title: Role of DNA topology in uptake of polyplex molecules by dendritic cells.
abstract: Dendritic cells (DCs) are an attractive target for DNA vaccines as they are potent antigen presenting cells. This study demonstrated how non-viral gene delivery to DCs involving complexes of poly-l-lysine (PLL) and plasmid DNA (pDNA) (polyplexes) showed dependence on DNA vector topology. DNA topology is of importance from both production and regulatory viewpoints. In our previous study with CHO cells we demonstrated that polyplex uptake was dependent on DNA topology whereby complexes containing supercoiled (SC) pDNA were smaller, more resistant to nucleases and more effectively condensed by PLL than open circular (OC) and linear-pDNA complexes. In this study polyplex uptake in DCs was measured qualitatively and quantitatively by confocal microscopy along with gene expression studies and measurement of DC phenotype. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Quantification studies revealed that by 1h following uptake 15% (±2.59% relative standard error [RSE]) of SC-pDNA polyplexes were identified to be associated (fluorescent co-localisation) with the nucleus, in comparison to no nuclear association identified for OC- and linear-pDNA complexes. By 48 h following uptake, 30% (±1.82% RSE) of SC-pDNA complexes associated with the nucleus in comparison to 16% (±4.40% RSE) and 12% (±6.97% RSE) of OC- and linear-pDNA polyplexes respectively. Confocal microscopy images showed how DNA and PLL remained associated following uptake by dual labelling. Polyplex (containing 20 μg pDNA) gene expression (plasmid encoded lacZ [β-galactosidase] reporter gene) in DCs was greatest for SC-pDNA polyplexes at 14.12% unlike that of OC- (9.59%) and linear-pDNA (7.43%). DCs express cell surface markers which contribute towards antigen presentation. Polyplex gene expression did not alter DC phenotype through surface marker expression. This may be due to the pDNA dose employed (20μg) as other studies have used doses as high as 200 μg pDNA to induce DC phenotypic changes. Although no change in DC phenotype occurred, this could be advantageous in terms of biocompatibility. Collectively these results indicate that DNA topology is an important parameter for DC vector design, particularly pDNA in the SC conformation in regards to DNA vaccination studies.
authors.affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
authors.forename: Arjun
authors.initials: A
authors.lastname: Dhanoya
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Chain
authors.forename: Eli
authors.initials: E
authors.lastname: Keshavarz-Moore
mesh_terms: D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry* / Q000493:pharmacokinetics; D004278:DNA, Superhelical / Q000737:chemistry / Q000493:pharmacokinetics; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D005822:Genetic Vectors / Q000737:chemistry* / Q000493:pharmacokinetics; D006801:Humans; D011107:Polylysine / Q000737:chemistry / Q000493:pharmacokinetics*; D014162:Transfection / Q000379:methods*; D019444:Vaccines, DNA / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D004278:DNA, Superhelical; D019444:Vaccines, DNA; D011107:Polylysine; D004247:DNA