title: OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA.
abstract: Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application.
authors.affiliation: 1 Institute of Virology, Campus Benjamin Franklin, Charité Medical School, Berlin, Germany .
authors.forename: Mario
authors.initials: M
authors.lastname: Mietzsch
authors.affiliation: 1 Institute of Virology, Campus Benjamin Franklin, Charité Medical School, Berlin, Germany .
authors.forename: Vincent
authors.initials: V
authors.lastname: Casteleyn
authors.affiliation: 1 Institute of Virology, Campus Benjamin Franklin, Charité Medical School, Berlin, Germany .
authors.forename: Stefan
authors.initials: S
authors.lastname: Weger
authors.affiliation: 2 Department of Pediatrics, University of Florida College of Medicine , Gainesville, Florida.
authors.forename: Sergei
authors.initials: S
authors.lastname: Zolotukhin
authors.affiliation: 1 Institute of Virology, Campus Benjamin Franklin, Charité Medical School, Berlin, Germany .
authors.forename: Regine
authors.initials: R
authors.lastname: Heilbronn
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics* / Q000627:therapeutic use; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans
chemical_list: D036022:Capsid Proteins