A Poisson-multinomial model for quantifying gene therapy vector integrity using quadplex digital PCR

Abstract

Quantitative characterization of gene therapy vectors is critical for confirming product safety and efficacy. While digital PCR (dPCR) strategies are widely used to estimate sequence integrity, accurate statistical interpretation of multiplexed assays remains a persistent challenge due to limitations of existing analytical models. Here, we describe a quadplex droplet dPCR approach for quantitative characterization of vector integrity across four targets within a single reaction. We adapt a Poisson-multinomial model to enable quantitation of sequence integrity and genetic linkage across all combinations of the four targeted regions. We evaluate both the capabilities and limitations of this approach and provide practical guidance for multiplexed assay design and data interpretation.