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A novel method for quantitation of AAV genome integrity using duplex digital PCR

2023; Public Library of Science; Volume: 18; Issue: 12 Linguagem: Inglês

10.1371/journal.pone.0293277

1932-6203

Lauren Tereshko, Xiaohui Zhao, Jake Gagnon, Tin-Chi Lin, Trevor Ewald, Yu Wang, Marina Feschenko, Cullen L. Schmid,

Molecular Biology Techniques and Applications

Recombinant adeno-associated virus (rAAV) vectors have become a reliable strategy for delivering gene therapies. As rAAV capsid content is known to be heterogeneous, methods for rAAV characterization are critical for assessing the efficacy and safety of drug products. Multiplex digital PCR (dPCR) has emerged as a popular molecular approach for characterizing capsid content due to its high level of throughput, accuracy, and replicability. Despite growing popularity, tools to accurately analyze multiplexed data are scarce. Here, we introduce a novel statistical model to estimate genome integrity from duplex dPCR assays. This work demonstrates that use of a Poisson-multinomial mixture distribution significantly improves the accuracy and quantifiable range of duplex dPCR assays over currently available models.