Tissue-specific Calibration of Real-time PCR Facilitates Absolute Quantification of Plasmid DNA in Biodistribution Studies

Abstract

Analysis of the tissue distribution of plasmid DNA after administration of nonviral gene delivery systems is best accomplished using quantitative real-time polymerase chain reaction (qPCR), although published strategies do not allow determination of the absolute mass of plasmid delivered to different tissues. Generally, data is expressed as the mass of plasmid relative to the mass of genomic DNA (gDNA) in the sample. This strategy is adequate for comparisons of efficiency of delivery to a single site but it does not allow direct comparison of delivery to multiple tissues, as the mass of gDNA extracted per unit mass of each tissue is different. We show here that by constructing qPCR standard curves for each tissue it is possible to determine the dose of intact plasmid remaining in each tissue, which is a more useful parameter when comparing the fates of different formulations of DNA. We exemplify the use of this tissue-specific qPCR method by comparing the delivery of naked DNA, cationic DNA complexes, and neutral PEGylated DNA complexes after intramuscular injection. Generally, larger masses of intact plasmid were present 24 hours after injection of DNA complexes, and neutral complexes resulted in delivery of a larger mass of intact plasmid to the spleen.

Self-Crosslinking Lipopeptide/DNA/PEGylated Particles: A New Platform for DNA Vaccination Designed for Assembly in Aqueous Solution

“…From the tissue-calibrated qPCR assay carried out previously in our laboratories, intact plasmid was detected in the lymph node after administration of both complexes, at similarly low levels (<0.05% of the administered dose). 21 A reason for these discordant results in our and published studies, aside from the different routes of administration used, may be the considerable difference in particle surface charge of the PEGylated liposomes used by Carstens et al. 53 and our PEGylated complexes.…”

“… 20 and Ho et al. 21 ( Figure 1 A). These LPs offer the advantage of ease of manufacture because they can readily self-assemble with plasmid DNA in aqueous solution, circumventing the need for organic-based solvent preparations, limiting the chance of carry-over of the unwanted solvent.…”

“…Additionally, the tissue-calibrated qPCR assay also showed no significant differences in the distribution of LP/DNA and DSPE-PEG 2000 /LP/DNA to the spleen or liver. 21 Because a similar tissue distribution profile is observed post-administration of the two DNA complexes at both 30 min and 24 hr, it is possible that the significant differences in transgene expression and immunogenicity exhibited between the two particulates are due to the subtle differences in the degree of localization or mobility within the injected muscle. It should be noted that, 30 min after administration of all three formulations, it was evident that <5% of the administered dose of DNA or DNA complexes was retained as functional plasmid in the muscle (by qPCR).…”

“…Tissue-calibrated qPCR was carried out according to the procedure outlined by Ho et al. 21 Briefly, mice received an intramuscular injection of the DNA complexes using pCMV-luc. Thirty minutes after injection, tissues were harvested and homogenized in DNAzol reagent (Thermo Fisher Scientific, Carlsbad, CA) using a handheld TissueTearor homogenizer (Biospec Products, Bartlesville, OK) or with a Pellet Pestles motor grinder (Sigma-Aldrich, St. Louis, MO) and incubated overnight with proteinase K (QIAGEN, Venlo, Netherlands) (100 μg/mL) at room temperature.…”