575. In Vivo Evaluation of Supercoiled Plasmids

Abstract

Top of pageAbstract Supercoiled plasmids are an important component of gene-based drugs. Their applications include gene vaccine and gene therapeutic agents or intermediates in the production of recombinant viruses. These applications require very high quality product with low levels of residual contaminants such as genomic DNA, RNA, host protein and endotoxin. While a variety of methods for the production of supercoiled plasmids for clinical applications have been developed, there is no consensus as to the effect of plasmid quality on biological activity. We have produced two plasmids using four production methodologies based on reversed-phase ion-pairing (PolyFlo) and hydrophobic interaction chromatography, either alone or in tandem processes. The plasmids were analyzed using standard molecular biological techniques as well as more rigorous biophysical techniques. Further, the plasmids were evaluated in vivo in models for gene vaccination and gene therapy. In addition, the gene therapy study included a component to evaluate the degree of plasmid supercoiling and biological activity. Analysis of the plasmids produced by the four methods showed minimal measurable differences using molecular biological methods. Biophysical characterization revealed more significant differences among the preparations. However, there were significant differences in the in vivo response in both models. In the gene vaccination study, preparations using a tandem process of PolyFlo used in two separation modes provided significantly higher antibody responses to an HIV gag antigen. By contrast, PolyFlo as the single chromatographic step resulted in a significant decrease in the rate of tumor growth when compared to two tandem preparations. These data indicate that the process by which supercoiled plasmids are made can influence biological activity and further that the same process might not be ideal for gene vaccine or gene therapy applications. These data emphasize the need for more rigorous methods to evaluate the biological activity of supercoiled plasmids.