title: Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system.
abstract: Scalable production of rAAV vectors remains a major obstacle to the clinical application of this prototypical gene therapy vector. A recently developed baculovirus-based production protocol (M. Urabe et al., 2002, Hum. Gene Ther. 13, 1935-1943) found limited applications due to the system's design. Here we report a detailed analysis of the stability of the original baculovirus system components BacRep, BacVP, and transgene cassette-containing BacGFP. All of the baculovirus helpers analyzed were prone to passage-dependent loss-of-function deletions resulting in considerable decreases in rAAV titers. To alleviate the instability and to extend the baculovirus platform to other rAAV serotypes, we have modified both Rep- and Cap-encoding components of the original system. The modifications include a parvoviral phospholipase A2 domain swap allowing production of infectious rAAV8 vectors in vivo. Alternatively, an infectious rAAV8 (or rAAV5) vector incorporating the AAV2 VP1 capsid protein in a mosaic vector particle with AAV8 capsid proteins was produced using a novel baculovirus vector. In this vector, the level of AAV2 VP1 expression is controlled with a "riboswitch," a self-cleaving ribozyme controlled by toyocamycin in the "ON" mode. The redesigned baculovirus system improves our capacity for rAAV manufacturing by making this production platform more applicable to other existing serotypes.
authors.affiliation: Powell Gene Therapy Center, Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Erik
authors.initials: E
authors.lastname: Kohlbrenner
authors.forename: George
authors.initials: G
authors.lastname: Aslanidi
authors.forename: Kevin
authors.initials: K
authors.lastname: Nash
authors.forename: Stanislav
authors.initials: S
authors.lastname: Shklyaev
authors.forename: Martha
authors.initials: M
authors.lastname: Campbell-Thompson
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Byrne
authors.forename: Richard O
authors.initials: RO
authors.lastname: Snyder
authors.forename: Nicholas
authors.initials: N
authors.lastname: Muzyczka
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Warrington
authors.forename: Sergei
authors.initials: S
authors.lastname: Zolotukhin
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002213:Capsid / Q000737:chemistry; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007313:Insecta; D051379:Mice; D008957:Models, Genetic; D010741:Phospholipases A / Q000737:chemistry; D054467:Phospholipases A2; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D049452:Green Fluorescent Proteins; D010741:Phospholipases A; D054467:Phospholipases A2