Figure 1.

Constructing a Stable Single Vaccinia Carrier for rAAV Production

(A) Schematic illustration of vaccinia carriers. W5 and W8 carry AAV trans factors. The hypothetical W5/W8 carrier shows the long direct repeat sequences of Rep52-Rep78 and VP2-VP1 sequences that would have destabilized the vaccinia carrier. VW22 contains Rep52*, the codon-optimized variant of Rep52, and VP1*, the codon-optimized variant of VP1. The arrow stands for p7.5 promoter. (B and C) Immunoblot analysis of Rep (B) and Cap (C) proteins from lysates that were harvested at 24 hr post-VV infection. Mouse anti-Rep and mouse anti-VP1, VP2, and VP3 were used as detecting antibodies. (D) Southern blot analysis of rAAV vector genome replication in vaccinia-Ad-mediated rAAV production system. The Hirt DNA was extracted at 24 hr post-VW22-infection, and the rAAV vector genomes were detected with EGFP-specific probe. The bands corresponding to the linear monomer (M), dimer (D), and tetramer (T) of rAAV genomes are indicated. (E) rAAV vector yield comparison between VW22 and W5 + W8. rAAV vectors were harvested 36 hr post vaccinia carrier infection. The rAAV vector yields were measured by qPCR. (F) Test of the stability of VW22. VW22 were consecutively passed 12 times in BSC-1 cells and tested for the ability to complement rAAV packaging. The x axis indicated the passages of VW22 used for rAAV package. The y axis showed the vector yield. For the experiments in (B)–(F), HeLa-S3 cells were infected with wtAd (MOI = 1) and Ad/AAV-CMV-EGFP (MOI = 50), and VVs were introduced 16 hr later. *p < 0.01. Bars represent the means of three independent experiments.