Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates
Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates
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DOI：
10.1016/j.jconrel.2005.10.026
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发表时间：
2006-02-21
影响因子：
10.8
通讯作者：
Lebleu, B
中科院分区：
文献类型：
--
作者：
Abes, S;Williams, D;Lebleu, B
空间阻断寡核苷酸(ON)的剪接纠正可能会导致重要的临床应用，但需要有效的递送到细胞核。在阳性读出实验中，短寡聚赖氨酸尾巴的接合被用来传递正确的肽核酸(PNA)序列，在该实验中，荧光素酶报告基因的表达严格要求杂交到隐蔽的剪接位点。在这个模型体系中，我们研究了细胞摄取的机制和a(Lys)(8)-PNA-Lys结构的效率。细胞摄取是温度依赖的，并导致结合物在细胞质小泡中的隔离，符合内化的内吞机制。因此，只有在氯喹或0.5M蔗糖等内体干扰剂存在的情况下，才能实现显着和序列特异性的剪接纠正。这些内小体干扰剂不影响游离PNA的活性，也不增加(Lys)(8)-PNA-Lys的摄取。(C)2005 Elsevier B.V.保留所有权利。
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. (C) 2005 Elsevier B.V. All rights reserved.