Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown

被引：506

作者：

Wittrup, Anders ^{[1
,2
]}

Ai, Angela ^{[1
]}

Liu, Xing ^{[1
]}

Hamar, Peter ^{[1
,3
]}

Trifonova, Radiana ^{[1
]}

Charisse, Klaus ^{[4
]}

Manoharan, Muthiah ^{[4
]}

Kirchhausen, Tomas ^{[1
,5
,6
]}

Lieberman, Judy ^{[1
,6
]}

机构：

[1] Harvard Univ, Sch Med, Boston Childrens Hosp, Program Cellular & Mol Med, Boston, MA 02163 USA

[2] Lund Univ, Dept Clin Sci, Sect Oncol & Pathol, Lund, Sweden

[3] Semmelweis Univ, Inst Pathophysiol, H-1085 Budapest, Hungary

[4] Alnylam Pharmaceut, Cambridge, MA USA

[5] Harvard Univ, Sch Med, Dept Cell Biol, Boston, MA USA

[6] Harvard Univ, Sch Med, Dept Pediat, Boston, MA 02115 USA

来源：

NATURE BIOTECHNOLOGY | 2015年 / 33卷 / 08期

基金：

美国国家卫生研究院; 瑞典研究理事会;

关键词：

INTRACELLULAR TRAFFICKING; RNAI THERAPEUTICS; DELIVERY; NANOPARTICLES; AUTOPHAGY; PROTEIN; CELLS; GLYCOSPHINGOLIPIDS; MATURATION; REPORTER;

D O I：

10.1038/nbt.3298

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA release occurred invariably from maturing endosomes within similar to 5-15 min of endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release. Gene knockdown occurred within a few hours of release and required <2,000 copies of cytosolic siRNAs. The ability to detect cytosolic release of siRNAs and understand how it is regulated will facilitate the development of rational strategies for improving the cytosolic delivery of candidate drugs.

引用

页码：870 / +

页数：9

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