Folate Receptor-Mediated siRNA Delivery to Cancer Cells

描述（由申请人提供）：小干扰RNA （siRNA）的转录后基因沉默已经发展成为一种高效和序列特异性的下调任何靶基因的有力工具。原则上，siRNA可能成为开发下一代高效、低毒副作用的抗病毒和抗癌药物的基础。然而，目前还没有治疗上可接受的siRNA递送方法。虽然叶酸受体（FR）介导的功能药物通过内吞作用递送到癌细胞已被证明对FR阳性癌细胞是有效的和高度特异性的，但叶酸和siRNA之间的直接偶联化学直到现在还没有容易实现。利用最近在Huang （PI）实验室开发的RNA生物偶联方法，本研究将开发一种新的基于叶酸受体的siRNA递送策略，针对表达fr的癌细胞中的特定靶基因。结合最近在fr介导的药物传递和RNA干扰（RNAi）等不同领域的进展，结合PI在化学/RNA和Dr. Quo （co-Pi）在细胞生物学方面的专业知识，我们将合成叶酸偶联siRNA并研究它们的细胞传递和功能，针对癌细胞标记物尿激酶纤溶酶原激活物受体（uPAR）。我们将验证一个假设，即通过浮子共轭siRNA下调癌细胞中uPAR的表达将有效抑制与肿瘤生长相关的uPAR的细胞活性。叶酸sirna的递送效率和特异性将在KB癌细胞（人鼻咽部表皮样癌细胞系）上进行分析。RNAi效应将通过确定mRNA和蛋白水平上uPAR的表达以及uPAR的细胞功能分析来评估。我们期望在KB细胞中递送叶酸sirna对抗uPAR将比目前其他可用的方法更有效和特异性。因此，期望通过uPAR沉默抑制肿瘤生长。拟议研究的结果可能会导致fr介导的siRNA递送针对表达fr的癌细胞中的特定靶基因的一般策略和方法的发展，因此可能直接导致癌症治疗应用。

DESCRIPTION (provided by applicant): Posttranscriptional gene silencing by small interfering RNA (siRNA) has evolved into a powerful tool for down regulation of any target gene(s) with both high efficiency and sequence specificity. In principle, siRNA may become the basis for developing the next generation of antiviral and anti-cancer agents with high potencies and low side toxicities. However, no therapeutically acceptable delivery methods of siRNA are currently available. Although folate receptor (FR)-mediated delivery of functional agents by endocytosis to cancer cells has been shown to be efficient and highly specific towards FR-positive cancer cells, the chemistry of direct coupling between folate and siRNA has not been readily achievable until now. Capitalizing on the recent RNA bio-conjugation methods developed in the Huang (PI) laboratory, this proposed research will develop a novel folate receptor-based siRNA delivery strategy against specific target genes in FR-expressing cancer cells. Integrating the recent advances in such diverse areas as FR-mediated drug delivery and RNA interference (RNAi) with the expertise of the PI in chemistry/RNA and Dr. Quo (co-Pi) in cell biology, we will synthesize folate-conjugated siRNA and investigate their cellular delivery and functions, targeting a cancer cell marker, urokinase plasminogen activator receptor (uPAR). We will test the hypothesis that down- regulation uPAR expression in cancer cells by floate-conjugated siRNA will effectively inhibit the cellular activity of uPAR associated with tumor growth. Both the delivery efficiency and specificity of Folate-siRNA will be analyzed using KB cancer cells (a human nasopharyngeal epidermoid carcinoma cell line). RNAi effects will be assessed by determining the expression of uPAR at mRNA and protein levels and by cellular functional analysis of uPAR. We expect that the delivery of Folate-siRNA against uPAR in KB cells will be more efficient and specific than other current available methods. As a consequence, tumor growth suppression through uPAR silencing is expected. Results from the proposed research will likely lead to the development of general strategies and methods for FR-mediated delivery of siRNA against specific target genes in FR-expressing cancer cells and, therefore, may lead directly to cancer therapeutic applications.