1. Volume 8, Issue 3, Pages 475-484 (September 2003)
Novel interfering bifunctional molecules against the CCR5 coreceptor are efficient inhibitors of HIV-1 infection 
José Luis Abad, Manuel A González, Gustavo del Real, Emilia Mira, Santos Mañes, Fernando Serrano, Antonio Bernad 
Molecular Therapy 
Volume 8, Issue 3, Pages (September 2003)
DOI: /S (03)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Surface CCR5 expression inhibitory constructs. (A) Schematic representation of wt CCR5 receptor and structure of CCR5 truncated mutants generated for this study. 1TM, 2TM, and 4TM constructs are analogous to 1TMK, 2TMK, and 4TMK, respectively, with the difference that they lack the KDEL tetrapeptide. (B) Structure of RANTES-based constructs designed to function as intracellular CCR5 ligands. These constructs and those in (A) were cloned into the pcDNA3 vector. (C) Intrakine expression analysis in transfected 293T cells. A single cell sample was used for simultaneous evaluation of intra- and extracellular expression for each intrakine. The plot shows extracellular intrakine expression as determined by ELISA of 293T supernatants. Supernatant and cell lysates were collected simultaneously. n.d., not detectable (ELISA sensitivity 3 pg/ml). Bottom inset shows Western blot analysis of intracellular intrakine expression (30 μg total protein was loaded in each well). Arrow indicates mobility (6.5 kDa) of wt RANTES.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Effects of CCR5 truncated mutants and intrakine constructs on surface CCR5 expression. Plots show CCR5 inhibition in 293 cells cotransfected with 200 ng of a CCR5 expression plasmid (pcCCR5) and plasmids encoding six truncated variants of (A) CCR5- or (B) RANTES-derived intrakines, at two molar ratios (1:4 and 1:9).
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Effects of inhibitors of surface CCR5 expression in the MCF-7 growth factor-induced chemokine receptor transactivation model. MCF-7 cells were transduced with control vector or the same retroviral vector encoding CCR5 inhibitory constructs and loaded onto the top of a vitronectin-coated chamber. Cells were incubated with a chemotactic stimulus (IGF alone or plus RANTES; 37°C, 4 h). The top of the membrane was washed and the bottom stained to determine the number of transmigrated cells by direct counting of five representative microscopic fields per well. (A) Microphotographs of stained membranes showing a representative example of transmigrated MCF-7 cells transduced with control or RT-4TMK retroviral vectors. (B) The graph shows the ability of different CCR5 inhibitory constructs to decrease IGF-1 + RANTES-induced chemotaxis in MCF-7 cells (gray bars, left scale). Black points (right scale) plot the ratio between percentage chemotaxis inhibition and percentage transduced cells (both relative to total cell number). A 0.9 ratio indicates that only 10% of transduced cells transmigrated through the membrane.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Optimized stimulation protocol to evaluate CCR5 inhibitory constructs in human lymphocytes. (A) Arrows at top show stimulation procedure and time (days) for each step of the experiment. t = 0 indicates start of stimulation of freshly purified PBL. Retroviral transduction and sorting of GFP+ PBL were done on days 2 and 7 after start of stimulation, respectively. Bottom graph shows evolution of CCR5 expression. CCR5 expression was measured at start of PBL stimulation, after retroviral transduction, before sorting, and 4 days after sorting. Insets, FACS plots (x axis, mean fluorescence intensity; y axis, cell number) show CCR5 expression profiles for control retroviral vector (pLZR-IRES/gfp)-transduced PBL. (B) Histograms show retroviral transduction (top) and FACS sorting (bottom) of PBL transduced with retroviral bicistronic vectors encoding CCR5 inhibitory molecules. Gray histograms correspond to control stimulated untransduced PBL. Data are from a representative transduction/sorting experiment. (C) Postsorting analysis of CCR5 inhibitor expression in PBL transduced with pLZR-RTK-IRES/gfp (ELISA), pLZR-RT4TMK-IRES/gfp, pLZR-4TMK-IRES/gfp, pLZR-RzR5-76-IRES/gfp, and pLZR-RTK-RzR5-76-IRES/gfp (RT-PCR). RANTES-KDEL ELISA data were normalized for 5 × 105 transduced cells/ml; n.d., not detectable (assay sensitivity 3 pg/ml). Bottom shows RT-PCR expression analysis of the other four CCR5 inhibitors. C−, no template; C+, 1 pg of the corresponding retroviral plasmid; −RT, no reverse transcriptase added; +RT, cDNA; gfp, control retroviral vector. β-Actin amplification was used to normalize sample load.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
CCR5 expression inhibition in transduced PBL. (A) Human lymphocytes were transduced with CCR5 inhibitory constructs. The plot shows surface CCR5 expression inhibition kinetics in sorted PBL; the first measurement was made 48 h after sorting. Culture medium was replaced at day 2 postsorting. Data correspond to a representative experiment. CCR5 inhibitors used were as follows: RTK, RTK-RzR5-76, RT-4TMK, RzR5-76, 4TMK. (B) CCR5 expression inhibition at day 3 postsorting (day 8 posttransduction) in PBL transduced with the inhibitory constructs, relative to control vector-transduced cells. Medium was not renewed between sorting and FACS analysis of CCR5 surface expression in transduced lymphocytes. Data are from four independent experiments using lymphocytes from four healthy donors.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Inhibition of HIV-1 replication in human PBL expressing CCR5 inhibitory molecules. (A) Gray bars (right scale) show relative p24 levels produced by transduced PBL inoculated with NL-4 (X4) strain HIV-1, compared to control vector-transduced PBL (white bar). Black bars (left scale) show mean inhibition of R5 (BaL) strain replication in PBL transduced with CCR5 inhibitors, compared to control cells. Data are from three unrelated healthy donors at day of maximum BaL p24 production from PBL transduced with control vector (pLZR-IRES/gfp). (B) Progression of p24 production, indicating evolution of BaL HIV-1 replication in control and CCR5 inhibitor-transduced PBL from one donor. (C) The plot shows linear correlation (R = 0.96) between CCR5 inhibition and reduction of HIV-1 (R5 strain) replication in PBL transduced with the inhibitor molecules. Data are from Figs. 5B and 6A. Symbols used for CCR5 inhibitors in B and C are as for Fig. 5A; control vector, open square.
Molecular Therapy 2003 8, DOI: ( /S (03) )
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions