title: Growth factors and stromal support generate very efficient retroviral transduction of peripheral blood CD34+ cells from Gaucher patients.
abstract: We have achieved high-efficiency gene transfer into nonmobilized peripheral blood (PB) CD34+ cells from patients with Gaucher's disease using a clinically acceptable retroviral supernatant transduction protocol. In our studies, bone marrow (BM) and PB CD34+ cells were transduced using a high titer (10(8) particles/mL) retroviral supernatant once a day for 4 consecutive days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), with or without an irradiated allogeneic BM stromal layer. The growth factors alone resulted in 29% +/- 10% gene transfer of PB CD34+ clonogenic cells in contrast with 71% +/- 17% gene transfer efficiency using stroma with the growth factors; a 2.5-fold increase. The increase in gene transfer efficiency was less prominent when BM CD34+ cells were used (40% +/- 16% without and 57% +/- 8% with stroma, a 1.5-fold increase). The overall transduction efficiency of both PB and BM CD34+ cells was lower when the cells were transduced over a stromal cell layer without added growth factors. The combination of IL-3, IL-6, and SCF with stroma transduced 75% of primitive long-term culture initiating cells (PB LTC-ICs) in comparison with 34% of LTC-ICs when IL-3, IL-6, and SCF were used without stromal support. Using this clinically acceptable supernatant/cytokines/stroma transduction protocol, correction of the glucocerebrosidase (GC) deficiency in the progeny cells of PBLTC-ICs from Gaucher's-disease patients has been accomplished. Efficient transduction of the PB CD34+ cells using this transduction protocol may allow repeated delivery of "GC-corrected" hematopoietic stem and progenitor cells to Gaucher's-disease patients.
authors.affiliation: Molecular and Medical Genetics Section, National Institutes of Neurological Disorders and Stroke (NINDS), Bethesda, MD 20892, USA.
authors.forename: L C
authors.initials: LC
authors.lastname: Xu
authors.forename: S
authors.initials: S
authors.lastname: Kluepfel-Stahl
authors.forename: M
authors.initials: M
authors.lastname: Blanco
authors.forename: R
authors.initials: R
authors.lastname: Schiffmann
authors.forename: C
authors.initials: C
authors.lastname: Dunbar
authors.forename: S
authors.initials: S
authors.lastname: Karlsson
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34; D001483:Base Sequence; D001853:Bone Marrow / Q000502:physiology*; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003238:Connective Tissue / Q000502:physiology*; D046508:Culture Techniques / Q000379:methods; D005776:Gaucher Disease / Q000097:blood* / Q000473:pathology; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification; D005962:Glucosylceramidase / Q000096:biosynthesis* / Q000235:genetics; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000473:pathology / Q000821:virology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008969:Molecular Sequence Data; D011533:Proviruses / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012190:Retroviridae / Q000235:genetics / Q000302:isolation & purification; D019089:Stem Cell Factor; D014162:Transfection
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D005962:Glucosylceramidase