title: Transplantation and gene transfer of the human glucocerebrosidase gene into immunoselected primate CD34+Thy-1+ cells.
abstract: In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, Rockville, MD 20850, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Donahue
authors.forename: E R
authors.initials: ER
authors.lastname: Byrne
authors.forename: T E
authors.initials: TE
authors.lastname: Thomas
authors.forename: M R
authors.initials: MR
authors.lastname: Kirby
authors.forename: B A
authors.initials: BA
authors.lastname: Agricola
authors.forename: S E
authors.initials: SE
authors.lastname: Sellers
authors.forename: G
authors.initials: G
authors.lastname: Gaudernack
authors.forename: S
authors.initials: S
authors.lastname: Karisson
authors.forename: P M
authors.initials: PM
authors.lastname: Lansdorp
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells; D018801:CD2 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D004274:DNA, Recombinant / Q000032:analysis; D003673:Defective Viruses / Q000235:genetics / Q000302:isolation & purification; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification; D005962:Glucosylceramidase / Q000235:genetics*; D006085:Graft Survival; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D018189:Immunomagnetic Separation; D016131:Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology / Q000821:virology; D008253:Macaca mulatta; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D018800:Thy-1 Antigens / Q000032:analysis*; D014182:Transplantation, Autologous
chemical_list: D018952:Antigens, CD34; D018801:CD2 Antigens; D016827:CD8 Antigens; D004274:DNA, Recombinant; D018800:Thy-1 Antigens; D005962:Glucosylceramidase