title: Transduction of retrovirus-mediated NeoR gene into CD34+ cells purified from granulocyte colony-stimulating factor (G-CSF)-mobilized infant and cord blood.
abstract: To help establish an effective gene therapy protocol for patients with congenital metabolic diseases, we evaluated retrovirus-mediated transduction and long-term (LT) expression of the NeoR gene in cryopreserved and thawed CD34+ cells purified from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) of infant and cord blood (CB). The results were compared with those in bone marrow (BM) CD34+ cells. The final purity of the CD34+-enriched fraction from PB, CB, and BM, based on FACS analysis, was 88 +/- 14%, 73 +/- 13%, and 68 +/- 19% (mean +/- SEM), respectively. Cells were then cultured for 96 hours with supernatant containing the vector in the presence of interleukin (IL)-3, IL-6, and stem cell factor (SCF). The average efficiency of gene transfer into mobilized PB (n = 5) or CB CD34+ cells (n = 6) was significantly higher than that into BM CD34+ cells, as measured by G418-resistant colony-forming units for granulocyte/macrophage (CFU-GM; 59% or 58% vs. 39%; p < 0.05) and PCR-positive CFU-GM (83% or 79% vs. 53%; p < 0.05). When the evaluation was made in an LT culture system with irradiated allogeneic marrow stroma, these efficiencies were, respectively, 74% or 61% vs. 34% (p < 0.005 or < 0.02) for G418-resistant CFU-GM at week 5 of long-term culture, and 88% or 83% vs. 63% (p < 0.05) for PCR-positive CFU-GM. Fluorometric examination was performed for cell-cycle analysis before and after culture, and the results showed that the fraction of cycling cells was largest in freshly prepared BM (18%), whereas only a small portion of PB (4.6%) and CB (2%) was cycling. However, this value was 17% in BM, 22% in PB, and 13% in CB after culture. These results suggest that mobilized PB from small children and CB cells are suitable and realistic targets for clinical gene therapy and that tandem transduction procedures can be achieved by combining CB and PB.
authors.affiliation: Department of Pediatrics, University of Tokushima, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Abe
authors.forename: M
authors.initials: M
authors.lastname: Ito
authors.forename: Y
authors.initials: Y
authors.lastname: Okamoto
authors.forename: H J
authors.initials: HJ
authors.lastname: Kim
authors.forename: Y
authors.initials: Y
authors.lastname: Takaue
authors.forename: K
authors.initials: K
authors.lastname: Yasutomo
authors.forename: A
authors.initials: A
authors.lastname: Makimoto
authors.forename: T
authors.initials: T
authors.lastname: Yamaue
authors.forename: Y
authors.initials: Y
authors.lastname: Kawano
authors.forename: T
authors.initials: T
authors.lastname: Watanabe
authors.forename: T
authors.initials: T
authors.lastname: Shimada
authors.forename: Y
authors.initials: Y
authors.lastname: Kuroda
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis*; D001853:Bone Marrow; D002453:Cell Cycle; D002469:Cell Separation; D002675:Child, Preschool; D004351:Drug Resistance / Q000235:genetics*; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006098:Granulocytes / Q000378:metabolism; D006801:Humans; D007223:Infant; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism; D009355:Neomycin; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics; D019089:Stem Cell Factor / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; D007377:Interleukin-3; D015850:Interleukin-6; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D009355:Neomycin