Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates

• Published in: Journal of controlled release Vol. 110; no. 3; pp. 595 - 604
• Main Authors: Abes, Saïd, Williams, Donna, Prevot, Paul, Thierry, Alain, Gait, Michael J., Lebleu, Bernard
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 21.02.2006; Elsevier
• Subjects: Biological and medical sciences; Cell Membrane - genetics; Cell Membrane - metabolism; Conjugation; Endocytosis; Endosomes - genetics; Endosomes - metabolism; General pharmacology; HeLa Cells; Humans; Medical sciences; Oligolysine; Peptide Nucleic Acids - genetics; Peptide Nucleic Acids - metabolism; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; PNA; RNA Splicing - genetics; Splicing correction; Oligolysine; PNA; Endocytosis; Conjugation; Splicing correction; Pharmaceutical technology; Splicing; Efficiency; Conjugated compound; Endosome
• ISSN: 0168-3659; 1873-4995
• DOI: 10.1016/j.jconrel.2005.10.026

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.