title: Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells.
abstract: Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus, 35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells, primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures, and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells, 5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly, the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated, indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders.
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency and University of British Columbia, Vancouver, BC, Canada.
authors.forename: Franck E
authors.initials: FE
authors.lastname: Nicolini
authors.forename: Suzan
authors.initials: S
authors.lastname: Imren
authors.forename: Il-Hoan
authors.initials: IH
authors.lastname: Oh
authors.forename: R Keith
authors.initials: RK
authors.lastname: Humphries
authors.forename: Philippe
authors.initials: P
authors.lastname: Leboulch
authors.forename: Mary E
authors.initials: ME
authors.lastname: Fabry
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Nagel
authors.forename: Connie J
authors.initials: CJ
authors.lastname: Eaves
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D002454:Cell Differentiation; D002478:Cells, Cultured; D004912:Erythrocytes / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D005312:Fetal Blood / Q000166:cytology*; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics*; D049452:Green Fluorescent Proteins; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D008099:Liver / Q000166:cytology / Q000196:embryology*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D019076:Transgenes / Q000235:genetics*
chemical_list: D018952:Antigens, CD34; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005914:Globins