1. Volume 15, Issue 1, Pages 76-85 (January 2007)
Lentiviral Vector Transduction of a Dominant-negative Rev Gene Into Human CD34+ Hematopoietic Progenitor Cells Potently Inhibits Human Immunodeficiency Virus-1 Replication 
Ingrid Bahner, Teiko Sumiyoshi, Mercy Kagoda, Robin Swartout, Denise Peterson, Karen Pepper, Fred Dorey, Jacob Reiser, Donald B Kohn 
Molecular Therapy 
Volume 15, Issue 1, Pages (January 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Schematic diagram of lentiviral vectors. All of the vectors are SIN vectors because of deletions in the U3 region of the long terminal repeat (ΔU3) and contain the packaging signal (ψ), the complete RRE to facilitate RNA export and the central polypurine tract (cPPT) to facilitate nuclear entry of the pre-integration complex. Transcription of the transgene is controlled internally by the U3 region of the MND retroviral vector (MND). The control vector CCL-ME contains the eGFP transgene, the vector CCL-MhuM contains the huM10 transgene, and the vector CCL-MhuMIE contains both the huM10 and the eGFP transgene linked via an internal ribosome entry site.
Molecular Therapy  , 76-85DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Titers of lentiviral vectors using different packaging constructs. (a) Comparison of the titers of the CCL-ME and CCL-MhuMIE vectors using different packaging constructs. Vector supernatants were analyzed for the number of GFP-transducing virions on HEK293 cells by serial dilution. The number of transduced HEK cells was determined by FACS and used to calculate TU/ml. The figure compares the mean GFP titers produced by CCL-ME (gray bars) to the mean GFP titers produced by CCL-MhuMIE (white bars) for each packaging plasmid. The SDs are indicated by the error bars (n=5). (b) Comparison of the level of GAG protein produced by the different packaging plasmid in the presence or absence of CCL-MhuMIE. Western blots using an anti-p24 GAG primary antibody were analyzed by densitometry. The top panel shows the amount of p24 GAG protein detected, with the blot re-developed with antibody to extracellular signal-regulated kinase2 as a loading control. The bar graph in the bottom panel compares the amount p24 GAG protein produced from the different packaging plasmid in the presence (white bars) or absence (gray bars) of the huM10 transgene. The amount of p24 GAG is graphed as the ratio of p24 GAG to the loading control protein extracellular signal-regulated kinase2. CEM cells infected with HIV-1 IIIB served as a positive control.
Molecular Therapy  , 76-85DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Analysis of Transduction of human CD34+ cells by lentiviral vectors. Human CD34+ cells were transduced with either the CCL-ME, CCL-MhuMIE, or CCL-MhuM lentiviral vectors and analyzed for the gene transfer efficiency, hematopoietic progenitor growth, and for CD4-expression. (a) Gene transfer efficiencies determined by FACS analysis of eGFP expression in cells after 7–10 days of bulk culture and by quantitative-PCR of CFU grown in methylcellulose for 14 days. The % of cells expressing eGFP (% eGFP+ cells) is shown with white bars. The percentage CFU positive for vector sequences (eGFP and/or huM10) were calculated from analysis of 50 colonies in each experiment. The mean values of all experiments were graphed (hatched bars). (b, c) Numbers and types of hematopoietic colonies grown from transduced CD34+ cells from (b) bone marrow and (c) cord blood. The SDs are indicated by the error bars (n=4). The white bars represent the number of granulocytic/monocytic colonies (CFU-GM), the hatched bars represent the number of burst forming colonies of the erythroid lineage (BFU-E), and the stippled bars represent the number of colonies that produced cells of the granulocytic, monocytic, erythroid, and megakaryocytic lineages (CFU-GEMM). (d) Transduced CD34+ cells were grown in bulk culture for 10 days and then analyzed by FACS for the total percentage with expression of eGFP (% eGFP+), for the total percentage expressing CD4 (% CD4+), and the percentage of the transduced cells that expressed CD4 (CD4+/eGFP+). CD34+ cells were mock transduced (black bar), transduced with CCL-ME (gray bar), or CCL-MhuMIE (white bar).
Molecular Therapy  , 76-85DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
HIV-1 challenge of cultures derived from transduced human CD34+ cells. (a) HIV-1 viral replication was measured in cultures derived from transduced human bone marrow CD34+ cells a, (c), or (b) cord blood cells. The amount of HIV-1 p24 GAG protein produced by the culture transduced with the control vector CCL-ME (filled circle, solid line) was compared to cultures transduced with the vector CCL-MhuM (open symbols, hatched line).
Molecular Therapy  , 76-85DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Schematic flow chart of assay to detect lentiviral vector mobilization. 
Molecular Therapy  , 76-85DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Assay to detect lentiviral vector mobilization. HIV-1 IIIB infection of (a) primary and (b) secondary CEM cells. Primary CEM cells were transduced with SIN vectors, CCL (circles), and SMPU (squares), and with a non-SIN vector, HR′ (triangle). Each type of vector either carried the eGFP transgene alone (solid symbol, solid line) or the eGFP and huM10 transgene together (open symbol, hatched line). Transduced and naïve CEM (filled diamond, solid line) were infected with HIV-1 IIIB and analyzed for the numbers of viral particles produced in each culture by measuring the amount of extracellular p24. HIV-1 GAG protein (20 ng) produced after 22 days by the primary cells were used to infect the naïve secondary cells and serially collected sample of cell culture supernatant were analyzed for the amount of extracellular p24 GAG protein. Quantitative-PCR analysis of the secondary indicator CEM cells infected with 20 ng of (c) HIV-1 p24 GAG or with 5 ml of the (d) primary culture medium. Cell pellets of the secondary indicator CEM cells were harvested on day 22 of the experiment and analyzed with by quantitative-PCR for the eGFP and the huM10 sequences. The numbers of vector copies per cell (eGFP: gray bar, huM10: white bar) were calculated by using a standard consisting of a CEM clone that contains one copy of SMPU-MhuMIE per cell.
Molecular Therapy  , 76-85DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions