title: Impact of lysine-affinity chromatography on supercoiled plasmid DNA purification.
abstract: Gene therapy and DNA vaccination cover a variety of applications using viral and non-viral vectors as vehicles of choice for treatment of genetic or acquired diseases. Recently, most therapeutic applications have been performed with non-viral biological agents preparations highly enriched in supercoiled plasmid molecules and it has been concluded that this isoform is more efficient at gene transfection than open circular isoform. This work describes for the first time a new strategy that uses lysine-chromatography to efficiently eliminate Escherichia coli impurities as well as other ineffective plasmid isoforms present in a complex clarified lysate to purify and obtain pharmaceutical-grade supercoiled plasmid DNA. The quality control tests indicated that the levels of impurities in the final plasmid product were below the generally accepted specifications. Furthermore, the delivery of the purified product to eukaryotic cells, the cell uptake and transfection efficiency were also analyzed. The results showed that the transfection efficiency reached with the application of the supercoiled plasmid conformation, purified with lysine-agarose, was higher than the values achieved for other plasmid topologies. Therefore, this study presents a new enabling technology to obtain the completely purified non-viral vector, able to act with good efficiency as gene therapy delivery vehicle in several diseases like cancer.
authors: CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal.
authors: A
authors: A
authors: Sousa
authors: F
authors: F
authors: Sousa
authors: J A
authors: JA
authors: Queiroz
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli; D005822:Genetic Vectors / Q000737:chemistry / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D018613:Microscopy, Confocal; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*; D014162:Transfection
chemical_list: D004278:DNA, Superhelical; D008239:Lysine