Polyplexes Traffic through Caveolae to the Golgi and Endoplasmic Reticulum en Route to the Nucleus
Polyplexes Traffic through Caveolae to the Golgi and Endoplasmic Reticulum en Route to the Nucleus
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DOI：
10.1021/mp200583d
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发表时间：
2012-05-01
影响因子：
4.9
通讯作者：
Sullivan, Millicent O.
中科院分区：
文献类型：
--
作者：
Reilly, Meghan J.;Larsen, John D.;Sullivan, Millicent O.
参与非病毒基因载体内化及其随后向细胞核运输的细胞机制直接影响其治疗效率。因此，确定关键的内吞途径和细胞器有助于多聚体成功转移到细胞核，这为改进载体设计提供了新的机会。先前，我们发现编码已知参与染色质激活的序列的组蛋白H3尾部肽与聚亚胺（PEI）表现出协同基因传递活性。含有H3和PEI的多聚体对溶酶体转运的内吞途径的依赖性降低，对通过高尔基体逆行转运的抑制剂的敏感性增强。因此，我们试图确定是否通过高尔基体和/或内质网（ER）的囊泡摄取和运输先于核递送。通过使用一组化学内吞抑制剂，我们确定H3多聚体比PEI多聚体更大程度地利用了空洞途径。小泡菌介导的内吞作用被发现是H3/PEI-pDNA多聚体表达基因的有效途径，与先前对聚合物介导的基因传递的研究一致。此外，在仅5分钟的孵化期后，多聚体基本上在内质网内共定位，并利用逆行高尔基到内质网的途径，其水平与感染期间已知通过这些途径运输的病原体相似。本研究的结果扩大了我们对空洞多丛如何转运到细胞核的理解，并为高尔基-内质网途径在转染中的作用提供了新的证据。这些发现提出了新的设计标准和机会，以战略性地靶向非病毒基因传递载体。
The cellular machinery involved in the internalization of nonviral gene carriers and their subsequent trafficking to the nucleus directly impacts their therapeutic efficiency. Hence, identifying key endocytic pathways and organelles that contribute to the successful transfer of polyplexes to the nucleus generates new opportunities for improving carrier design. Previously, we showed that histone H3 tail peptides encoding a sequence known to participate in chromatin activation exhibit synergistic gene delivery activity with poly(ethylenimine) (PEI). Polyplexes containing H3 and PEI exhibited a reduced dependence on endocytic pathways that trafficked to lysosomes, and had enhanced sensitivity to an inhibitor associated with retrograde trafficking through the Golgi apparatus. Thus, we sought to determine whether caveolar uptake and transport through the Golgi and/or endoplasmic reticulum (ER) preceded nuclear delivery. By the use of a panel of chemical endocytic inhibitors, we determined that H3 polyplexes utilized caveolar pathways to a greater degree than PEI polyplexes. Caveolae-mediated endocytosis was found to be a productive route for gene expression by the H3/PEI-pDNA polyplexes, consistent with previous studies of polymer-mediated gene delivery. Additionally, the polyplexes substantially colocalized within the ER after only 5 min of incubation, and utilized retrograde Golgi-to-ER pathways at levels similar to pathogens known to traffic by these routes during infection. The results of this study have expanded our understanding of how caveolar polyplexes are trafficked to cell nuclei, and provide new evidence for the role of Golgi-ER pathways in transfection. These findings suggest new design criteria and opportunities to stragetically target nonviral gene delivery vehicles.