Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor.
Intracellular delivery of an antisense oligonucleotide via endocytosis of a G protein-coupled receptor.
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DOI：
10.1093/nar/gkq534
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发表时间：
2010-10
影响因子：
14.9
通讯作者：
Juliano RL
中科院分区：
文献类型：
--
作者：
Ming X;Alam MR;Fisher M;Yan Y;Chen X;Juliano RL
胃泌素释放肽受体（GRPR）是G蛋白偶联受体超家族的一员，已被用于成像和治疗剂的受体介导靶向；在这里，我们将其应用扩展到寡核苷酸递送。将剪接转移的反义寡核苷酸与炸弹蛋白（BBN）肽结合，并在稳定转染荧光素酶基因（被异常剪接的内含子中断）的表达GRPR的PC3细胞中检测其细胞内递送。与未修饰的寡核苷酸相比，BBN偶联物产生了显著更高的荧光素酶表达，这种增加被过量的BBN肽逆转。动力学研究揭示了受体介导的饱和内吞作用和非饱和胞饮作用的结合，以摄取缀合物。饱和摄取的Km值与药理反应的EC50值相似，表明受体介导的内吞作用是反应的主要因素。使用药物和分子内吞抑制剂表明，该偶联物利用网格蛋白、肌动蛋白和动力蛋白依赖途径进入PC3细胞。bbn偶联物部分定位于与Rab7或Rab9相关的膜囊泡中，表明它被转运到晚期核内体和反式高尔基网络。这些观察结果表明，bbn寡核苷酸缀合物通过GRPR介导的内吞作用进入细胞，然后转运到深层膜室。
Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The Km value for saturable uptake was similar to the EC50 value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments.
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影响因子：
4.9
作者：
Juliano R;Bauman J;Kang H;Ming X
通讯作者：
Ming X
影响因子：
64.8
作者：
Castanotto, Daniela;Rossi, John J.
通讯作者：
Rossi, John J.
影响因子：
2.9
作者：
Kang, SH;Cho, MJ;Kole, R
通讯作者：
Kole, R
有效递送反义和siRNA寡核苷酸的机制和策略。
影响因子：
14.9
作者：
Juliano, Rudy;Alam, Md. Rowshon;Dixit, Vidula;Kang, Hyumin
通讯作者：
Kang, Hyumin
影响因子：
4.9
作者：
Gao K;Huang L
通讯作者：
Huang L