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

Abstract

Top of pageAbstract Scalable production of rAAV vectors remains a major obstacle to the clinical application of this protypical gene therapy vector. A recently developed baculovirus-based production protocol (Urabe et al) 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 passaging-dependent, loss-of-function deletions resulting in considerable decreases in rAAV titers. To alleviate the instability problem, we have modified the Rep-encoding component by splitting it into two separate vectors. Furthermore, we have examined the expression limits of the remaining components of this system in order to optimize its application to AAV vector production. In addition, to successfully employ this system to pseudotyped AAV vectors, we have introduced a novel modular approach of parvoviral phospholipase A2 (PLA2) domain swapping that allows for baculovirus production of infectious AAV8 based vectors. The novel chimeric rAAV2/8 vector, produced in Sf9 cells, incorporated AAV2 PLA2 into AAV8 capsid structure and was characterized by robust transduction in vivo. Finally, using the novel riboswitch-controlled gene expression system, we have constructed a universal Bac helper vector for producing infectious mosaic rAAV vector of any given serotype, as demonstrated for rAAV5 and rAAV8. The re-designed baculovirus system improves our capacity for rAAV production by making this AAV platform more applicable to other existing serotypes.