Development of high-throughput analytical methods for characterization and quantitation of rAAV genome integrity.

Recombinant AAV (rAAV) vectors are a leading viral vector for gene therapy. Viral genome (Vg) titer is the primary method to determine potency of rAAV and dosing in preclinical/clinical studies. However, the rAAV genome comprises a heterogeneous population. These particles not only contain the intact genome but also include numerous truncated species, which likely lack functionality and may induce adverse effects. Consequently, the Vg titer does not accurately reflect the integrity of the rAAV genome. Currently, there is no reliable quantitative method available. In this study, we demonstrate that there is a disconnect between Vg titer and the activity of rAAV by using multiple vectors and high-throughput imaging assays. Importantly, we have developed a novel, high-throughput RNA-DNA hybrid capture-multiplex meso scale discovery (MSD) method for characterizing the integrity of the rAAV genome. This method quantifies the intact versus truncated genomes of both the plus and minus strands individually with high sensitivity and specificity. The integrity data generated by our novel method exhibits a strong correlation with the activity of the rAAV. We anticipate that our new method will significantly improve preclinical/clinical studies, enhance vector design, and increase delivery efficiency. Furthermore, this method can be used to characterize and quantitate RNA and DNA in various fields.