1. Volume 12, Issue 6, Pages 1157-1167 (December 2005)
Inhibition of Simian/Human Immunodeficiency Virus Replication in CD4+ T Cells Derived from Lentiviral-Transduced CD34+ Hematopoietic Cells 
Stephen E. Braun, Fay Eng Wong, Michelle Connole, Gang Qiu, Lorrin Lee, Jackie Gillis, Xiaobin Lu, Laurent Humeau, Vladimir Slepushkin, Gwendolyn K. Binder, Boro Dropulic, R. Paul Johnson 
Molecular Therapy 
Volume 12, Issue 6, Pages (December 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Schematic diagram of the lentiviral vector VRX494. The vector contains the antisense HIV envelope (reverse arrow) situated with the Rev-responsive element (RRE; gray box) between the splice-donor (SD) and splice-acceptor (SA) sites and transcriptionally regulated by the HIV-1 LTR (open box), eGFP (right hatch) translated from the spliced message, and the cis-acting lentiviral elements Ψ packaging signal (bold line) and central polypurine tract (cppt; triple line).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
CEMx174-VRX494 cells (closed symbols) and CEMx174-GFP cells (open symbols) were challenged with (A) HIV-1 NL4-3, (B) SIVmac239, (C) SHIV 89.6P, (D) SHIV HXBC2P 3.2, (E) SHIV DH12R, or (F) SHIV DH12. The m.o.i. were 0.01 (square) and TCID50/cell (diamond) for (A) and (B) and 1 ng p27 per 106 cells (square) and 0.1 ng Gag p27 per 106 cells (diamond) for (C–F). Transduced cells were infected and followed for 2 weeks. Viral replication was assessed by ELISA determination of HIV-1 Gag p24 or SIV Gag p27 concentrations in cell-free culture supernatants. Results shown are representative of two experiments for each virus.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Transduction of rhesus CD34+ bone marrow cells with VRX494. (A) Expression of eGFP in rhesus CD34+ bone marrow cells 48 h after 1, 2, 3, and 4 days of cytokine stimulation and transduction with VRX494 at m.o.i. of 25 IU per cell. Analysis of CD34+ and GFP expression was performed using a forward- and side-scatter gate that included both normal and activated cells. The percentage of CD34+ cells that were GFP+ and the mean fluorescence intensity are shown in the upper right quadrants. (B) Effect of the number of cycles of transduction on the percentage of GFP+ CD34+ cells. Rhesus CD34+ cells were transduced with VRX494 (filled symbols) or LZRS-GFP (open symbols) for the indicated number of cycles at m.o.i. of 25 or 3–8, respectively. (C) Effect of m.o.i. on the percentage of GFP+ CD34+ cells. Rhesus CD34+ cells were transduced four times at the indicated m.o.i. over 4 days with either VRX494 (filled symbols) or LZRS-GFP (open symbols). The diamond, triangle, circle, and square symbols indicate separate experiments.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Expression of GFP in T cells derived from VRX494-transduced CD34+ cells. (A) T cells derived from VRX494-transduced rhesus CD34+ bone marrow cells. Left: CD4 versus CD8 expression in CD3+ lymphocytes. CD4+ CD8− cells, in the lower right quadrant, were gated. Right: Histogram of GFP expression in CD3+ CD4+ CD8− cells derived from VRX494-transduced CD34+ rhesus bone marrow cells. (B) Effect of the number of cycles of transduction on the percentage of GFP+ CD4+ T cells derived from CD34+ cells when transduced with VRX494 (filled) or LZRS-GFP (open) at m.o.i. 25 or 3 to 8, respectively. (C) Effect of m.o.i. on the percentage of GFP+ CD4+ T cells derived from CD34+ cells transduced four times over 4 days with VRX494 (filled) or LZRS-GFP (open) vectors. The diamond, triangle, circle, and square symbols indicate separate experiments.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
CD4+ T cells derived from VRX494-transduced CD34+ cells (filled) or from nontransduced CD34+ cells (open) were challenged with (A) SHIV 89.6P or (B) SHIV HXBC2P 3.2 at 1 (square) and 0.1 (diamond) ng Gag p27 per 1 ± 106 cells. The CD4+ T cells were infected and followed for 2 weeks. Viral replication was assessed by SIV Gag p27 ELISA. Results shown are representative of two experiments for each virus.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Southern analysis of T cell clones. T cell clones were isolated from T cells derived in vitro from VRX494-transduced CD34+ cells. Genomic DNA from the clones was digested with NcoI (a restriction enzyme with one recognition site in VRX494), separated by electrophoresis, blotted to membranes, and probed with GFP. The sizes of the bands are indicated. Multiple bands are indicative of multiple lentiviral integrations. Six representative clones of 16 total are shown.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Expression of VRX494 is responsive to the expression of Tat. (A) VRX494-transduced CEMx174 cells were infected with HIV-1 NL4-3 at 0.1 TCID50 per cell. After 4 days, cells were stained with a PE-conjugated antibody to HIV-1 Gag and analyzed by flow cytometry for expression of GFP and p24 Gag. R2 and R6 describe the gates for uninfected cells. R3 and R7 describe the gates for HIV-1-infected cells. (B) VRX494-transduced 293T cells were transiently transfected with control, Tat, Rev, or both Tat and Rev expression vectors. RNA levels for HIV envelope inhibitor (Antisense Env) and for GFP were determined by real-time RT-PCR. Expression of GFP was determined by flow cytometry. Results show the average ± standard deviation for three samples.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

9. FIG. 8
Mechanism of antisense inhibition. Analysis for adenosine modifications in RNA–RNA hybrids in VRX494-transduced CEMx174 cells after infection with HIV-1 NL4-3. RNA samples were harvested from the cells on day 3. Potential hybrid sequences were amplified by RT-PCR and digested with DraI. Modifications to the recognition sequence result in the loss of the DraI restriction site (TTT^AAA).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions