Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes.
Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes.
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DOI：
10.1093/nar/gkr089
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发表时间：
2011-06
影响因子：
14.9
通讯作者：
Bennett CF
中科院分区：
文献类型：
--
作者：
Koller E;Vincent TM;Chappell A;De S;Manoharan M;Bennett CF
单链反义寡核苷酸（SSO）用于调节动物模型中的基因表达，并且正在作为潜在的治疗方法进行研究。为了更好地理解为什么合成的 SSO 会在与靶 RNA 相同的细胞内位置积累，我们分离出了一种新型小鼠肝细胞 SV40 大 T 抗原癌细胞系 MHT，该细胞系在培养数年中仍保持有效吸收 SSO 的能力。序列特异性反义效应在低纳摩尔浓度下得到证实。 SSO 在细胞中的积累与时间和浓度有关。至少有两种不同的细胞途径导致 SSO 在细胞中积累：一种是导致溶酶体中积累的非生产性途径，另一种是 SSO 获得目标 RNA 的功能性摄取途径。我们证明，布雷菲德菌素 A 和氯喹会抑制功能性摄取（通过靶标 mRNA 的序列特异性减少来定义）。衔接蛋白 AP2M1 的 siRNA 抑制剂可阻断功能性摄取，但网格蛋白或小窝蛋白不会阻断。此外，我们记录了用 AP2M1 siRNA 治疗小鼠会阻断肝组织的功能性摄取。 SSO 的功能性摄取似乎是由一种新型的网格蛋白和小窝蛋白独立的内吞过程介导的。
Single-stranded antisense oligonucleotides (SSOs) are used to modulate the expression of genes in animal models and are being investigated as potential therapeutics. To better understand why synthetic SSOs accumulate in the same intracellular location as the target RNA, we have isolated a novel mouse hepatocellular SV40 large T-antigen carcinoma cell line, MHT that maintains the ability to efficiently take up SSOs over several years in culture. Sequence-specific antisense effects are demonstrated at low nanomolar concentrations. SSO accumulation into cells is both time and concentration dependent. At least two distinct cellular pathways are responsible for SSO accumulation in cells: a non-productive pathway resulting in accumulation in lysosomes, and a functional uptake pathway in which the SSO gains access to the targeted RNA. We demonstrate that functional uptake, as defined by a sequence-specific reduction in target mRNA, is inhibited by brefeldin A and chloroquine. Functional uptake is blocked by siRNA inhibitors of the adaptor protein AP2M1, but not by clathrin or caveolin. Furthermore, we document that treatment of mice with an AP2M1 siRNA blocks functional uptake into liver tissue. Functional uptake of SSO appears to be mediated by a novel clathrin- and caveolin-independent endocytotic process.
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DOI：
10.1073/pnas.91.24.11762
发表时间：
1994-11-22
影响因子：
11.1
作者：
DEAN, NM;MCKAY, R
通讯作者：
MCKAY, R
影响因子：
5.4
作者：
Kolokoltsov, Andrey A.;Deniger, Drew;Davey, Robert A.
通讯作者：
Davey, Robert A.
DOI：
10.1073/pnas.77.7.3957
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
ALBERTS, AW;CHEN, J;SPRINGER, J
通讯作者：
SPRINGER, J
影响因子：
6.5
作者：
NESTLE, FO;MITRA, RS;NICKOLOFF, BJ
通讯作者：
NICKOLOFF, BJ
影响因子：
5.8
作者：
Geary, Richard S.;Wancewicz, Ed;Bennett, Frank
通讯作者：
Bennett, Frank