title: Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies.
abstract: Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)(4) (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)(4)-PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure-activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs.
authors.affiliation: UMR 5235 CNRS, Université Montpellier 2, Place Eugene Bataillon, 34095 Montpellier cedex 5, France.
authors.forename: Rachida
authors.initials: R
authors.lastname: Abes
authors.forename: Hong M
authors.initials: HM
authors.lastname: Moulton
authors.forename: Philippe
authors.initials: P
authors.lastname: Clair
authors.forename: Sung-Tae
authors.initials: ST
authors.lastname: Yang
authors.forename: Said
authors.initials: S
authors.lastname: Abes
authors.forename: Kamran
authors.initials: K
authors.lastname: Melikov
authors.forename: Paul
authors.initials: P
authors.lastname: Prevot
authors.forename: Derek S
authors.initials: DS
authors.lastname: Youngblood
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Iversen
authors.forename: Leonid V
authors.initials: LV
authors.lastname: Chernomordik
authors.forename: Bernard
authors.initials: B
authors.lastname: Lebleu
mesh_terms: D000577:Amides / Q000737:chemistry; D015119:Aminocaproic Acid / Q000737:chemistry; D001120:Arginine / Q000737:chemistry*; D001692:Biological Transport; D011992:Endosomes / Q000378:metabolism; D006367:HeLa Cells; D006493:Heparin / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008081:Liposomes / Q000737:chemistry; D009025:Morpholines / Q000737:chemistry; D009841:Oligonucleotides / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010756:Phosphoric Acids / Q000737:chemistry; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D000577:Amides; D008081:Liposomes; D009025:Morpholines; D009841:Oligonucleotides; D010455:Peptides; D010756:Phosphoric Acids; D006493:Heparin; D001120:Arginine; C011067:phosphoramidic acid; D015119:Aminocaproic Acid
references.citation: Mol Cell Biol. 2003 Dec;23(24):9318-26
references.pmid: 14645541
references.citation: Bioconjug Chem. 2004 Mar-Apr;15(2):290-9
references.pmid: 15025524
references.citation: J Biol Chem. 2002 Aug 30;277(35):32157-64
references.pmid: 12065580
references.citation: Traffic. 2001 Sep;2(9):612-21
references.pmid: 11555415
references.citation: Mol Ther. 2008 Jun;16(6):1120-8
references.pmid: 18443602
references.citation: J Gene Med. 2006 Feb;8(2):207-16
references.pmid: 16285002
references.citation: Biochem Soc Trans. 2007 Aug;35(Pt 4):775-9
references.pmid: 17635146
references.citation: Adv Drug Deliv Rev. 2008 Mar 1;60(4-5):517-29
references.pmid: 18037527
references.citation: Antisense Nucleic Acid Drug Dev. 1997 Jun;7(3):187-95
references.pmid: 9212909
references.citation: J Biol Chem. 2003 Jan 3;278(1):585-90
references.pmid: 12411431
references.citation: Biochemistry. 1984 Mar 27;23(7):1532-8
references.pmid: 6722105
references.citation: Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):13003-8
references.pmid: 11087855
references.citation: Int J Pharm. 2007 Nov 1;344(1-2):96-102
references.pmid: 17600642
references.citation: J Control Release. 2006 Dec 1;116(3):304-13
references.pmid: 17097177
references.citation: Nucleic Acids Res. 2007;35(13):4495-502
references.pmid: 17584792
references.citation: J Virol. 2007 Jun;81(11):5637-48
references.pmid: 17344287
references.citation: Gene Ther. 2006 Oct;13(19):1373-81
references.pmid: 16724091
references.citation: Biochim Biophys Acta. 1985 Jan 10;812(1):55-65
references.pmid: 23008845
references.citation: J Biol Chem. 2001 Feb 2;276(5):3254-61
references.pmid: 11024024
references.citation: Bioconjug Chem. 2007 Jan-Feb;18(1):50-60
references.pmid: 17226957
references.citation: Antimicrob Agents Chemother. 2007 Jul;51(7):2470-82
references.pmid: 17485503
references.citation: Mol Ther. 2007 Sep;15(9):1587-92
references.pmid: 17579573
references.citation: Bioconjug Chem. 2007 Jul-Aug;18(4):1325-31
references.pmid: 17583927
references.citation: Arch Virol. 2008;153(5):929-37
references.pmid: 18369525
references.citation: Nucleic Acids Res. 2007;35(15):5182-91
references.pmid: 17670797