits human immunodefi