title: Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide.
abstract: Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5-10 microM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 muM concentration of the R6Pen-PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen-PNA705 structure-function relationship have also been evaluated.
authors.affiliation: UMR 5235 CNRS, Université Montpellier 2, Place Eugene Bataillon, 34095 Montpellier cedex 5, France.
authors.forename: Saïd
authors.initials: S
authors.lastname: Abes
authors.forename: John J
authors.initials: JJ
authors.lastname: Turner
authors.forename: Gabriela D
authors.initials: GD
authors.lastname: Ivanova
authors.forename: David
authors.initials: D
authors.lastname: Owen
authors.forename: Donna
authors.initials: D
authors.lastname: Williams
authors.forename: Andrey
authors.initials: A
authors.lastname: Arzumanov
authors.forename: Philippe
authors.initials: P
authors.lastname: Clair
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
authors.forename: Bernard
authors.initials: B
authors.lastname: Lebleu
mesh_terms: D021581:Active Transport, Cell Nucleus; D001120:Arginine / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D012326:RNA Splicing / Q000187:drug effects*
chemical_list: D002352:Carrier Proteins; D020135:Peptide Nucleic Acids; C521937:R6-penetratin; D002738:Chloroquine; D001120:Arginine