1. Volume 7, Issue 3, Pages 304-316 (March 2003)
A SIN lentiviral vector containing piga cdna allows long-term phenotypic correction of CD34+-derived cells from patients with paroxysmal nocturnal hemoglobinuria 
David Robert, François-Xavier Mahon, Emmanuel Richard, Gabriel Etienne, Hubert de Verneuil, François Moreau-Gaudry 
Molecular Therapy 
Volume 7, Issue 3, Pages (March 2003)
DOI: /S (03)00011-X
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Schematic representations of SIN lentiviral vector constructs. (A) Different transfer vector constructs. Abbreviations: PIGA cDNA, 1542-bp coding sequence of PIGA gene; cPPT, central polypurine tract; CTS, central termination sequence; eGFP, enhanced green fluorescent protein gene; Δgag/RRE, truncated gag region containing the rev-responsive element; hEF1α, human elongation factor 1α promoter; ψ, packaging signal; SA, splice acceptor site; SD, splice donor site; ΔU3, U3 region deleted of TATA box and enhancer sequences; WPRE, woodchuck hepatitis posttranscriptional regulatory element. (B) Southern blot analysis of GPI-deficient K562 (K) and Jurkat (J) cell lines transduced with TEPW vector shows appropriate single proviral band with the expected size (2.2 kb).
Molecular Therapy 2003 7, DOI: ( /S (03)00011-X)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Gene transfer by TEPW vector into GPI-deficient hematopoietic cell lines restores GPI anchor and overcomes resistance to aerolysin. GPI-deficient K562 (A) and Jurkat (B) cell lines were transduced with the TEPW vector at a m.o.i. of 0.4 to 12. Five days after transduction, cells were stained with anti-CD59–FITC monoclonal antibody or fluorescent aerolysin, which binds directly GPI anchor, for analysis by flow cytometry. The open bars represent the percentage of total cells expressing CD59 and the solid bars, the percentage of total cells expressing fluorescent aerolysin. (C) A semiquantitative Southern blot analysis was performed on genomic DNA from untransduced (0) and TEPW-transduced GPI-deficient K562 at various m.o.i. (0.4, 1.2, 4, and 12) as described for Fig. 1. One hundred fifty, 50, and 15 pg of TEPW plasmid were used with 14 μg of normal DNA corresponding to 5, 1.5, and 0.5 copies per cell of the transgene, respectively.
Molecular Therapy 2003 7, DOI: ( /S (03)00011-X)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Lentiviral-mediated gene transfer by TEPW vector and expression into GPI-deficient K562 cell line. Transduced cells were harvested 5 days after a single round of transduction with the TEPW or TEEW vector at a m.o.i. of 4 to 400 and analyzed by FACS for CD59 (CD59–FITC), GPI (fluorescent aerolysin; FLAER), or GFP expression. The mean fluorescence intensity (MFI) of CD59+, GPI+, or GFP+ cells is indicated. These MFI are also indicated for the normal phenotype K562 cell line.
Molecular Therapy 2003 7, DOI: ( /S (03)00011-X)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Lentiviral-mediated gene transfer by TEPW vector overcomes resistance to aerolysin. Transduced GPI-deficient K562 (A) and Jurkat cells (B) were exposed to 10−8 M aerolysin for 3 h at 37°C. Cells were stained with propidium iodide to assess viability and analyzed by flow cytometry: nonviable cells were those exhibiting fluorescence. Open bars represent the percentage of nonviable cells without aerolysin exposure and solid bars the percentage of nonviable cells after 10−8 M aerolysin exposure. Values represent means ± standard errors of three independent experiments.
Molecular Therapy 2003 7, DOI: ( /S (03)00011-X)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Lentiviral-mediated gene transfer by the TEPW vector into PNH-phenotype CD34+ hematopoietic progenitor stem cells restores PIGA function. After 18 h of cytokine prestimulation, CD34+ cells were transduced twice at a m.o.i. of 200 ((A) mPB CD34+-derived cells) or 400 ((B) mPB CD34+- and (C) BM CD34+-derived cells). Five days after the last transduction, cells were stained with anti-CD34–PC5 (y axes) and anti-CD59–FITC (x axes) monoclonal antibodies and analyzed by flow cytometry. The percentage of cells in each quadrant is indicated.
Molecular Therapy 2003 7, DOI: ( /S (03)00011-X)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Restoration of PIGA function in genetically modified PNH-phenotype CD34+ hematopoietic progenitor stem cells is stable during hematopoietic differentiation. FACS analyses of mPB CD34+-derived cells transduced with the TEPW vector at a m.o.i. of 400 × 2, showing CD59 (x axes) expression in erythroid (A), myeloid (B), and megakaryocytic (C) cells (y axes), 12 days after the last transduction. The percentage of cells in each quadrant is indicated.
Molecular Therapy 2003 7, DOI: ( /S (03)00011-X)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Lentiviral vector TEPW restores PIGA function in PNH-phenotype T lymphocytes. BMMCs from a PNH patient were maintained for 7 days under T lymphoid expansion culture conditions (rhIL-2 and phytohemagglutinin A) with 0.3 × 10−9 M aerolysin. Then, cells were transduced with the TEPW vector at a m.o.i. of 200. Five days after the transduction, cells were stained with anti-CD3–PC5 and anti-CD59–FITC monoclonal antibodies and analyzed by flow cytometry.
Molecular Therapy 2003 7, DOI: ( /S (03)00011-X)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

9. FIG. 8
Lentiviral-mediated gene transfer into colony-forming progenitor cells is stable. Five days after the last transduction, CFC assays were performed with mPB CD34+ cells transduced with TEPW vector at a m.o.i. of 200 or 400 × 2. (A) Total colonies of BFU-E, CFU-E, and CFU-GM were counted after 14 days in culture. The values were calculated for 3 × 103 CD34+ cells plated in methylcellulose. (B) Representative FACS analyses of two individual erythroid colonies, stained with anti-glycophorin A–APC (y axes) and anti-CD59–FITC (x axes) monoclonal antibodies: PNH-phenotype colony on the left and normal phenotype on the right. (C and D) Transduction efficiency of erythroid colonies as detected by PCR analysis of individual colonies derived from mPB CD34+ cells transduced with the TEPW vector. (C) PCR analysis (representative examples) of randomly picked individual colonies. Abbreviations: NC, negative control; PC, positive control; NT, nontransduced colonies; PCR +, PCR-positive colony; PCR −, PCR-negative colony. (D) This table summarizes the results of immunostaining and PCR for each colony in the two experiments.
Molecular Therapy 2003 7, DOI: ( /S (03)00011-X)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

10. FIG. 9
Lentiviral-mediated gene transfer by TEPW vector allows long-term phenotypic correction of PNH-phenotype mPB CD34+ derived cells. CD59 expression was first evaluated after transduction (day 7) by flow cytometry after immunostaining with anti-CD59–FITC monoclonal antibody. Then, long-term liquid culture was initiated on a murine adherent stromal cell layer and maintained for 5 weeks. Nonadherent cells from weekly semi-depopulations of long-term culture of TEPW-transduced cells were analyzed for CD59 expression.
Molecular Therapy 2003 7, DOI: ( /S (03)00011-X)
Copyright © 2003 The American Society of Gene Therapy Terms and Conditions