title: A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform.
abstract: Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 10(14) rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 10(5) vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.
authors.affiliation: Department of Pediatrics, Powell Gene Therapy Center, University of Florida , Gainesville, Florida, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Adamson-Small
authors.affiliation: Department of Pediatrics, Powell Gene Therapy Center, University of Florida , Gainesville, Florida, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Potter
authors.affiliation: Department of Pediatrics, Child Health Research Institute, University of Florida , Florida, USA.
authors.forename: Darin J
authors.initials: DJ
authors.lastname: Falk
authors.affiliation: Department of Pediatrics, Powell Gene Therapy Center, University of Florida , Gainesville, Florida, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Cleaver
authors.affiliation: Department of Pediatrics, Powell Gene Therapy Center, University of Florida , Gainesville, Florida, USA.
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Byrne
authors.affiliation: Department of Pediatrics, Powell Gene Therapy Center, University of Florida , Gainesville, Florida, USA.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Clément