title: Improved nuclear delivery of antisense 2'-Ome RNA by conjugation with the histidine-rich peptide H5WYG.
abstract: BACKGROUND
  Antisense oligonucleotides are promising medicines for treating various diseases, although their efficiency still requires high doses. Their delivery in the cytosol and nucleus to reach their mRNA targets would increase their efficiency at the same time as reducing the dose.
  
  
  METHODS
  We conjugated the histidine-rich peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) at the 5'-end of the RNase H-incompetent antisense 2'-O-methyl-phosphodiester oligonucleotide (2'-Ome RNA705) targeting aberrant splicing of luciferase pre-mRNA in HeLa pLuc705 cells. H5WYG was also conjugated with 2'-Ome-RNA705 labelled by fluorescein at the 3'-end. Then, H5WYG-2'-Ome-RNA705 conjugate and 2'-Ome-RNA705 were formulated with lipofectamine to favor their uptake in HeLa pLuc705 cells.
  
  
  RESULTS
  Confocal microscopy showed that, after 4 h and overnight incubation, the presence of fluorescein-labelled 2'-Ome-RNA705 in the cytosol and nucleus was enhanced when the oligonucleotide was conjugated with H5WYG. We found that H5WYG-2'-Ome-RNA705 increased the splicing redirection and restoration of a functional luciferase mRNA. Luciferase activity and luciferase mRNA levels in these cells were 6.6- and two-fold higher, respectively, with H5WYG-2'-Ome-RNA705 than with 2'-Ome-RNA705.
  
  
  CONCLUSIONS
  The results of the present study show that the conjugation of 2'-Ome antisense RNA to peptide H5WYG is a good strategy for improving its cytosol delivery, accumulation in the nucleus and antisense activity.
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR4301, Inserm and Université d'Orléans, Orléans, France.
authors.forename: Ulysse
authors.initials: U
authors.lastname: Asseline
authors.forename: Cristine
authors.initials: C
authors.lastname: Gonçalves
authors.forename: Chantal
authors.initials: C
authors.lastname: Pichon
authors.forename: Patrick
authors.initials: P
authors.lastname: Midoux
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D020320:Oligoribonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing; D014162:Transfection
chemical_list: C112056:H5WYG peptide; D020320:Oligoribonucleotides, Antisense; D010455:Peptides; D008156:Luciferases
keywords: antisense oligonucleotides; endosome escape; fusogenic peptide; histidine; mRNA; splicing