Stable transduction of quiescent T cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins by Cecilia Frecha, Caroline Costa, Didier Nègre, Emmanuel Gauthier, Stephen J. Russell, François-Loïc Cosset, and Els Verhoeyen Blood Volume 112(13):4843-4852 December 15, 2008 ©2008 by American Society of Hematology

Efficient pseudotyping of MLV- and HIV-derived vectors with wild-type or mutant F and H MV-gps. Efficient pseudotyping of MLV- and HIV-derived vectors with wild-type or mutant F and H MV-gps. (A) Schematic presentation of wild-type or mutant F and H gps from the Edmonston MV strain. The double vertical lines (//) separate the transmembrane sequences (TM) from the cytoplasmic domain (cyt-tail). The single line (/) indicates that part of TM is not shown. HΔ15, HΔ20, and HΔ24 represent H mutants with progressive deletions of the cytoplasmic tail. H97E contains a single amino acid mutation at position 97 in the TM region of wt H. FΔ30 contains only 3 residual amino acids of the F wt cytoplasmic tail. (B) 293T cells were incubated with serial dilutions of GFP-encoding MLV or HIV vectors that were pseudotyped with Hwt, HΔ15, HΔ20, HΔ24, or H97E in combination with Fwt or FΔ30. Control incubations were performed with RDTR gp–pseudotyped vectors. Cells were analyzed for GFP expression 72 hours after transduction by FACS analysis. Titers are expressed as TU/mL. Data are shown as means plus or minus SD; n = 6. Cecilia Frecha et al. Blood 2008;112:4843-4852 ©2008 by American Society of Hematology

Codisplay of cytoplasmic tail deletion mutants of H and F gps on HIV vectors results in high titers. 293T cells were transfected with HIV-gagpol, HIV-GFP vector, and the combination of H and F gps: Hwt/Fwt, HΔ24/FΔ30, Hwt/FΔ30, and HΔ24/Fwt. Codisplay of cytoplasmic tail deletion mutants of H and F gps on HIV vectors results in high titers. 293T cells were transfected with HIV-gagpol, HIV-GFP vector, and the combination of H and F gps: Hwt/Fwt, HΔ24/FΔ30, Hwt/FΔ30, and HΔ24/Fwt. RDTR was used as a control gp. (A) Images show GFP expression and syncytia formation of 293T producer cells 24 hours after transfection. Scale bar represents 1000 μm. (B) At 24 and 48 hours after transfection, cells were fixed and stained with Giemsa staining solution, and syncytia formation (percentage cell fusion) was quantified. (C) 293T cells were incubated with serial dilutions of GFP-encoding HIV vectors pseudotyped with Hwt/Fwt, HΔ24/FΔ30, Hwt/FΔ30, HΔ24/Fwt, or RDTR gps. Vector supernatant was harvested at 24 or 48 hours after transfection. Cells were analyzed for GFP expression 72 hours after transduction by FACS. Titers are expressed as TU/mL (means ± SD; n = 6). (D) HIV-GFP vectors were pseudotyped with Hwt/Fwt, HΔ24/FΔ30, Hwt/FΔ30, and HΔ24/Fwt and purified over a sucrose cushion. Subsequently, viral gps, Hwt and HΔ24, were detected by Western blot with an antibody recognizing H ecto-domain; incubation with an anti-HIV p24 antibody was used to reveal HIV capsid (CA). (E) HIV-GFP vectors were pseudotyped with Hwt/Fwt, HΔ24/FΔ30, Hwt/FΔ30, and HΔ24/Fwt. A cell-binding assay was performed using the different vector preparations, concentrated by low-speed centrifugation. Both 293T cells (CD46+, SLAM−) and control CHO cells (CD46−, SLAM−) were incubated with the indicated HIV vector pseudotypes for 1 hour. After washing, bound viral particles were detected using conformational antibodies directed against the ectodomain of H gp (anti-H) or F gp (anti-F). Data shown are representative of 3 experiments. Cecilia Frecha et al. Blood 2008;112:4843-4852 ©2008 by American Society of Hematology

HΔ24/FΔ30-pseudotyped LVs conserve the tropism of Edmonston MV strain. HΔ24/FΔ30-pseudotyped LVs conserve the tropism of Edmonston MV strain. (A) SLAM and CD46 surface staining (open histograms) of the different cell lines (CHO, 293T, Jurkat, Raji, and B95a). Closed histograms represent staining with the isotype IgG control. (B) The different cell lines were transduced with a HΔ24/FΔ30-pseudotyped HIV-GFP vector at an MOI of 1 in the absence of antibody (no antibody) or after 2-hour incubation with an anti-CD46 antibody that blocks H binding to CD46 receptor or, as control, with anti-SLAM antibody prior to transduction. In the case of B95-a cells, a specific anti-SLAM antibody was used to block the SLAM receptor, whereas anti-CD46 antibody was used as a control prior to transduction. Cells were analyzed for GFP expression 72 hours after transduction by FACS analysis. Data shown here are representative of 4 independent experiments. (C) 293T cells were transduced with 2 different shRNA LVs to knock down hCD46 (293T-CD46KD). Open histograms represent the expression of CD46 in 293T or 293T-CD46KD. Closed histograms represent staining with the isotype IgG control. Both cell lines were subsequently transduced with HΔ24/FΔ30-pseudotyped HIV-GFP vector at MOI = 1. Dot blots represent the percentage of GFP+ cells 3 days after transduction, as analyzed by FACS. Cecilia Frecha et al. Blood 2008;112:4843-4852 ©2008 by American Society of Hematology

