Successful Production of Pseudotyped rAAV Vectors Using a Modified Baculovirus Expression System

摘要

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.