Knockdown of a G protein-coupled receptor through efficient peptide-mediated siRNA delivery.
Knockdown of a G protein-coupled receptor through efficient peptide-mediated siRNA delivery.
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DOI：
10.1016/j.jconrel.2012.05.017
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发表时间：
2012-08
期刊：
影响因子：
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通讯作者：
Jan Hoyer;I. Neundorf
中科院分区：
文献类型：
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作者：
Jan Hoyer;I. Neundorf
近年来，siRNA的治疗应用因其特异性调节基因表达的潜力而成为药物研究的焦点。然而，寡核苷酸必须克服一系列细胞外和细胞内障碍，这就是为什么迫切需要帮助克服这些障碍的递送系统。将核酸转运到细胞膜外的一种有前途的方法是使用细胞穿透肽（CPP），它能够自主穿过质膜。最近，我们合成了截短的人降钙素（hCT）的分支衍生物，并确定它们是非共价基因传递的有效载体。在这里，我们描述了两种新型分支 hCT 衍生物，它们通过与脂肪酸或内体溶解肽序列缀合而优化，以实现 siRNA 的有效细胞内递送。作为靶点，我们选择人类 NPY Y1 受体 (NPY1R)，它属于 G 蛋白偶联受体家族，因此构成了与各种疾病相关的复杂治疗靶点的模型。例如，Y1 受体表达的敲低为骨质疏松症提供了潜在的治疗方法。我们提出了一种读出系统，可以对蛋白质以及 mRNA 水平上受体表达的诱导敲低进行定量。这项研究的结果是，我们可以证明本文提出的细胞穿透肽有效地将 siRNA 转运到 HEK-293 细胞中，而不诱导细胞毒性，并且敲低率与通过脂转染获得的敲低率相当。
In recent years, therapeutic applications of siRNAs have come into the focus of pharmaceutical research owing to their potential to specifically regulate gene expression. However, oligonucleotides have to overcome a series of extracellular and intracellular barriers which is why delivery systems helping to overcome these barriers are desperately needed. A promising approach to transport nucleic acids beyond cellular membranes is the use of cell-penetrating peptides (CPPs), which are able to autonomously cross the plasma membrane. Recently, we synthesized branched derivatives of truncated human calcitonin (hCT) and identified them as efficient vehicles for non-covalent gene delivery. Here we describe two novel branched hCT-derivatives that are optimized for efficient intracellular delivery of siRNA by conjugation with either a fatty acid or an endosomolytic peptide sequence. As target we chose the human NPY Y1receptor (NPY1R), which belongs to the family of G protein-coupled receptors and thus constitutes a model for complex therapeutic targets related to various disorders. For instance, knockdown of Y1receptor expression offers a potential therapy for osteoporosis. We present a read-out system that allows for the quantitation of the induced knockdown of receptor expression on the protein as well as on the mRNA level. As a result of this study, we could show that the herein presented cell-penetrating peptides effectively transport siRNA into HEK-293 cells without inducing cytotoxicity and that the knockdown rates are comparable to those obtained by lipofection.