title: Development of inducible EIAV-based lentiviral vector packaging and producer cell lines.
abstract: Large-scale production of gene therapeutics comprising equine infectious anaemia virus (EIAV) -based lentiviral vectors (LVs) would benefit from the development of producer cell lines enabling the generation of larger quantities of vector than achievable by transient systems. Such cell lines would contain three vector components (Gag/Pol, VSV-G envelope and genome expression constructs). As the vesicular stomatitis virus (VSV-G) envelope protein is cytotoxic, its expression must be regulated. It is also desirable to regulate Gag/Pol expression to minimise metabolic burden on the cell. The Tet repressor (TetR) system was selected to regulate expression of VSV-G and Gag/Pol, necessitating the introduction of a fourth construct, encoding TetR, into the cell line. We have generated an inducible packaging cell line that shows tight control of the packaging components, and high-titre vector production on transient transfection of the EIAV genome. The cell line is stable for at least 7 weeks in the absence of selective pressure. To verify that this packaging cell line can support the generation of producer cell lines it was transfected stably with an EIAV genome cassette encoding ProSavin; a gene therapeutic for Parkinson's disease. Producer cell lines were generated, which on induction, yielded ProSavin with titres comparable to the transient system.
authors.affiliation: Oxford BioMedica (UK) Ltd, Medawar Centre, Robert Robinson Avenue, The Oxford Science Park, Oxford, UK. H.Stewart@oxfordbiomedica.co.uk
authors.forename: H J
authors.initials: HJ
authors.lastname: Stewart
authors.forename: M A
authors.initials: MA
authors.lastname: Leroux-Carlucci
authors.forename: C J M
authors.initials: CJ
authors.lastname: Sion
authors.forename: K A
authors.initials: KA
authors.lastname: Mitrophanous
authors.forename: P A
authors.initials: PA
authors.lastname: Radcliffe
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002999:Clone Cells; D004318:Doxycycline / Q000494:pharmacology; D015685:Fusion Proteins, gag-pol / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D042822:Genomic Instability / Q000235:genetics; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D016366:Open Reading Frames; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase; D012097:Repressor Proteins / Q000235:genetics* / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D014759:Viral Envelope Proteins / Q000235:genetics*; D019065:Virus Assembly / Q000235:genetics; D014779:Virus Replication
chemical_list: D000900:Anti-Bacterial Agents; D015685:Fusion Proteins, gag-pol; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D012097:Repressor Proteins; D014759:Viral Envelope Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D012194:RNA-Directed DNA Polymerase; D004318:Doxycycline