title: Efficient lentiviral transduction and transgene expression in primary human B cells.
abstract: Primary human B cells are an attractive target for gene-therapeutic applications, but have been found to be relatively resistant toward transduction with lentiviral vectors (LVVs), even though a number of different envelope pseudotypes were tested. Moreover, low transgene expression in primary human B cells has impeded the use of LVVs for this target cell. We investigated the transduction potential of gibbon-ape leukemia virus (GALV) Env-pseudotyped LVVs for primary human B cells. By establishing optimized transduction kinetics and multiplicities of infection, we were able to regularly obtain transduction efficiencies of more than 50% in CD40L-activated B cells. Noteworthy, with the use of GALV-pseudotyped LVVs we could achieve a more than 10-fold higher yield of transduced activated B cells in direct comparison with LVVs pseudotyped with measles virus glycoproteins. Phenotyping of transduced primary B cells revealed a majority of memory B cells, a long-lived phenotype, presumed to be well suited for enduring therapeutic interventions. Finally, by combining the enhancer (Eμ) and the matrix/scaffold-attachment regions (MARs) of the human immunoglobulin heavy chain with the promoter of spleen focus-forming virus (SFFV) we aimed at generating a novel LVV particularly suitable for B cell transgenesis. We show that the optimized vector facilitated significantly higher transgene expression in various B cell lines and, more importantly, primary human B cells (mean factor of three). In summary, we have established a novel protocol for the efficient lentiviral transduction of primary human B cells and have improved transgene expression in B cells by a specific vector modification.
authors.affiliation: Research Department of Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Mock
authors.forename: Regine
authors.initials: R
authors.lastname: Thiele
authors.forename: Almut
authors.initials: A
authors.lastname: Uhde
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
authors.forename: Stefan
authors.initials: S
authors.lastname: Horn
mesh_terms: D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D019013:CD40 Antigens / Q000378:metabolism; D002478:Cells, Cultured; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics*; D045170:Matrix Attachment Regions / Q000235:genetics; D011401:Promoter Regions, Genetic; D013155:Spleen Focus-Forming Viruses / Q000235:genetics; D014161:Transduction, Genetic
chemical_list: D019013:CD40 Antigens; D007143:Immunoglobulin Heavy Chains