Efficient lentiviral transduction and transgene expression in primary human B cells.
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Efficient lentiviral transduction and transgene expression in primary human B cells. / Mock, Ulrike; Thiele, Regine; Uhde, Almut; Fehse, Boris; Horn, Stefan.
in: HUM GENE THER METHOD, Jahrgang 23, Nr. 6, 6, 2012, S. 408-415.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Efficient lentiviral transduction and transgene expression in primary human B cells.
AU - Mock, Ulrike
AU - Thiele, Regine
AU - Uhde, Almut
AU - Fehse, Boris
AU - Horn, Stefan
PY - 2012
Y1 - 2012
N2 - 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 (E?) 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.
AB - 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 (E?) 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.
KW - Humans
KW - Cells, Cultured
KW - Promoter Regions, Genetic
KW - Transduction, Genetic
KW - HEK293 Cells
KW - Antigens, CD40/metabolism
KW - B-Lymphocytes/cytology/immunology/metabolism
KW - Genetic Vectors/genetics/metabolism
KW - Immunoglobulin Heavy Chains/genetics
KW - Leukemia Virus, Gibbon Ape/genetics
KW - Matrix Attachment Regions/genetics
KW - Spleen Focus-Forming Viruses/genetics
KW - Humans
KW - Cells, Cultured
KW - Promoter Regions, Genetic
KW - Transduction, Genetic
KW - HEK293 Cells
KW - Antigens, CD40/metabolism
KW - B-Lymphocytes/cytology/immunology/metabolism
KW - Genetic Vectors/genetics/metabolism
KW - Immunoglobulin Heavy Chains/genetics
KW - Leukemia Virus, Gibbon Ape/genetics
KW - Matrix Attachment Regions/genetics
KW - Spleen Focus-Forming Viruses/genetics
M3 - SCORING: Journal article
VL - 23
SP - 408
EP - 415
JO - HUM GENE THER METHOD
JF - HUM GENE THER METHOD
SN - 1946-6536
IS - 6
M1 - 6
ER -
