Improving AAV vector yield in insect cells by modulating the temperature after infection.
Vectors based on adeno-associated viruses (AAV) are sought for therapeutic gene delivery because of their ability to transduce a variety of tissues with no significant immunological response. Production using the baculovirus expression vector (BEV)/insect cell system has the potential to meet the needs for pre-clinical and clinical trials. In this co-infection system, three baculoviruses are used to produce the AAV vector. A strategy aimed at increasing encapsidation/maturation of the viral vector involved varying the temperature over the course of the process. Cultures were subjected to temperature changes at various times pre- and post-infection (up to 24 h post-infection). It was found that raising the culture temperature to 30 degrees C at the time of infection nearly tripled the infectious titer. In fact, increasing the temperature to 30 degrees C at any time in the process investigated resulted in an increase in titer. Also, raising the culture to 33 degrees C or lowering the temperature to 24 degrees or 21 degrees C resulted in lower titers. The rise in infectious titer was also confirmed by an increase in DNase resistant particles (DRPs). Varying the temperature, however, did not affect the total amount of capsids significantly. Therefore increasing the culture temperature resulted in better encapsidation as determined by the ratio of capsids to DRPs to infectious particles. It is believed that an increase in early proteins and possibly a quicker cascade of baculovirus infection events resulted in this increased packaging efficiency.
Aucoin
Marc G
MG
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montréal, Quebec, Canada.
Perrier
Michel
M
Kamen
Amine A
AA
D000256:Adenoviridae / Q000502:physiology*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D024861:Genetic Enhancement / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D018411:Spodoptera / Q000166:cytology / Q000235:genetics* / Q000821:virology*; D013696:Temperature; D014776:Virus Cultivation / Q000379:methods*