title: Critical factors influencing stable transduction of human CD34(+) cells with HIV-1-derived lentiviral vectors.
abstract: Lentiviral vectors have been proposed as a more efficient alternative to Moloney murine leukemia virus-based retroviral vectors for transduction of human hematopoietic progenitors and stem cells. These studies were designed to evaluate the conditions that influence transduction frequency of CD34(+) progenitors, with the goal of optimizing efficiency of stable gene transfer with lentiviral vectors. CD34(+) human cord blood cells and 293 cells were transduced with a human immunodeficiency virus (HIV)-1 derived lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein and carrying an internal human cytomegalovirus promoter driving enhanced green fluorescent protein (eGFP) expression. Using fluorescence-activated cell sorting analysis of eGFP, we observed pseudotransduction beginning at the time of vector addition and lasting up to 24 h in CD34(+) cells and up to 72 h in 293 cells. Integrase-defective lentiviral vector caused transient eGFP expression for up to 10 days in CD34(+) cells and for up to 14 days in 293 cells. Protamine sulfate conferred no increase in transduction efficiency of CD34(+) cells on fibronectin-coated plates. Transduction frequency was related directly to vector concentration and not to multiplicity of infection across the ranges tested. First- and second-generation lentiviral vectors transduced CD34(+) cells equally, demonstrating a lack of dependence on HIV-1 accessory proteins. These findings will be useful for the optimal utilization of this new class of vectors for transduction of human hematopoietic stem cells.
authors.affiliation: Department of Molecular Microbiology and Immunology, University of Southern California Medical School, Los Angeles, CA 90033, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Haas
authors.forename: S S
authors.initials: SS
authors.lastname: Case
authors.forename: G M
authors.initials: GM
authors.lastname: Crooks
authors.forename: D B
authors.initials: DB
authors.lastname: Kohn
mesh_terms: D018952:Antigens, CD34 / Q000235:genetics*; D002460:Cell Line; D002469:Cell Separation; D003587:Cytomegalovirus / Q000235:genetics; D005312:Fetal Blood / Q000166:cytology; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006494:Heparin Antagonists / Q000494:pharmacology; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011479:Protamines / Q000494:pharmacology; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D005353:Fibronectins; C028391:G protein, vesicular stomatitis virus; D006494:Heparin Antagonists; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011479:Protamines; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins