Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates.
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.
Abes
Saïd
S
UMR 5124 CNRS, CC 086, Université Montpellier 2, Montpellier, France.
Williams
Donna
D
Prevot
Paul
P
Thierry
Alain
A
Gait
Michael J
MJ
Lebleu
Bernard
B
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*
D020135:Peptide Nucleic Acids; C078138:peptide nucleic acid, T10-lysine