A Chemopreventive Strategy Based on Edible MicroRNAs Produced in Plants

描述（由申请人提供）：结直肠癌（CRC）是美国第三大最常诊断的癌症，也是癌症死亡的主要原因。主要治疗是手术，特别是在CRC的早期阶段，手术可以治愈。然而，疾病的复发是一个主要问题，并且通常是致命的。因此，开发新的CRC化学预防策略仍然是一个高度优先事项。微小RNA（miRNAs）是一类新兴的治疗剂，具有显著的用于治疗癌症的翻译潜力。许多不同形式的癌症，包括CRC，与一种或多种作为肿瘤抑制因子的miRNA的损失或减少的积累有关。在动物模型中，缺失的肿瘤抑制miRNA的恢复可以防止疾病的发生、进展和/或扩散，这表明这些小RNA具有很有希望的治疗作用。然而，目前缺乏用于全身递送治疗性miRNA的有效方法是治疗性miRNA的关键障碍。 它们在癌症治疗中的应用。拟议的试点项目将测试一种新的化学预防策略，用于基于摄入已被生物工程化以产生治疗性miRNA的植物的miRNA替代疗法。这项工作建立在我们有希望的初步结果的基础上，这些结果表明，口服加入三种肿瘤抑制miRNAs（miR-34 a，-143和-145）的混合物的植物RNA，在ApcMin/+小鼠CRC模型中具有显着的化学预防活性，这些miRNAs是用植物miRNAs的3 '-甲基化特征合成的。在拟议的可行性研究的目标1中，我们将确定当口服递送至ApcMin/+小鼠时在抑制肿瘤发生方面最有效的三种miRNA的组合。研究设计是在肿瘤形成之前或之后（分别为预防性和治疗性方案）向小鼠饲喂掺有合成的肿瘤抑制剂miRNA的各种组合的植物RNA，并通过测定肿瘤负荷、施用的治疗性miRNA的水平以及治疗与对照组织中选择的miRNA靶基因的表达来确定治疗的影响。尽管在我们的初步研究和Aim 1中使用的商业合成的miRNA允许快速测试特定的miRNA替换策略，但它们对于翻译到人类来说过于昂贵。因此，在目标2中，我们将建立产生高水平的如目标1中确定的肿瘤抑制miRNA的最有效组合的转基因植物系，并测定当通过定制饮食饲喂ApcMin/+小鼠时它们的影响。在可食用植物中产生治疗性miRNA的概念在基础、翻译和临床应用中具有显著的潜力，并且为目前可用的合成RNA产生方法提供了具有成本效益的替代方案。我们的小组是独特的定位，以开展这一项目的基础上互补的专业知识的调查。万斯博士是植物小RNA生物学的先驱，也是使用植物中产生的基因工程miRNA专利技术的主要共同发明人，而佩纳博士则拥有多年的经验和良好的记录使用小鼠模型系统进行CRC研究。

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is the third most commonly diagnosed cancer and a leading cause of cancer deaths in the US. The primary treatment is surgery, which can be curative especially in early stages of CRC. However, recurrence of the disease is a major problem, and is often fatal. Thus, development of new chemopreventive strategies for CRC remains a high priority. MicroRNAs (miRNAs) are an emerging class of therapeutic agents with significant translational potential for the treatment of cancer. Many different forms of cancer, including CRC, are associated with loss or reduced accumulation of one or more miRNAs that function as tumor suppressors. In animal models, restoration of the missing tumor suppressor miRNA prevents the initiation, progression and/or spread of the disease, suggesting a promising therapeutic role for these small RNAs. However, the current absence of an efficient method for systemic delivery of therapeutic miRNAs is a critical barrier to their use in cancer therapies. The proposed pilot project will test a novel chemopreventive strategy for miRNA replacement therapy based on ingestion of plants that have been bioengineered to produce therapeutic miRNAs. The work builds on our promising preliminary results showing that oral administration of plant RNA spiked with a cocktail of three tumor-suppressor miRNAs (miR-34a, -143, and -145), synthesized with the 3'-methylation characteristic of plant miRNAs, has significant chemopreventive activity in the ApcMin/+ mouse model of CRC. In Aim 1 of the proposed feasibility study, we will determine the combination of the three miRNAs that is most effective in suppressing tumorigenesis when delivered orally to ApcMin/+ mice. The research design is to feed plant RNA spiked with various combinations of synthesized tumor suppressor miRNAs to the mice either before or after tumors have developed (preventive and therapeutic regimens, respectively) and determine the impact of the treatment by assaying tumor burden, level of administered therapeutic miRNAs and expression of select miRNA target genes in treatment versus control tissues. Although the commercially synthesized miRNAs used in our pilot study and in Aim 1 allow rapid testing of specific miRNA replacement strategies, they are prohibitively expensive for translation to humans. Therefore, in Aim 2, we will establish transgenic plant line(s) producing high levels of the most effective combination(s) of tumor suppressor miRNAs as determined in Aim 1, and assay their impact when fed to ApcMin/+ mice via a custom diet. The concept of producing therapeutic miRNAs in edible plants has significant potential in basic, translational and clinical applications and provides a cost-effective alternative to currently available synthetic RNA production methods. Our group is uniquely positioned to undertake this project based on complementary expertise of the investigators. Dr. Vance is a pioneer in small RNA biology in plants and is primary co-inventor on patented technology for the use of genetically engineered miRNAs produced in plants, and Dr. Pena has many years of experience and a strong record of achievement in CRC research using mouse model systems.