title: Generation and characterization of adeno-associated virus producer cell lines for research and preclinical vector production.
abstract: Adeno-associated virus (AAV) producer cell lines represent an effective method for large-scale production of AAV vectors. We set out to evaluate and characterize the use of an abbreviated protocol to generate "masterwells" (MWs; a nonclonal cell population) as a platform for research and preclinical vector production. In this system, a single plasmid containing three components, the vector sequence, the AAV rep, and cap genes, and a selectable marker gene is stably transfected into HeLaS3 cells. Producer cell lines generating an AAV2 vector expressing a secreted form of human placental alkaline phosphatase (SEAP) have been created. Several MWs showed vector yields in the 5×10(4) to 2×10(5) DNase-resistant particles/cell range, and the productivity was stable over >60 population doublings. Integrated plasmid copy number in three high-producing MWs ranged from approximately 12 to 50; copies were arranged in a head-to-tail configuration. Upon infection with adenovirus, rep/cap copy number was amplified approximately 100-fold and high yield appeared to be dependent on the extent of amplification. Rep/cap gene expression and vector packaging both reached a peak at 48 hr postinfection. AAV2-SEAP vector was produced in 1-liter shaker culture and purified for assessment of vector quality and potency. The data showed that the majority of the capsids from the MWs contained vector DNA (≥70%) and that purified vector was free of replication-competent AAV. In vitro and in vivo analyses demonstrated that potency of the producer cell-derived vector was comparable to vector generated via the standard transfection method.
authors.affiliation: Genzyme, a Sanofi company , Framingham, MA 01701-9322, USA.
authors.forename: John
authors.initials: J
authors.lastname: Martin
authors.forename: Amy
authors.initials: A
authors.lastname: Frederick
authors.forename: Yuxia
authors.initials: Y
authors.lastname: Luo
authors.forename: Robert
authors.initials: R
authors.lastname: Jackson
authors.forename: Michelle
authors.initials: M
authors.lastname: Joubert
authors.forename: Bruce
authors.initials: B
authors.lastname: Sol
authors.forename: Francis
authors.initials: F
authors.lastname: Poulin
authors.forename: Eric
authors.initials: E
authors.lastname: Pastor
authors.forename: Donna
authors.initials: D
authors.lastname: Armentano
authors.forename: Samuel
authors.initials: S
authors.lastname: Wadsworth
authors.forename: Karen
authors.initials: K
authors.lastname: Vincent
mesh_terms: D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins