title: Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy.
abstract: In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)(4) peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)(4) peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine 2000. We show that stearyl-(RxR)(4) mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)(4) or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine 2000, we show that stearyl-(RxR)(4) is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)(4) complexed with 2'-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)(4) promotes dose-dependent splice correction in parity with (RxR)(4)-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)(4) an intriguing vector for future in vivo experiments.
authors.affiliation: Laboratory of Molecular Biotechnology, Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia. tlehto@ut.ee
authors.forename: Taavi
authors.initials: T
authors.lastname: Lehto
authors.forename: Rachida
authors.initials: R
authors.lastname: Abes
authors.forename: Nikita
authors.initials: N
authors.lastname: Oskolkov
authors.forename: Julia
authors.initials: J
authors.lastname: Suhorutsenko
authors.forename: Dana-Maria
authors.initials: DM
authors.lastname: Copolovici
authors.forename: Imre
authors.initials: I
authors.lastname: Mäger
authors.forename: Joana R
authors.initials: JR
authors.lastname: Viola
authors.forename: Oscar E
authors.initials: OE
authors.lastname: Simonson
authors.forename: Kariem
authors.initials: K
authors.lastname: Ezzat
authors.forename: Peter
authors.initials: P
authors.lastname: Guterstam
authors.forename: Elo
authors.initials: E
authors.lastname: Eriste
authors.forename: C I Edvard
authors.initials: CI
authors.lastname: Smith
authors.forename: Bernard
authors.initials: B
authors.lastname: Lebleu
authors.lastname: Samir El Andaloussi
authors.forename: Ulo
authors.initials: U
authors.lastname: Langel
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D002463:Cell Membrane Permeability; D049109:Cell Proliferation; D006224:Cricetinae; D003412:Cricetulus; D004337:Drug Carriers / Q000737:chemistry*; D005434:Flow Cytometry; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008055:Lipids; D008156:Luciferases / Q000235:genetics; D009696:Nucleic Acids / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000737:chemistry*; D054735:Phosphorothioate Oligonucleotides / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D013229:Stearic Acids / Q000737:chemistry*; D014162:Transfection
chemical_list: D004337:Drug Carriers; D008055:Lipids; C086724:Lipofectamine; D009696:Nucleic Acids; D010455:Peptides; D054735:Phosphorothioate Oligonucleotides; D013229:Stearic Acids; C015462:polyarginine; C031183:stearic acid; D008156:Luciferases