A mechanistic study on the cellular uptake, intracellular trafficking, and antisense gene regulation of bottlebrush polymer-conjugated oligonucleotides.
A mechanistic study on the cellular uptake, intracellular trafficking, and antisense gene regulation of bottlebrush polymer-conjugated oligonucleotides.
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DOI：
10.1039/d2cb00149g
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发表时间：
2023-02-08
影响因子：
4.1
通讯作者：
Sun, Tingyu
中科院分区：
文献类型：
--
作者：
Zhang, Lei;Wang, Yuyan;Chen, Peiru;Wang, Dali;Zhang, Zheyu;Wang, Ruimeng;Kang, Xi;Fang, Yang;Lu, Hao;Cai, Jiansong;Ren, Mengqi;Dong, Sijia;Zhang, Ke;Sun, Tingyu
我们开发了一种以瓶刷聚合物-反义寡核苷酸（ASO）偶联物形式的非阳离子转染载体。这些药物被称为pacDNA（聚合物辅助DNA压实），在体内表现出改善的生物制药特性和反义效力，同时抑制非反义副作用。尽管如此，人们对pacDNA的细胞摄取、亚细胞运输和基因敲低的机制仍然缺乏了解。在这里，我们发现pacDNA主要通过清除剂受体介导的内吞作用和巨噬细胞作用进入人非小细胞肺癌细胞（NCI-H358），并通过细胞内溶酶体途径运输。尽管转染某些游离ASOs会导致KRAS mRNA依赖核糖核酸酶H1 （RNase H）降解，但pacDNA在蛋白水平上显著降低靶基因表达（KRAS），而在mRNA水平上则不会。此外，pacDNA的反义活性不依赖于ASO的化学修饰，这表明pacDNA一直起着空间阻滞剂的作用。我们发现一种瓶刷状聚合物-反义寡核苷酸偶联物（称为pacDNA）通过清道夫受体介导的内吞作用和微胞吞作用进入癌细胞，通过内溶酶体途径运输，并发挥空间阻滞剂的作用。
We have developed a non-cationic transfection vector in the form of bottlebrush polymer-antisense oligonucleotide (ASO) conjugates. Termed pacDNA (polymer-assisted compaction of DNA), these agents show improved biopharmaceutical characteristics and antisense potency in vivo while suppressing non-antisense side effects. Nonetheless, there still is a lack of the mechanistic understanding of the cellular uptake, subcellular trafficking, and gene knockdown with pacDNA. Here, we show that the pacDNA enters human non-small cell lung cancer cells (NCI-H358) predominantly by scavenger receptor-mediated endocytosis and macropinocytosis and trafficks via the endolysosomal pathway within the cell. The pacDNA significantly reduces a target gene expression (KRAS) in the protein level but not in the mRNA level, despite that the transfection of certain free ASOs causes ribonuclease H1 (RNase H)-dependent degradation of KRAS mRNA. In addition, the antisense activity of pacDNA is independent of ASO chemical modification, suggesting that the pacDNA always functions as a steric blocker. We show that a bottlebrush polymer-antisense oligonucleotide conjugate (termed pacDNA) enters cancer cells by scavenger receptor-mediated endocytosis and micropinocytosis, trafficks via the endolysosomal pathway, and functions as a steric blocker.
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影响因子：
5.3
作者：
DeVos, Sarah L.;Goncharoff, Dustin K.;Miller, Timothy M.
通讯作者：
Miller, Timothy M.
影响因子：
11.8
作者：
Macia, Eric;Ehrlich, Marcelo;Kirchhausen, Tomas
通讯作者：
Kirchhausen, Tomas
DOI：
10.1073/pnas.1605731113
发表时间：
2016-09-27
影响因子：
11.1
作者：
Hammond, Suzan M.;Hazell, Gareth;Wood, Matthew J. A.
通讯作者：
Wood, Matthew J. A.
影响因子：
5.5
作者：
Hakem, IF;Lal, J;Bockstaller, MR
通讯作者：
Bockstaller, MR
影响因子：
168.9
作者：
Cirak, Sebahattin;Arechavala-Gomeza, Virginia;Guglieri, Michela;Feng, Lucy;Torelli, Silvia;Anthony, Karen;Abbs, Stephen;Garralda, Maria Elena;Bourke, John;Wells, Dominic J.;Dickson, George;Wood, Matthew J. A.;Wilton, Steve D.;Straub, Volker;Kole, Ryszard;Shrewsbury, Stephen B.;Sewry, Caroline;Morgan, Jennifer E.;Bushby, Kate;Muntoni, Francesco
通讯作者：
Muntoni, Francesco