HΔ24/FΔ30-displaying LVs outperform VSV-G-LVs for the transduction of IL-7–prestimulated adult T cells. HΔ24/FΔ30-displaying LVs outperform VSV-G-LVs for the transduction of IL-7–prestimulated adult T cells. (A) Surface expression of SLAM or CD46 receptors on unstimulated PBLs or on PBLs stimulated for 3 days with anti-CD3+ anti-CD28 antibodies in the presence of IL-2. (B) PBLs were stimulated for 3 days with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and subsequently transduced with HΔ24/FΔ30- or VSV-G–pseudotyped HIV-GFP vectors at the indicated vector doses. Transduced cells were kept in culture for 3 days in the presence of IL-2 and the percentage of GFP expression was monitored by FACS (means ± SD; n = 3). (C,E) The surface expression of SLAM or CD46 receptors on resting adult or cord blood CD3+ T cells, respectively, immediately upon isolation by negative antibody-mediated selection or upon incubation of these T cells with rIL-7 for 3 days. Purified resting adult (D) or cord blood (F) CD3+ T cells were stimulated for 3 days with rIL-7 and subsequently transduced with HΔ24/FΔ30- or VSV-G–pseudotyped HIV-GFP vectors in the presence of rIL-7 at the indicated vector doses. Transduced cells were maintained in culture for 3 days and GFP expression was then monitored by FACS analysis (means ± SD; n = 5). Cecilia Frecha et al. Blood 2008;112:4843-4852 ©2008 by American Society of Hematology

The HΔ24/FΔ30-displaying LVs efficiently transduce quiescent adult T cells. The HΔ24/FΔ30-displaying LVs efficiently transduce quiescent adult T cells. (A) Highly purified resting adult T cells were isolated by negative antibody-mediated selection to avoid cell activation and then transduced with HΔ24/FΔ30- or VSV-G–pseudotyped HIV-GFP vectors at an MOI of 10 or 30 in the absence of exogenous stimuli. Three days after transduction, surface staining for naive and memory subsets was performed by anti-CD45RA/anti-CD45RO double staining. In parallel, surface staining for the CD3+, CD4+, and CD8+ T-cell subsets was performed and the percentage of GFP expression for each of these T-cell subsets was analyzed by FACS (means ± SD; n = 5). Inset shows CD45RA versus SLAM expression on freshly isolated total CD3+ T cells (B) Resting T cells were transduced with HΔ24/FΔ30- or VSV-G–pseudotyped LVs at an MOI of 10. After 3 days, part of the transduced cell cultures was continued in the presence of rIL-7 for 3 more days to verify stable GFP expression. For both culture conditions, surface staining for naive and memory subsets was performed by anti-CD45RA/anti-CD45RO double staining. Dot blots represent the percentage of GFP+ cells in the CD45RA+ naive T cells (top row) and the percentage GFP+ cells in the CD45RO+ memory T cells (bottom row). Data are representative of 6 experiments. (C) Resting T cells were transduced with HΔ24/FΔ30- or VSV-G–pseudotyped LVs at an MOI of 30. Surface staining for naive T cells was performed by anti-CD45RA staining. Dot blot represents the GFP+ CD45RA+ naive T cells (top right quadrant R2) and the GFP+ CD45RA− memory T cells (bottom right quadrant R4). For each gate, R1, R2, R3, and R4, the expression of CD62L is shown. Data are representative of 3 experiments. (D) Surface expression of SLAM or CD46 receptors on resting adult CD3+ cells after transduction with HΔ24/FΔ30- or VSV-G–pseudotyped lentivectors at an MOI of 10. (E) Resting adult T cells were incubated with or without anti-SLAM or anti–CD46 receptor–blocking antibodies for 3 hours and subsequently transduced with HΔ24/FΔ30-LVs or VSV-G-LVs at an MOI of 10. The percentage of GFP+ cells was analyzed by FACS 3 days after transduction. Cecilia Frecha et al. Blood 2008;112:4843-4852 ©2008 by American Society of Hematology

Comparison of cell-cycle entry and activation of adult T cells following exposure to HΔ24/FΔ30-LVs or VSV-G-LVs. Comparison of cell-cycle entry and activation of adult T cells following exposure to HΔ24/FΔ30-LVs or VSV-G-LVs. Resting adult CD3+ T cells were transduced with HΔ24/FΔ30- or VSV-G–displaying LVs, or no vector, and as a control IL-7–prestimulated T cells were transduced with HΔ24/FΔ30-displaying LVs. (A) Cell-cycle progression was monitored by simultaneously visualizing the RNA (pyronin-Y) and DNA (7-AAD) content of the T cells at day 3 of incubation. The percentages of cells in the G0/G1a, G1b, and S/G2/M phase of the cell cycle are indicated in the dot blots. (B) Surface staining for CD69, HLA-DR, and CD71 versus GFP+ cell expression is shown at day 3 after transduction. The percentage of activation marker–expressing GFP+ cells is indicated in the top right quadrant; the percentage of GFP+ cells not expressing the activation marker is indicated in the bottom right quadrant. Cecilia Frecha et al. Blood 2008;112:4843-4852 ©2008 by American Society of Hematology