Development and fit-for-purpose validation of a qPCR assay for biodistribution assessment of plasmid DNA following electrode-needle device-assisted intramuscular electroporation.

Abstract

Reliable biodistribution assessment is essential for the nonclinical evaluation of plasmid DNA (pDNA)-based therapeutics. Here, we developed and performed fit-for-purpose validation of a TaqMan-based quantitative PCR (qPCR) assay for the absolute quantification of pDNA following electrode-needle device-assisted intramuscular electroporation. The assay showed high target specificity, linear quantitative performance over six orders of magnitude, and acceptable accuracy and precision under standardized genomic DNA matrix conditions. Using this assay, we characterized the biodistribution of the SARS-CoV-2 spike-encoding DNA vaccine pSARS2-S in Sprague-Dawley rats following intramuscular administration with or without electroporation. Plasmid DNA was detected predominantly in the injection-site region, particularly in muscle and overlying subcutaneous tissue, whereas substantially lower levels were observed in non-target organs. At the terminal sampling time point, pDNA levels in the evaluated major organs, including the gonads, were below the lower limit of quantification (LLOQ), while low-level residual pDNA remained detectable in subcutaneous tissue. These findings characterize the tissue distribution and persistence of pSARS2-S under the established delivery condition. Overall, this study establishes a reproducible, fit-for-purpose qPCR assay and analytical workflow for quantitative nonclinical biodistribution assessment of pSARS2-S.