Validation of a Göttingen Minipig Gene Therapy Biodistribution Assay for AVGN7 Using Droplet Digital Polymerase Chain Reaction

Minipig models are rapidly gaining popularity in gene therapy preclinical studies. This includes biodistribution studies that are required by regulatory agencies, but are poorly described in the literature. This is particularly true for ddPCR assays with enhanced sensitivity over traditional "real-time" PCR. We, therefore, validated a novel duplex ddPCR assay for quantifying AVGN7, an AAV gene therapeutic featuring a codon-optimized human Smad7 gene and the porcine RPP30 reference gene, in Göttingen minipig tissues. The DNA extraction methods were optimized to ensure maximum recovery of target gene. Validation parameters included accuracy and precision, limit of detection, ruggedness/robustness, selectivity, recovery, dilutional linearity and stability. The assay was sensitive to 10 cp/µg gDNA while intra- and inter-accuracy and precision were demonstrated for the dynamic range of 25,000-5 cp/µL where the inter-assay total error was ≤ 35.5% for AVGN7 and ≤ 4.3% for RPP30. Moreover, ruggedness was assessed over time using multiple analysts, instruments and reagent lots, demonstrating high reliability and reproducibility. Linearity was established across 1,000-fold dilutions and stability after 6 freeze-thaw cycles and 24 h at room temperature. Selectivity was evaluated in unspiked and post-spiked samples using multiple lots of different tissue matrices. High recovery of AVGN7 during nucleic acid extraction was also demonstrated for all tissues. These data not only validate the assay for tracking and quantifying AVGN7 in preclinical studies with Göttingen minipigs, but additionally address challenges of using pig models in general while providing a procedural road map for developing other AAV biodistribution assays.