553. Considering the Wild Type Adeno-Associated Virus of Serotype 2 Genome as a Recombinant AAV in the Baculovirus/Sf9 Cells System

Abstract

In 2010, a study on the infectivity of wild type Adeno-Associated Virus (WT AAV2) compared to recombinant AAV (rAAV) vectors have been published. The wild type AAV2 had an up to hundred fold higher infectivity compared to rAAVs.With the aim to decipher if this higher infectivity is due to the WT AAV2 production system, or only to the overall nature of the DNA encapsidated in the AAV capsid we have used the baculovirus/Sf9 system to produce rAAV, with the recombinant genome being the Wt AAV2 genome.However, expressing WT AAV2 genome in the baculovirus system has shown an early activity of the p5 promoter leading to the expression of Rep78. The expression of this protein resulted in an excision of the WT AAV2 genome during the first steps of Baculovirus stock formation.The absence of detection of the Rep68 and Rep40 proteins tended point to reduced or impaired splicing of the rep transcripts in the baculovirus/insect cells system. To put this into evidence, we have characterized the activity of the p5 promoter in the baculovirus/Sf9 context and studied the kinetics of expression of the Rep78 protein.