title: High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.
abstract: Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.
authors.affiliation: Clinical Gene Therapy Branch, National Center for Human Genome Research, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Bunnell
authors.forename: L M
authors.initials: LM
authors.lastname: Muul
authors.forename: R E
authors.initials: RE
authors.lastname: Donahue
authors.forename: R M
authors.initials: RM
authors.lastname: Blaese
authors.forename: R A
authors.initials: RA
authors.lastname: Morgan
mesh_terms: D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015496:CD4-Positive T-Lymphocytes / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D017931:DNA Primers; D015342:DNA Probes; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D019868:Kanamycin Kinase; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012190:Retroviridae; D013045:Species Specificity; D014161:Transduction, Genetic
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase