Abstract:
Recombinant adeno-associated virus (rAAV) has been a vector of choice for in vivo gene delivery with many clinical trials for the treatment of human diseases. The clinical use of rAAV in gene therapy requires a precise quantification method for viral vector products. The current single channel ddPCR method provides incomplete pictures regarding rAAV genome integrity, i.e. complete vs. partial genome. In this study, we applied multiplexing 2D ddPCR to thoroughly characterize a rAAV product by providing new information on intact viral genome titer. Particularly, we designed the amplicon sites targeting both ends of the viral genome to ensure capturing the full-length viral genome in the analysis. We showed that the selections of appropriate PCR amplification sites are critical to precisely evaluate the viral genome integrity. Together with the application of linkage analysis using QuantaSoft software, we can accurately calculate the intact viral genome titer and the percentage of full-length rAAV among all genome-containing species in the product. Therefore, this new method can serve as quick characterization and titer assays for both process development and final product release with improved robustness, accuracy and precision.
Author: Tam Duong, PhD, Scientist II, R&D