Three-Dimensional Linkage Analysis With Digital PCR For Genome Integrity And Identity Of Recombinant Adeno-Associated Virus

By Tam Duong, Michele Firmo, Chien-Ting Li, Bingnan Gu, and Peng Wang

Duong, T., Firmo, M., Li, CT. et al. Three-dimensional linkage analysis with digital PCR for genome integrity and identity of recombinant adeno-associated virus. Sci Rep 15, 2154 (2025). https://doi.org/10.1038/s41598-024-77378-7

Recombinant adeno-associated virus (rAAV) is a leading vector for in vivo gene delivery, with many clinical trials underway. Precise quantification of viral genome titer is crucial for determining optimal therapeutic dosage. Traditional single-channel droplet digital PCR (1D ddPCR) only provides partial information, lacking data on viral genome integrity. To address this, we developed a novel 3D ddPCR assay with advanced 3D linkage analysis. Our assay targets three amplicon sites: both ends of the viral genome and the center of the therapeutic gene of interest (GOI). This provides a more comprehensive assessment of rAAV products, including both quantity (viral genome titer) and quality (distinguishing intact, full-length genomes with the correct GOI from partial genomes). Because digital PCR randomly partitions DNA, we developed a mathematical model to distinguish true linked DNA molecules (full-length/intact) from false/unlinked molecules (fragmented/partial). This AAV 3D linkage analysis workflow characterizes genomic integrity and intact titer for rAAV gene therapy products. We evaluated our 3D linkage model using DNA mixing experiments and a case study with multiple rAAV samples. Specifically, we mixed seven DNA fragments representing various AAV viral genome populations (three single partials, three double partials, and one full-length genome) and tested 37 scenarios. The workflow's accuracy in determining 3D linkage percentages was validated against the known input DNA percentages. This comprehensive AAV analytical package provides insights into viral genome titer, integrity, and identity. This cost-effective approach, comparable to 1D or 2D dPCR, has the potential to advance rAAV use in cell and gene therapy for human diseases.