RNA Therapeutics for Targeted Treatment of Colon Cancer

 描述：目前迫切需要开发治疗晚期和转移性结肠癌的新策略，因为它们对传统化疗的反应有限。在这项提案中，我们将应用一种新的基于RNA的策略来提供治疗性的miRNA和siRNA用于结肠癌的靶向治疗。我们(郭斌的团队)最近发现miR-627是由骨化三醇诱导的，并且是骨化三醇抑制结肠癌的关键介质(胃肠病学，2013,145(2)：437-46)。我们进一步证明了miR-627的靶点是组蛋白去甲基酶JMJD1A，这是一种具有促进生长活性的表观遗传调节因子。靶向miR-627和JMJD1A的siRNA在体外和体内(裸鼠移植瘤)均能显著抑制结肠癌的生长。我们假设，如果我们能够使用miR-627或siRNA的靶向递送来直接抑制JMJD1A，那么就有可能绕过与骨化三醇相关的副作用，实现肿瘤抑制。该合作研究员(郭培轩)的团队发现了一个异常稳定的phi29 PRNA三向连接(3WJ)基序，可用作构建具有非常高的化学和热力学稳定性的多价RNA纳米颗粒的支架(自然纳米技术，2011，6：658-67；Nano Today，2012，7：245-257)。得到的RNA纳米颗粒具有不同的siRNA、miRNA和肿瘤靶向模块(如RNA适配子)，这些模块保留了它们在体外和体内用于基因沉默和癌症靶向的折叠和独立功能。这些RNA纳米粒子在8M尿素中耐变性，在体外和体内超低浓度下不解离。向小鼠尾静脉系统注射表明，它们保持完好和 与癌症有很强的结合力，不会在肝、肺或其他重要器官积聚。与裸露的siRNA相比，这种纳米技术方法将RNA纳米颗粒的半衰期提高了10倍。3WJ-PRNA纳米粒无毒，表现出良好的药理特性，包括生物分布、药代动力学(稳定性、半衰期和清除率)和不可检测的免疫反应(分子治疗，2011，19：1312-22)。本研究项目的目标是构建由RNA组成的多价纳米粒子，以获得增强或协同治疗结肠癌的疗效。我们将结合(1)针对肿瘤抗原EpCAM或A33 scFv的EpCAM适配子，以与结肠癌特定细胞表面抗原A33特异性结合，从而导致RNA纳米颗粒内化到结肠癌细胞中；以及(2)治疗模块miR-627和/或JMJD1A siRNA，以沉默JMJD1A的表达。重点将放在特定的肿瘤靶向和体内基因沉默治疗原发肿瘤和抑制转移的效率上。我们将系统地评估这一创新的RNA传递平台的生物分布、药代动力学和免疫反应(抗体诱导、干扰素、Toll样免疫和先天免疫、PKR效应和细胞因子诱导)的药理学特性。将评估多价RNA纳米颗粒用于增强化疗的联合效果。我们相信，我们基于RNA的方法将为结肠癌带来创新的治疗方法。

 DESCRIPTION: There is an urgent need to develop novel strategies for treatment of advanced and metastatic colon cancers since they have limited responses to conventional chemotherapies. In this proposal, we will apply a new RNA-based strategy to deliver therapeutic miRNA and siRNA for targeted colon cancer therapy. We (Bin Guo's group) have recently found that miR-627 is induced by calcitriol and acts as a key mediator of calcitriol's suppression of colon cancer (Gastroenterology, 2013, 145(2):437-46). We have further demonstrated that miR-627 targets histone demethylase JMJD1A, an epigenetic regulator that has growth-promoting activity. Both miR-627 and JMJD1A-targeting siRNA can significantly inhibit colon cancer growth in vitro and in vivo (nude mice xenografts). We hypothesize that if we could employ targeted delivery of miR-627 or siRNA to directly inhibit JMJD1A, it would be possible to bypass the side effects associated with calcitriol and achieve tumor suppression. The co-investigator's (Peixuan Guo) group has discovered an unusually stable phi29 pRNA three-way junction (3WJ) motif that can be used as a scaffold to construct multivalent RNA nanoparticles with very high chemical and thermodynamic stability (Nature Nanotechnology, 2011, 6:658-67; Nano Today, 2012, 7:245- 257). The resulting RNA nanoparticles harbor different siRNA, miRNA, and cancer targeting module (such as RNA aptamer) that retain their folding and independent functionalities for gene silencing and cancer targeting both in vitro and in vivo. These RNA nanoparticles are resistant to denaturation in 8 M urea and do not dissociate at ultra-low concentrations in vitro and in vivo. Systemic injection into the tail-vein of mice has revealed that they remain intact and strongly bind to cancers without accumulating in the liver, lungs or other vital organs. This nanotechnology approach has enhanced the half-life of the RNA nanoparticles by 10-fold compared to the bare siRNA. The 3WJ-pRNA nanoparticles are non-toxic and display favorable pharmacological profiles that include biodistribution, pharmacokinetics (stability, half-life, and clearance rate), and undetectable immune responses (Molecular Therapy, 2011, 19:1312-22). The goal of this research project is to construct multivalent nanoparticles composed of RNA, to obtain enhanced or synergistic therapeutic effects for the treatment of colon cancer. We will conjugate (1) EpCAM aptamer for targeting tumor antigen EpCAM or A33 scFv for specific binding to colon cancer specific cell surface antigen A33 resulting in the internalization of RNA nanoparticles into the colon cancer cells; and (2) therapeutic modules, miR-627 and/or JMJD1A siRNA, to silence the expression of JMJD1A. Emphasis will be on specific tumor targeting and efficiency of gene silencing in vivo for treatment of primary tumor as well as suppression of metastasis. We will systematically evaluate the pharmacological profiles concerning biodistribution, pharmacokinetics, and immune responses (antibody induction, and interferon, toll-like and innate immunity, PKR effect, and cytokine induction) of this innovative RNA delivery platform. The combined effect of the multivalent RNA nanoparticles for enhancing chemotherapy will be evaluated. We are confident that our RNA-based approach will result in an innovative therapeutic for colon cancer.