title: Construction of stable packaging cell lines for clinical lentiviral vector production.
abstract: Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 10(6) transducing units/ml can be harvested from the final producer clones, which can be increased to 10(8) TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.
authors.affiliation: 1] Division of Infection and Immunity, University College London, London, UK [2] National Institute for Biological Standards and Control, South Mimms, UK.
authors.forename: Khaled S
authors.initials: KS
authors.lastname: Sanber
authors.affiliation: Division of Infection and Immunity, University College London, London, UK.
authors.forename: Sean B
authors.initials: SB
authors.lastname: Knight
authors.affiliation: Division of Infection and Immunity, University College London, London, UK.
authors.forename: Sam L
authors.initials: SL
authors.lastname: Stephen
authors.affiliation: Division of Infection and Immunity, University College London, London, UK.
authors.forename: Ranbir
authors.initials: R
authors.lastname: Bailey
authors.affiliation: Division of Infection and Immunity, University College London, London, UK.
authors.forename: David
authors.initials: D
authors.lastname: Escors
authors.affiliation: DNA2.0, Menlo Park, CA94025, USA.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Minshull
authors.affiliation: Institute of Child Health, University College London, London, UK.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Santilli
authors.affiliation: Institute of Child Health, University College London, London, UK.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Thrasher
authors.affiliation: 1] Division of Infection and Immunity, University College London, London, UK [2] National Institute for Biological Standards and Control, South Mimms, UK.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Collins
authors.affiliation: Division of Infection and Immunity, University College London, London, UK.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takeuchi
mesh_terms: D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism; D054302:pol Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D011498:Protein Precursors; D014759:Viral Envelope Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D054302:pol Gene Products, Human Immunodeficiency Virus; C091950:pr160 gag-pol precursor protein, Human immunodeficiency virus 1; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1