title: Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential.
abstract: Listeriolysin O (LLO), a sulfhydryl-activated pore-forming protein from Listeria monocytogenes, was tested and utilized for promoting plasmid DNA (pDNA) delivery into the cytosol of cells in culture. To render pDNA-complexing capability to LLO, the unique cysteine 484 of LLO was conjugated to polycationic peptide protamine (PN) at a 1:1 molar ratio through a reversible, endosome-labile disulfide bond. The sulfhydryl-oxidized LLO construct, LLO-s-s-PN, completely lacked its pore-forming activity, yet regained its original activity upon reduction. The enhanced cytosolic delivery using this construct therefore relies on the requisite reduction of the disulfide bond in LLO-s-s-PN by endogenous cellular reducing capacity. Condensed PN/pDNA complexes incorporating LLO-s-s-PN were tested for their enhanced gene delivery capability monitoring reporter gene expression in HEK293, RAW264.7, P388D1 cell lines and bone-marrow-derived macrophages in the presence of serum. Dramatic enhancement was observed for all tested complexes with varying weight ratios. The effect was most prominent at 0.64-0.80 (w/w) of PN/pDNA upon replacing 1-4% of PN with LLO-s-s-PN, resulting in approximately three orders of magnitude higher luciferase expression compared to PN/pDNA without apparent toxicity. These results demonstrate that incorporation of endosomolytic LLO into pDNA delivery systems in a controlled fashion is a promising approach of enhancing delivery into the cytosol of target cells in gene delivery strategies.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065, USA.
authors.forename: G
authors.initials: G
authors.lastname: Saito
authors.forename: G L
authors.initials: GL
authors.lastname: Amidon
authors.forename: K-D
authors.initials: KD
authors.lastname: Lee
mesh_terms: D000818:Animals; D001427:Bacterial Toxins; D001854:Bone Marrow Cells / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003470:Culture Media; D003600:Cytosol / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage*; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011479:Protamines; D051381:Rats; D013438:Sulfhydryl Compounds; D014162:Transfection / Q000379:methods*
chemical_list: D001427:Bacterial Toxins; D003470:Culture Media; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D008164:Luminescent Proteins; D011479:Protamines; D013438:Sulfhydryl Compounds; D049452:Green Fluorescent Proteins; D004247:DNA; D008156:Luciferases; C038705:hlyA protein, Listeria monocytogenes