A Baculovirus expression vector system for the production of recombinant Adeno-associated virus for gene therapy applications

Vectors based on Adeno-associated virus (AAV) have emerged as the leading candidates for gene therapy applications to treat monogenic diseases. Its low immunogenicity in humans, strong and long term gene expression and varying cell and tissue tropisms bid promising prospect to their utilization in future medicine. However, the lack of a flexible and efficient recombinant AAV (rAAV) production platform stands in the way of utilizing their full potential. Conventional production methods have mostly been based on the infection of adherent mammalian cell cultures, which has proven to be too expensive and labor intensive to scale up. Recently, the use of suspension insect cell cultures and the baculovirus expression vector system (BEV) for the generation of rAAV has gained more traction. Current challenges with this application of the BEV lie in the instability of the baculovirus leading to excision of the transgene from the genome and the requirement of multiple baculovirus constructs to facilitate the production of rAAV. This project aims to develop a simplified and flexible approach for the reliable generation of rAAV particles using the BEVS. (1) The initial focus will be on the development of a genomically stable Baculovirus, which will remain stable over several passages with consistent transgene expression. (2) Subsequently, this baculovirus will then be developed to encompass all the elements required for rAAV production in insect cells to eliminate the need for co-infection. (3) Additionally, fundamental research will be performed aiming to identify which elements of the baculovirus supply the helper functions for AAV production.