title: High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency [correction of imunodeficiency] virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter.
abstract: Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (< or =24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high levels of transduction (over 80%) and gene expression in vivo after a single exposure to viral supernatant. These results demonstrate that lentiviral vectors are highly effective for gene transfer to human HSC, and that SFFV regulatory sequences can be successfully incorporated to enhance the long-term expression of a transgene in primary human hematopoietic cells in vivo.
authors.affiliation: Molecular Immunology Unit, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK.
authors.forename: Christophe
authors.initials: C
authors.lastname: Demaison
authors.forename: Kathryn
authors.initials: K
authors.lastname: Parsley
authors.forename: Gaby
authors.initials: G
authors.lastname: Brouns
authors.forename: Michaela
authors.initials: M
authors.lastname: Scherr
authors.forename: Karin
authors.initials: K
authors.lastname: Battmer
authors.forename: Christine
authors.initials: C
authors.lastname: Kinnon
authors.forename: Manuel
authors.initials: M
authors.lastname: Grez
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Thrasher
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D004742:Enhancer Elements, Genetic; D005312:Fetal Blood / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D016513:Mice, SCID; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011687:Purines / Q000737:chemistry; D013155:Spleen Focus-Forming Viruses / Q000235:genetics; D020079:Terminal Repeat Sequences; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D016207:Cytokines; C028391:G protein, vesicular stomatitis virus; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011687:Purines; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins