title: Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer.
abstract: For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.
authors.affiliation: Chiron Corporation, Center for Gene Therapy, San Diego, California 92121, USA.
authors.forename: P L
authors.initials: PL
authors.lastname: Sheridan
authors.forename: M
authors.initials: M
authors.lastname: Bodner
authors.forename: A
authors.initials: A
authors.lastname: Lynn
authors.forename: T K
authors.initials: TK
authors.lastname: Phuong
authors.forename: N J
authors.initials: NJ
authors.lastname: DePolo
authors.forename: D J
authors.initials: DJ
authors.lastname: de la Vega
authors.forename: J
authors.initials: J
authors.lastname: O'Dea
authors.forename: K
authors.initials: K
authors.lastname: Nguyen
authors.forename: J E
authors.initials: JE
authors.lastname: McCormack
authors.forename: D A
authors.initials: DA
authors.lastname: Driver
authors.forename: K
authors.initials: K
authors.lastname: Townsend
authors.forename: C E
authors.initials: CE
authors.lastname: Ibañez
authors.forename: N C
authors.initials: NC
authors.lastname: Sajjadi
authors.forename: J S
authors.initials: JS
authors.lastname: Greengard
authors.forename: M D
authors.initials: MD
authors.lastname: Moore
authors.forename: J
authors.initials: J
authors.lastname: Respess
authors.forename: S M
authors.initials: SM
authors.lastname: Chang
authors.forename: T W
authors.initials: TW
authors.lastname: Dubensky
authors.forename: D J
authors.initials: DJ
authors.lastname: Jolly
authors.forename: S L
authors.initials: SL
authors.lastname: Sauter
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005169:Factor VIII / Q000235:genetics; D005822:Genetic Vectors; D006467:Hemophilia A / Q000628:therapy; D006801:Humans; D012190:Retroviridae / Q000235:genetics*; D019065:Virus Assembly
chemical_list: D017931:DNA Primers; D005169:Factor VIII