(58a) Increasing Adeno-Associated Virus Specific Yield in Insect Cell Culture by Increasing Temperature

Vectors based on adeno-associated viruses (AAV) are sought for therapeutic gene delivery. Recently the baculovirus expression vector system (BEVS) system has been studied for the mass production of AAV vectors to fulfill the needs for pre-clinical and clinical trials. In this co-infection system, three baculoviruses are used to produce the AAV vector: BacCap contains the gene for the capsid proteins, BacRep contains the gene for the replication proteins and BacITR contains the AAV vector genome. The capsid proteins self-assemble into virus-like particles when they are expressed in the insect cells and remain non-functional without the presence of the replication proteins and the vector genome. The latter are essential for the formation of an infectious AAV particle that is able to transduce cells. Genomes are thought to be encapsidated through a maturation process whereby the vector genome is inserted into pre-formed capsids. A strategy aimed at increasing encapsidation/maturation of the viral vector that involved changes in culture temperature was investigated. Cultures were grown at 27ºC followed by a temperature change at a time pre- or post-infection (up to 24 hours post-infection). It was found that increases in culture temperature up to 30ºC resulted in increases in the number of genomes encapsidated and infectious particles produced. Surprisingly, the number of capsids did not increase with an increase in culture temperature. Furthermore, it was found that an increase in culture temperature was most beneficial when it occurred before 12 hpi. These results are readily achievable at larger scale since instantaneous shifts are not necessary to achieve the beneficial effect.