Abes Saïd, Williams Donna, Prevot Paul, Thierry Alain, Gait Michael J, Lebleu Bernard
UMR 5124 CNRS, CC 086, Université Montpellier 2, Montpellier, France.
J Control Release. 2006 Feb 21;110(3):595-604. doi: 10.1016/j.jconrel.2005.10.026. Epub 2005 Dec 27.
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.
通过空间位阻寡核苷酸（ON）进行剪接校正可能会带来重要的临床应用，但需要有效地递送至细胞核。短寡聚赖氨酸尾巴的缀合已被用于在阳性读出测定中递送校正肽核酸（PNA）序列，在该测定中，荧光素酶报告基因的表达严格要求ON与隐蔽剪接位点杂交。我们在该模型系统中研究了细胞摄取机制以及（Lys）8-PNA-Lys构建体的效率。细胞摄取是温度依赖性的，并导致缀合物隔离在细胞质囊泡中，这与内化的内吞机制一致。因此，仅在内体破坏剂如氯喹或0.5M蔗糖存在下才能实现显著的序列特异性剪接校正。这些内体破坏剂不影响游离PNA的活性，也不增加（Lys）8-PNA-Lys的摄取。
