title: Optimized large-scale production of high titer lentivirus vector pseudotypes.
abstract: The goal of the present study was to develop an efficient transient transfection method for large-scale production of high titer lentivirus vector stocks of eight different pseudotypes. The envelope genes used for this purpose were those from VSV-G, Mokola, Rabies, MLV-Ampho, MLV-10A1, LCMV-WE, and LCMV-Arm53b. All envelopes were cloned into phCMV, which yielded lentivirus vector titers one, two, or three orders of magnitude higher than the original plasmids for the Rabies, MLV-10A1, and MLV-Ampho envelopes, respectively. When these newly constructed envelope expression plasmids were used for packaging, treatment with sodium butyrate resulted in almost five-fold increase in titers for some of the pseudotypes, had no effect for others (VSV-G and Rabies), and negatively impacted titers for the LCMV-derived pseudotypes. Production of vectors in serum-free media yielded titers only slightly lower than those obtained in the presence of serum. The efficiency of concentrating vector supernatants by ultracentrifugation or ultrafiltration was compared, with higher recovery efficiencies for the latter method, but the highest titers for most pseudotypes were obtained by ultracentrifugation. The best conditions for each individual pseudotype yielded lentivirus vector stocks with titers above 1 x 10(9) tu/mL for most pseudotypes, and higher than 1 x 10(10) tu/mL for VSV-G.
authors.affiliation: Department of Surgery, The Children's Hospital of Philadelphia, Abramson Research Center, 3615 Civic Center Blvd., Philadelphia, PA 19104, USA. msesteves@partners.org
authors.forename: Miguel
authors.initials: M
authors.lastname: Sena-Esteves
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Tebbets
authors.forename: Sabine
authors.initials: S
authors.lastname: Steffens
authors.forename: Timothy
authors.initials: T
authors.lastname: Crombleholme
authors.forename: Alan W
authors.initials: AW
authors.lastname: Flake
mesh_terms: D002460:Cell Line; D029043:Ebolavirus / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology; D016086:Lentivirus / Q000235:genetics / Q000502:physiology*; D008217:Lymphocytic choriomeningitis virus / Q000235:genetics; D010957:Plasmids; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics; D014760:Viral Fusion Proteins / Q000235:genetics
chemical_list: D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins