Adenoviral vector design for high-level transgene expression in primitive human hematopoietic progenitors.
Adenoviral vector-mediated transient gene expression can provide new possibilities for ex vivo manipulation of quiescent hematopoietic stem cells (HSC). In order to define a suitable expression cassette for high levels of transgene expression in HSCs, we have studied the level of transgene expression in human CD34+CD38- cells using adenoviral vectors with various gene expression cassettes encoding the enhanced green fluorescence protein (EGFP) gene. CD34+ hematopoietic cells were cultured in serum-free medium with megakaryocyte growth and development factor (MGDF) alone for supporting the survival of primitive progenitors or with MGDF, c-kit ligand (KL) and flt3 ligand (FL) for inducing proliferation of primitive progenitors. With all the vectors tested, higher percentages of EGFP expressing cells were found in CD34+CD38- cells than those in CD34+CD38high cells from all donors tested. The phosphoglycerate kinase (PGK)-1 promoter was found to allow higher levels of EGFP expression than the human cytomegalovirus (HCMV) promoter in CD34+CD38- cells. Replacing the SV40 polyadenylation signal with the human beta-globin gene IVS2 and polyadenylation signal in the expression cassette (Ad5xPGK-EGFP-beta-globin) enhanced the level of EGFP expression markedly further. These results provide a guideline for the development of adenoviral vectors for gene expression in human primitive hematopoietic progenitor cells. Gene Therapy (2000) 7, 2132-2138.
Department of Molecular Medicine and Gene Therapy, Wallenberg Neuroscience Center, Lund University, Sölvegatan 17, 223 62 Lund, Sweden.
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D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000256:Adenoviridae / Q000235:genetics*; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D002478:Cells, Cultured; D015870:Gene Expression; D005809:Genes, Regulator; D005818:Genetic Engineering; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins; D009244:NAD+ Nucleosidase; D010735:Phosphoglycerate Kinase / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D013997:Time Factors; D014161:Transduction, Genetic
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D049452:Green Fluorescent Proteins; D005914:Globins; D010735:Phosphoglycerate Kinase; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1