1. Volume 15, Issue 8, Pages 1487-1494 (August 2007)
Characteristics of Lentiviral Vectors Harboring the Proximal Promoter of the vav Proto- oncogene: A Weak and Efficient Promoter for Gene Therapy 
Elena Almarza, Paula Río, Nestor W Meza, Montserrat Aldea, Xabier Agirre, Guillermo Guenechea, José C Segovia, Juan A Bueren 
Molecular Therapy 
Volume 15, Issue 8, Pages (August 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Comparative analysis of transgene expression conferred by lentiviral vectors (LVs) harboring the vav and the cytomegalovirus (CMV) promoters in hematopoietic and non-hematopoietic cell lines. (a) Comparative analyses of expression mediated by vav-LVs and CMV-LVs in Epstein-Barr virus–transformed B-lymphoblast cell lines (LCLs) and HeLa cells. The transduction efficiency and mean fluorescence intensity (MFI) were determined 7 days after transduction. (b) Analysis of MFI values in hematopoietic cell lines transduced with CMV-LVs (dark bars) and vav-LVs (white bars) and cultured for up to 50 days in vitro. Each bar represents data corresponding to 3, 10, 20, 30, 40, and 50 days in culture. The vector copy number in each cell line is indicated in the bottom of the figure. *Mean values of MFI between samples transduced with vav-LVs and CMV-LVs were significantly different at P < GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Analysis of transgene expression conferred by vav-derived lentiviral vectors (LVs) compared with other LVs frequently used in gene therapy. (a) Average mean fluorescence intensity (MFI) values corresponding to 293T, NIH-3T3, HL60, and K562 cell lines transduced with four LVs harboring the cytomegalovirus (CMV) (black bars), spleen focus-forming virus (SFFV) (striped bars), human phosphoglycerate kinase (hPGK) (dotted bars), and vav (white bars) promoters. Bars represent mean ± SE of MFI values obtained at 10, 30, and 50 days of culture. The vector copy number obtained in samples analyzed at day 50 of culture is indicated in the bottom of the figure. *Differences with respect to samples transduced with vav-LVs were significant at P < (b) Box-and-whisker plot with the distribution of the MFI values deduced from the analysis of the four cell lines shown in panel a analyzed at 10, 30, and 50 days of culture. Mean ± SD (+) and median and range (central line in the boxes) values are shown. (c) Analysis of the transgene transcription index (copy number of expressed RNA/integrated DNA copy) in cells transduced with the four LVs shown in panel a, after 50 days in culture.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Analysis of the expression stability mediated by vav-lentiviral vectors (vav-LVs) in mouse hematopoietic cells transplanted into irradiated recipients. Lineage-negative bone marrow cells (Ly5.2) transduced with high (2 transduction units (TU)/cell, gray symbols) and low (0.2 TU/cell, white symbols) multiplicities of infection (MOIs) of vav-cGFP-LVs were transplanted into myeloablated Ly5.1 mice. Peripheral blood samples from recipient mice were periodically analyzed to determine the level of engraftment and green fluorescent protein (GFP) expression in primary and secondary recipients. In the upper panel, percentages of donor Ly5.2 cells are shown. In the middle panel percentages of GFP+ cells inside the window of donor Ly5.2 cells are shown. Columns in the lower panel represent the mean fluorescence intensity (MFI) values of transduced cells inside windows of each lineage population: GR/MAC (smooth bars), B220 (striped bars), and CD3 (dotted bars). cGFP, cop GFP.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Analysis of the expression stability mediated by vav-lentiviral vectors in human hematopoietic cells transplanted into irradiated non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. CD34+ cord blood cells transduced with high (3 transduction units (TU)/cell, gray symbols) and low (0.3 TU/cell, white symbols) multiplicities of infection (MOIs) were transplanted into NOD/SCID mice. Bone marrow aspirates from transplanted mice were periodically analyzed to determine the level of human hematopoietic engraftment and the expression of the green fluorescent protein (GFP) marker gene. In the upper panel, percentages of donor human CD45+ cells are shown. In the middle panel, percentages of GFP+ cells inside the window of donor cells are indicated. Columns in the lower panel represent mean fluorescence intensity (MFI) values of transduced cells inside windows of each human lineage population: CD34+ (smooth bars), CD33+ (striped bars), and CD19+ (dotted bars).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Analysis of in vivo methylation of lentiviral vectors (LVs) harboring the vav promoter. Non-obese diabetic/severe combined immunodeficient mice were transplanted with transduced CD34+ cells, and bone marrow (BM) was harvested 130 days after transplantation. The level of methylation of the vav-LV promoter was then determined in DNA extracted from the BM of the animals. Each box represents a methylated (filled boxes) or non-methylated (open boxes) CpG dinucleotide. A, B, and D correspond to samples transduced at a multiplicity of infection (MOI) of 3 transducing units (TU)/cell, whereas F and J correspond to samples transduced at an MOI of 0.3 TU/cell. cGFP, cop green fluorescent protein.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Phenotypic correction of human Fanconi anemia cells (FA-A) transduced with vav-lentiviral vectors (vav-LVs) expressing FANCA. (a) Mytomycin C (MMC) survival curves of lymphoblast cell lines (LCLs) from healthy donor (HD) or FA-A patients transduced with enhanced green fluorescent protein (eGFP)–expressing or FANCA-expressing LVs. ▵: HD + SFFV-eGFP; ○: FA-A + SFFV-eGFP; ▪: FA-A + SFFV-FANCAieGFP; □: vav-FANCA. (b) Western blot analysis showing FANCA and FANCD2 expression in transduced HD or FA-A LCLs exposed to 33 nmol/l MMC (see panel a). The average LV copy number corresponding to cells transduced with spleen focus-forming virus (SFFV)- and vav-FANCA LVs were, respectively, 0.49 and 0.54 copies/cell. (c) Nuclear FANCD2 foci formation in LCLs from samples corresponding to panel b.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Schematic representation of the lentiviral vectors (LVs) used in the study. The figure shows the integrated proviral form of the constructs. (a) vav-cGFP-LV: 8,450-base-pair (bp) vector where cop green fluorescent protein (cGFP) is under the vav promoter; (b) CMV-cGFP-LV: 7,543-bp vector where cGFP is under the cytomegalovirus (CMV) promoter; (c) CMV-eGFP-LV: 7,418-bp vector where enhanced GFP (eGFP) is under the CMV promoter; (d) PGK-eGFP-LV: 7,377-bp vector where eGFP is under the human phosphoglycerate kinase (PGK) promoter; (e) SFFV-eGFP-LV: 7,987-bp vector where eGFP is under the spleen focus-forming virus (SFFV) promoter; (f) vav-FANCA-LV: 9,109-bp vector where FANCA is under the vav promoter; (g) SFFV-FANCAieGFP-LV: 12,438-bp vector where FANCA is under the SFFV promoter and eGFP is placed after an internal ribosome entry site sequence. Wpre, woodchuck hepatitis post-transcriptional regulatory element.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions