title: High efficiency gene transfer to human hematopoietic SCID-repopulating cells under serum-free conditions.
abstract: Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34(+) cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45(+) cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.
authors.affiliation: Molecular Virology Lab, Georg-Speyer-Haus, Frankfurt, Germany.
authors.forename: A J
authors.initials: AJ
authors.lastname: Schilz
authors.forename: G
authors.initials: G
authors.lastname: Brouns
authors.forename: H
authors.initials: H
authors.lastname: Knöss
authors.forename: O G
authors.initials: OG
authors.lastname: Ottmann
authors.forename: D
authors.initials: D
authors.lastname: Hoelzer
authors.forename: A A
authors.initials: AA
authors.lastname: Fauser
authors.forename: A J
authors.initials: AJ
authors.lastname: Thrasher
authors.forename: M
authors.initials: M
authors.lastname: Grez
mesh_terms: D000818:Animals; D016026:Bone Marrow Transplantation; D002478:Cells, Cultured / Q000637:transplantation; D003114:Colony-Forming Units Assay; D016895:Culture Media, Serum-Free; D005312:Fetal Blood / Q000166:cytology; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
chemical_list: D016895:Culture Media, Serum-Free; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins