title: Ex vivo selection for oncoretrovirally transduced green fluorescent protein-expressing CD34-enriched cells increases short-term engraftment of transduced cells in baboons.
abstract: In an effort to improve hematopoietic stem cell gene transfer rates using gibbon ape leukemia virus (GALV)-pseudotype retroviral vectors in baboons, we have studied preselection of transduced green fluorescent protein (GFP)-expressing CD34-enriched marrow cells. Three animals were transplanted with GFP-selected (GS) CD34-enriched marrow. To ensure engraftment, preselected GFP-positive cells were infused together with unselected neo-transduced cells. After transduction on fibronectin, cells were cultured for an additional 2 days to allow for expression of GFP. GFP-expressing cells were enriched by fluorescence-activated cell sorting and infused together with cells from the unselected fractions after myeloablative irradiation of the recipient. Three other animals were transplanted with GFP-transduced CD34-enriched cells without prior GFP selection (GU). At 4 weeks after transplant, the percentage of GFP-expressing white blood cells was significantly higher in the GS group (6.6%) than in the GU group (1.3%) (p < 0.002). The higher gene transfer levels in the animals transplanted with GS cells gradually declined, and by day 100 after transplant, gene transfer levels were similar in both groups. PCR analysis performed on genomic DNA isolated from peripheral blood cells demonstrated that the decline in GFP-positive cells was due to the loss of gene-marked cells and not due to loss of expression. These results show that transplantation of CD34-positive marrow cells selected for GFP-positive cells after transduction provides high levels of transduced granulocytes in the short term. However, using this experimental design with concomitant infusion of unselected cells and the use of oncoretroviral vectors, preenrichment of vector-expressing, transduced CD34-enriched cells does not improve long-term persistence and expression.
authors.affiliation: Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. hkiem@fhcrc.org
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Kiem
authors.forename: John E J
authors.initials: JE
authors.lastname: Rasko
authors.forename: Julia
authors.initials: J
authors.lastname: Morris
authors.forename: Laura
authors.initials: L
authors.lastname: Peterson
authors.forename: Peter
authors.initials: P
authors.lastname: Kurre
authors.forename: Robert G
authors.initials: RG
authors.lastname: Andrews
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics*; D001854:Bone Marrow Cells / Q000378:metabolism*; D016026:Bone Marrow Transplantation; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007700:Kinetics; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000097:blood / Q000235:genetics*; D051379:Mice; D008856:Microscopy, Fluorescence; D010215:Papio; D014161:Transduction, Genetic
chemical_list: D018952:Antigens, CD34; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins