title: Improved transfer of the leukocyte integrin CD18 subunit into hematopoietic cell lines by using retroviral vectors having a gibbon ape leukemia virus envelope.
abstract: Leukocyte adherence deficiency (LAD) is an inherited immunodeficiency disease caused by defects in the CD18 leukocyte integrin subunit. Transduction of CD18 into hematopoietic cells from children with LAD represents a potential therapy for this disorder. In an attempt to maximize transfer and expression of CD18, we evaluated retroviral vectors with and without the neomycin selectable marker, with a modified tRNA primer binding site designed to prevent inhibition of gene expression, and with two different viral envelope proteins produced by using the amphotropic retrovirus packaging cell line PA317 or the gibbon ape leukemia virus packaging cell line PG13. The vectors were tested using transducing K562/CD11b cells and LAD Epstein-Barr virus (EBV) B cells and measuring levels of cell-surface CD11/CD18 expression by fluorescence-activated cell sorter analysis. The best results were obtained with vectors made using PG13 packaging cells, for which about 25% of the K562 cells exposed once to the vectors expressed surface CD11b/CD18 and about 25% of the LAD EBV B cells exposed three times over a 3-day period to the vectors expressed surface CD11a/CD18. In contrast, transduction of cells under similar conditions with retroviral vectors produced using PA317 producer cells yielded less than 2% of the K562 cells and less than 4% of the LAD EBV B cells expressing the CD11/CD18 heterodimer on the cell surface. The presence or absence of the neomycin resistance gene or the modified tRNA primer had no effect on CD18 gene transfer rate or expression level. The increase in transduction with PG13 vectors correlated with Northern blotting and reverse transcription-polymerase chain reaction studies that indicated that both K562 cells and the LAD EBV B cells express transcripts for the gibbon ape leukemia virus receptor at higher levels than for the amphotropic virus receptor. These findings indicate that the transduction efficiency of retroviral packaging cell lines correlates with receptor gene expression in the target cells and that vectors made using PG13 cells may be efficacious for gene therapy for LAD and other diseases in which gene transfer to hematopoietic cells is required.
authors.affiliation: Medical Research Service, Seattle VA Medical Center, WA 98108, USA.
authors.forename: T R
authors.initials: TR
authors.lastname: Bauer
authors.forename: A D
authors.initials: AD
authors.lastname: Miller
authors.forename: D D
authors.initials: DD
authors.lastname: Hickstein
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D018845:CD11 Antigens / Q000032:analysis; D018821:CD18 Antigens / Q000096:biosynthesis / Q000235:genetics*; D002461:Cell Line, Transformed; D002648:Child; D003673:Defective Viruses / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D004854:Herpesvirus 4, Human; D006801:Humans; D019868:Kanamycin Kinase; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics / Q000502:physiology*; D018370:Leukocyte-Adhesion Deficiency Syndrome / Q000473:pathology*; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011991:Receptors, Virus / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics / Q000502:physiology; D014162:Transfection; D014759:Viral Envelope Proteins / Q000502:physiology*
chemical_list: D018845:CD11 Antigens; D018821:CD18 Antigens; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase