title: Delivery of antisense peptide nucleic acids to cells by conjugation with small arginine-rich cell-penetrating peptide (R/W)9.
abstract: Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.
authors.affiliation: Muséum National d'Histoire Naturelle, Structure et Instabilité des Génomes, Paris, France; INSERM, U1154, Paris, France; CNRS, UMR 7196, Paris, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Cordier
authors.affiliation: Muséum National d'Histoire Naturelle, Structure et Instabilité des Génomes, Paris, France; INSERM, U1154, Paris, France; CNRS, UMR 7196, Paris, France.
authors.forename: Fatima
authors.initials: F
authors.lastname: Boutimah
authors.affiliation: Muséum National d'Histoire Naturelle, Structure et Instabilité des Génomes, Paris, France; INSERM, U1154, Paris, France; CNRS, UMR 7196, Paris, France.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Bourdeloux
authors.affiliation: Muséum National d'Histoire Naturelle, Structure et Instabilité des Génomes, Paris, France; INSERM, U1154, Paris, France; CNRS, UMR 7196, Paris, France.
authors.forename: Florian
authors.initials: F
authors.lastname: Dupuy
authors.affiliation: Muséum National d'Histoire Naturelle, Structure et Instabilité des Génomes, Paris, France; INSERM, U1154, Paris, France; CNRS, UMR 7196, Paris, France.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Met
authors.affiliation: Muséum National d'Histoire Naturelle, Structure et Instabilité des Génomes, Paris, France; INSERM, U1154, Paris, France; CNRS, UMR 7196, Paris, France.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Alberti
authors.affiliation: Muséum National d'Histoire Naturelle, Structure et Instabilité des Génomes, Paris, France; INSERM, U1154, Paris, France; CNRS, UMR 7196, Paris, France.
authors.forename: François
authors.initials: F
authors.lastname: Loll
authors.affiliation: UPMC-Univ Paris 06, Laboratoire des BioMolécules, Université P. et M. Curie, Paris, France; CNRS, UMR 7203, Paris, France; ENS, UMR 7203, Département de Chimie, Ecole Normale Supérieure, Paris, France.
authors.forename: Gérard
authors.initials: G
authors.lastname: Chassaing
authors.affiliation: UPMC-Univ Paris 06, Laboratoire des BioMolécules, Université P. et M. Curie, Paris, France; CNRS, UMR 7203, Paris, France; ENS, UMR 7203, Département de Chimie, Ecole Normale Supérieure, Paris, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Burlina
authors.affiliation: Muséum National d'Histoire Naturelle, Structure et Instabilité des Génomes, Paris, France; INSERM, U1154, Paris, France; CNRS, UMR 7196, Paris, France.
authors.forename: Tula Ester
authors.initials: TE
authors.lastname: Saison-Behmoaras
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism; D003412:Cricetulus; D011992:Endosomes / Q000378:metabolism; D018390:Gene Targeting; D006025:Glycosaminoglycans / Q000378:metabolism; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics* / Q000493:pharmacokinetics; D020135:Peptide Nucleic Acids / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics* / Q000493:pharmacokinetics; D010455:Peptides; D010873:Pinocytosis
chemical_list: D057846:Cell-Penetrating Peptides; D006025:Glycosaminoglycans; D016376:Oligonucleotides, Antisense; D020135:Peptide Nucleic Acids; D010455:Peptides; D001120:Arginine