title: Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates.
abstract: Splicing correction by steric-blocking oligonucleotides (ON) might lead to important clinical applications but requires efficient delivery to cell nuclei. The conjugation of short oligolysine tails has been used to deliver a correcting peptide nucleic acid (PNA) sequence in a positive readout assay in which ON hybridization to the cryptic splice site is strictly required for the expression of a luciferase reporter gene. We have investigated the mechanism of cellular uptake and the efficiency of a (Lys)(8)-PNA-Lys construction in this model system. Cell uptake is temperature-dependent and leads to sequestration of the conjugate in cytoplasmic vesicles in keeping with an endocytic mechanism of internalization. Accordingly a significant and sequence-specific splicing correction is achieved only in the presence of endosome-disrupting agents as chloroquine or 0.5 M sucrose. These endosome-disrupting agents do not affect the activity of free PNA, and do not increase (Lys)(8)-PNA-Lys uptake.
authors.affiliation: UMR 5124 CNRS, CC 086, Université Montpellier 2, Montpellier, France.
authors.forename: Saïd
authors.initials: S
authors.lastname: Abes
authors.forename: Donna
authors.initials: D
authors.lastname: Williams
authors.forename: Paul
authors.initials: P
authors.lastname: Prevot
authors.forename: Alain
authors.initials: A
authors.lastname: Thierry
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gait
authors.forename: Bernard
authors.initials: B
authors.lastname: Lebleu
mesh_terms: D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D011992:Endosomes / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020135:Peptide Nucleic Acids / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*
chemical_list: D020135:Peptide Nucleic Acids; C078138:peptide nucleic acid, T10-lysine