Abstract:
Recombinant adeno-associated virus (rAAV) has emerged as the vector of choice for in vivo gene delivery, with numerous clinical trials underway for the treatment of various human diseases. Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer. This quantification process plays a pivotal role in establishing the optimal therapeutic dosage for a rAAV product, both in preclinical and clinical contexts. The conventional single-channel droplet digital PCR (ddPCR) method offers only partial information regarding the viral vector genome titer, lacking insights into its integrity, specifically whether it comprises a complete or partial genome. In our pursuit of further advancing rAAV analysis, we have developed a novel 3D ddPCR assay with advanced 3D linkage analysis. We have designed the three amplicon sites targeting both ends of the viral genome, as well as the center of key therapeutic gene of interest (GOI). This study aims to offer a more comprehensive and insightful assessment of rAAV products. Leveraging our 3D assay platform, we can determine not only the quantity of viral genome titer but also the quality, distinguishing between intact full-length viral genomes and partial ones, and even the identity of key GOI, therapeutic gene component. Importantly, we have developed algorithms to analyze 3D ddPCR linkage titer information. We rigorously tested our algorithms by conducting experiments involving the mixing of seven DNA fragments to represent various AAV viral genome populations, including 3 single partial, 3 double partial, and 1 full-length genomes. Across all 37 tested scenarios, we observed a significant correlation between the percentage of 3D linkage (representing full-length genomes) in the actual input and the model's output. Consequently, our comprehensive AAV analytical package not only offers insights into viral genome titer but also provides valuable information on its integrity and identity. This cost-effective approach, akin to the setup of traditional 1D or 2D dPCR, holds the potential to advance the application of rAAV in cell and gene therapy for the treatment of human diseases.
Author: Tam Duong, PhD, R&D Scientist II