Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells.
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.
Nicolini
Franck E
FE
Terry Fox Laboratory, British Columbia Cancer Agency and University of British Columbia, Vancouver, BC, Canada.
Imren
Suzan
S
Oh
Il-Hoan
IH
Humphries
R Keith
RK
Leboulch
Philippe
P
Fabry
Mary E
ME
Nagel
Ronald L
RL
Eaves
Connie J
CJ
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*
D018952:Antigens, CD34; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005914:Globins