title: Incorporation of a Nuclear Localization Signal in pH Responsive LAH4-L1 Peptide Enhances Transfection and Nuclear Uptake of Plasmid DNA.
abstract: The major intracellular barriers associated with DNA delivery using nonviral vectors are inefficient endosomal/lysosomal escape and poor nuclear uptake. LAH4-L1, a pH responsive cationic amphipathic peptide, is an efficient DNA delivery vector that promotes the release of nucleic acid into cytoplasm through endosomal escape. Here we further enhance the DNA transfection efficiency of LAH4-L1 by incorporating nuclear localizing signal (NLS) to promote nuclear importation. Four NLSs were investigated: Simian virus 40 (SV40) large T-antigen derived NLS, nucleoplasmin targeting signal, M9 sequence, and the reverse SV40 derived NLS. All peptides tested were able to form positively charged nanosized complexes with DNA. Significant improvement in DNA transfection was observed in slow-dividing epithelial cancer cells (Calu-3), macrophages (RAW264.7), dendritic cells (JAWSII), and thymidine-induced growth-arrested cells, but not in rapidly dividing cells (A549). Among the four NLS-modified peptides, PK1 (modified with SV40 derived NLS) and PK2 (modified with reverse SV40 derived NLS) were the most consistent in improving DNA transfection; up to a 10-fold increase in gene expression was observed for PK1 and PK2 over the unmodified LAH4-L1. Additionally PK1 and PK2 were shown to enhance cellular uptake as well as nuclear entry of DNA. Overall, we show that the incorporation of SV40 derived NLS, in particular, to LAH4-L1 is a promising strategy to improve DNA delivery efficiency in slow-dividing cells and dendritic cells, with development potential for in vivo applications and as a DNA vaccine carrier.
authors.affiliation: Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong , 21 Sassoon Road, Pokfulam, Hong Kong.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong , 21 Sassoon Road, Pokfulam, Hong Kong.
authors.forename: Wanling
authors.initials: W
authors.lastname: Liang
authors.affiliation: Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong , 21 Sassoon Road, Pokfulam, Hong Kong.
authors.forename: Yingshan
authors.initials: Y
authors.lastname: Qiu
authors.affiliation: School of Pharmacy, University of Camerino , Via S. Agostino 1, 62032 Camerino (MC), Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Cespi
authors.affiliation: School of Pharmacy, University of Camerino , Via S. Agostino 1, 62032 Camerino (MC), Italy.
authors.forename: Giovanni F
authors.initials: GF
authors.lastname: Palmieri
authors.affiliation: Institute of Pharmaceutical Science, King's College London , 150 Stamford Street, London SE1 9NH, United Kingdom.
authors.forename: A James
authors.initials: AJ
authors.lastname: Mason
authors.affiliation: Department of Pharmacology & Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong , 21 Sassoon Road, Pokfulam, Hong Kong.
authors.forename: Jenny K W
authors.initials: JK
authors.lastname: Lam
mesh_terms: D000072283:A549 Cells; D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D019913:Nuclear Localization Signals / Q000187:drug effects; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D000067996:RAW 264.7 Cells; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
chemical_list: D019913:Nuclear Localization Signals; D010455:Peptides
keywords: dendritic cells; gene delivery; nonviral vectors; nuclear localization signals; nuclear transport; pH responsive peptides