Intracellular processing of immunostimulatory CpG-siRNA: Toll-like receptor 9 facilitates siRNA dicing and endosomal escape.
Intracellular processing of immunostimulatory CpG-siRNA: Toll-like receptor 9 facilitates siRNA dicing and endosomal escape.
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DOI：
10.1016/j.jconrel.2013.06.007
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发表时间：
2013-09-28
期刊：
影响因子：
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通讯作者：
Kortylewski M
中科院分区：
文献类型：
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作者：
Nechaev S;Gao C;Moreira D;Swiderski P;Jozwiak A;Kowolik CM;Zhou J;Armstrong B;Raubitschek A;Rossi JJ;Kortylewski M
配备有CpG寡脱氧核糖核苷酸的切丁底物siRNA克服了细胞特异性siRNA递送中的主要障碍。CpG-siRNA分子被TLR 9+细胞主动内化，而不需要转染试剂，导致体外和体内RNA干扰。在这里，我们阐明了CpG-siRNA在靶细胞中加工的分子机制。我们表明，摄取到早期内体（EE）后不久，CpG和siRNA部分的共轭物在Dicer核酸内切酶的存在下解偶联。切割的siRNA分子从内体易位到内质网，在那里它们可以与RNA干扰机制相互作用。我们先前观察到，尽管TLR 9不参与CpG-siRNA摄取，但它对于诱导基因沉默是不可或缺的。为了解释TLR 9在CpG-siRNA的细胞内加工中的作用，我们使用了来自野生型和Tlr 9缺陷小鼠的原代巨噬细胞。缺乏TLR 9的巨噬细胞显示缀合物的CpG和siRNA部分的延长的内体共定位。然而，原位邻位连接试验表明，Tlr 9消融不干扰CpG-siRNA与Dicer的相互作用。使用用pH敏感染料标记的CpG-siRNA，我们最终确定巨噬细胞中TLR 9的缺乏导致siRNA在内体中的显著保留。因此，TLR 9促进CpG-siRNA解偶联后的关键步骤，即切割的siRNA的细胞质释放。这些发现表明，这类免疫刺激siRNA可能受益于某些内体免疫受体（如TLR 9）的激活，从而增强基因沉默和治疗功效。
Dicer-substrate siRNAs equipped with CpG oligodeoxyribonucleotides overcome the major hurdle in cell-specific siRNA delivery. The CpG-siRNA molecules are actively internalized by TLR9+ cells, without the need for transfection reagents, leading to RNA interference both in vitro and in vivo. Here, we elucidate the molecular mechanisms of CpG-siRNA processing in target cells. We show that shortly after uptake into early endosomes (EE), CpG and siRNA parts of the conjugate are uncoupled in the presence of Dicer endonuclease. Diced siRNA molecules are translocated from endosomes to endoplasmic reticulum, where they can interact with the RNA interference machinery. We previously observed that even though TLR9 is not involved in CpG-siRNA uptake, it is indispensable for induction of gene silencing. To explain the role of TLR9 in intracellular processing of CpG-siRNA, we used primary macrophages derived from wild-type and Tlr9-deficient mice. Macrophages lacking TLR9 showed extended endosomal colocalization of CpG and siRNA parts of the conjugate. However, Tlr9 ablation did not interfere with the interaction of CpG-siRNA with Dicer as shown by in situ proximity ligation assay. Using CpG-siRNA labeled with pH-sensitive dye, we finally identified that lack of TLR9 in macrophages resulted in significant retention of the siRNA in endosomes. Thus, TLR9 facilitates the critical step following CpG-siRNA uncoupling, which is cytoplasmic release of the diced siRNA. These findings suggest that the class of immunostimulatory siRNAs may benefit from activation of certain endosomal immune receptors, such as TLR9, in augmented gene silencing and therapeutic efficacy.
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影响因子：
46.9
作者：
Dassie, Justin P.;Liu, Xiu-ying;Thomas, Gregory S.;Whitaker, Ryan M.;Thiel, Kristina W.;Stockdale, Katie R.;Meyerholz, David K.;McCaffrey, Anton P.;McNamara, James O., II;Giangrande, Paloma H.
通讯作者：
Giangrande, Paloma H.
影响因子：
3.7
作者：
Li L;Zhu D;Huang L;Zhang J;Bian Z;Chen X;Liu Y;Zhang CY;Zen K
通讯作者：
Zen K
影响因子：
64.8
作者：
Chendrimada, TP;Gregory, RI;Shiekhattar, R
通讯作者：
Shiekhattar, R
影响因子：
64.8
作者：
Castanotto, Daniela;Rossi, John J.
通讯作者：
Rossi, John J.
影响因子：
64.5
作者：
通讯作者：
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