title: Rapid generation of stable cell lines expressing high levels of erythropoietin, factor VIII, and an antihuman CD20 antibody using lentiviral vectors.
abstract: Lentiviral vectors (LVs) are widely recognized as the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Using erythropoietin (EPO), recombinant factor VIII (fVIII), and an anti-CD20 antibody as model proteins, we demonstrate advantages of LV-based gene delivery to achieve high production levels by transduced cells. Highly productive cell clones were able to incorporate up to 100 vector copies per cellular genome, without selection or gene amplification, and were isolated without extensive screening of a large number of clones. The LV transgenes were shown to be distributed throughout the genome, as visualized by fluorescent in situ hybridization. High-expressing clones producing 100-200 pg/cell/day of EPO were isolated and characterized. EPO production was demonstrated for at least 5½ months of continuous culture without selection, during which all the clones displayed high levels of glycosylation despite production levels at 10-20 g/liter. To demonstrate the utility of LV technology for multiple classes of proteins, cell lines producing fVIII and an anti-CD20 antibody were also developed. Cell clones demonstrating high levels of fVIII (100 clot units/ml and anti-CD20 antibody as high as 40-100 pg/cell/day) were isolated and characterized. LV-transduced cells and plasmid-transfected cells were compared for protein production per transgene copy. LV-transduced cells produced significantly higher levels of protein per copy of transgene than plasmid-transfected cells did. This study demonstrates the utility of LV technology for rapid generation of highly productive and stable cell lines over conventional plasmid transfection methods, significantly decreasing the time, cost, and risk of the manufacture of proteins and other complex biological molecules.
authors.affiliation: Lentigen Corporation , Gaithersburg, MD 20878, USA.
authors.forename: Lajos
authors.initials: L
authors.lastname: Baranyi
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Doering
authors.forename: Gabriella
authors.initials: G
authors.lastname: Denning
authors.forename: Richard E
authors.initials: RE
authors.lastname: Gautney
authors.forename: Kyle T
authors.initials: KT
authors.lastname: Harris
authors.forename: H Trent
authors.initials: HT
authors.lastname: Spencer
authors.forename: Andre
authors.initials: A
authors.lastname: Roy
authors.forename: Hatem
authors.initials: H
authors.lastname: Zayed
authors.forename: Boro
authors.initials: B
authors.lastname: Dropulic
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000235:genetics* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000235:genetics* / Q000378:metabolism; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D000069283:Rituximab; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; C554270:EPO protein, human; D004921:Erythropoietin; D000069283:Rituximab; D005169:Factor VIII