title: Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector.
abstract: Human hematopoietic cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous inherited or acquired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with human CD34(+) cells transduced by an HIV-1-based self-inactivating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34(+) cells were transduced (up to 76%) at a low multiplicity of infection (MOI of 5) in the absence of cytokine prestimulation. Introduction of transduced hCD34(+) cells into irradiated recipients resulted in multilineage engraftment and stable transgene expression for 18 weeks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45(+) cells and up to 63% hCD45(+)/EGFP(+) cells. Analysis of extramedullar splenic reconstitution showed up to 13% hCD45(+) cells and up to 41% hCD45(+)/EGFP(+) cells. Analysis of human progenitor cells isolated from bone marrow of recipient animals showed equivalent percentages of EGFP(+) colony-forming cells (CFCs) by fluorescence microscopy and by PCR analysis of provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hematopoietic stem cell gene transfer and provide strong support for their future clinical evaluation.
authors.affiliation: Division of Infectious Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
authors.forename: J
authors.initials: J
authors.lastname: Gatlin
authors.forename: A
authors.initials: A
authors.lastname: Padgett
authors.forename: M W
authors.initials: MW
authors.lastname: Melkus
authors.forename: P F
authors.initials: PF
authors.lastname: Kelly
authors.forename: J V
authors.initials: JV
authors.lastname: Garcia
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis*; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics / Q000276:immunology; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage*; D006085:Graft Survival / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection; D019076:Transgenes / Q000276:immunology; D014407:Tumor Cells, Cultured; D014775:Virus Activation / Q000235:genetics*
chemical_list: D018952:Antigens, CD34