Novel extra cellular RNA-based combinatorial RNA inhibition therapy

描述（由申请人提供）：RNA分子分泌于细胞外空间（exRNAs），作为内分泌信号改变癌细胞的表型。在本应用中，我们将重点关注一类名为microrna的小非编码grna。我们提出了一种创新的治疗理念：通过增强肿瘤细胞中致癌microrna作为exRNAs的分泌或通过阻断分泌机制阻断抑制microrna在细胞外空间的递送，从而消耗肿瘤细胞中的致癌microrna。我们的主要目标是开发使用miRNA和siRNA联合的新策略，通过影响exrna的产生来最大化治疗效果。我们的建议集中在鉴定和靶向特异性肿瘤来源的exRNA和外泌体，从而导致新的治疗方法和改善治疗结果。在UH2期，我们将利用卵巢癌（OC）细胞系和来自OC患者肿瘤的细胞，通过基于分泌测量的siRNA文库全基因组筛选，通过对来自同一患者的肿瘤、血浆和腹水的“配对”样本进行小RNA深度测序，以及通过对先导exRNA候选物的功能研究，确定新的基于exRNA的癌症治疗先导候选物。在UH3阶段，我们将通过测试在UH2中鉴定的exRNA先导物在成熟的OC小鼠原位模型中的抗肿瘤功效以及它们在成熟的临床前方案中的安全性来实现临床前优化。探索性UH2期的目的是确定和验证用于临床前研究的新型exRNA治疗候选物的功能。在UH3期，我们将制定检测方法的验证方法，确定其在组织和血浆中的活性、药理学，建立动物模型的给药方案，设计合理的抗肿瘤途径。利用我们的纳米脂质体递送技术，我们还将进行靶标调节和疗效研究，以在建议的剂量范围内，为基于exrna的候选治疗方案的安全性提供明确的证据。我们的方法，通过使用一种新的调控exRNAs- miRNAs，可以大大提高癌症治疗的疗效。我们还将首次展示使用纳米脂质体- sirna - exrna靶向来产生癌症治疗的协同促进效应。由于卵巢癌的高死亡率，我们将重点放在卵巢癌上，但我们的发现对任何类型的癌症都有应用价值。这项提议带来了在miRNA （Calin博士）、siRNA （ood博士）、纳米颗粒递送（Lopez-Berestein博士）和外泌体生物学（O'Halloran博士）等领域工作了十多年的科学家的协同能力。该团队拥有专业知识和协同动力，通过进行高度创新的研究来实现新的exRNA治疗开发，并在过去开发了基于rna的治疗方法。

DESCRIPTION (provided by applicant): RNA molecules are secreted in extracellular spaces (exRNAs) and act as endocrine signals altering the phenotypes of cancer cells. In this application we will focus on the class of small non-codingRNAs named microRNAs. We propose an innovative therapeutic concept: the depletion of oncogenic microRNAs from malignant cells by enhancing their secretion as exRNAs or blocking suppressor microRNAs' delivery in extracellular space by blocking secretion mechanisms. Our main goal is to develop new strategies using combined miRNA and siRNA to maximize therapeutic benefit by affecting the production of exRNAs. Our proposal centers on the identification and targeting of specific tumor-derived exRNA and exosomes leading to novel therapies and improved therapeutic outcomes. In UH2 phase of the grant, using ovarian cancer (OC) cell lines and cells derived from OC patient tumors, we will identify novel exRNA-based cancer therapeutic lead candidates by siRNA library genome-wide screening based on secretion measurements, by small RNA deep sequencing in "paired" samples from tumors, plasma and ascites from the same patient, and by functional studies for the lead exRNA candidates. In the UH3 phase, we will achieve pre-clinical optimization by testing the exRNA lead(s) identified in the UH2 for antitumor efficacy in well-established OC murine orthotopic models and their safety using well-established preclinical protocols. The aims in exploratory UH2 phase are designed to identify and validate the function of novel exRNA therapeutic candidates for pre- clinical studies. In UH3 phase, we will formulate the methods for the validation of the assays to determine the activity, pharmacology in tissue and plasma to establish dosage schedules in animal models for the design of rational anti-tumor approaches. Using our nanoliposomal delivery technology, we will also conduct target modulation and efficacy studies to generate clear evidence regarding the safety of exRNA-based therapeutic candidates in the proposed dose range. Our approach, by using a new category of regulatory exRNAs- miRNAs, could substantially enhance therapeutic efficacy for cancer treatment. We will also demonstrate, for the first time, the use of nanoliposome-siRNA-exRNA targeting to generate a synergistic boosting effect for treatment of cancers. While we are focusing on OC due to the high mortality associated with this malignancy, our findings have applications for any type of cancer. This proposal brings synergistic capabilities of scientists working for over a decade in the fields of miRNA (Dr. Calin), siRNA (Dr. Sood), nanoparticle delivery (Dr. Lopez-Berestein), and exosome biology (Dr. O'Halloran). The team has the expertise and synergistic drive to achieve novel exRNA therapeutic development by performing highly innovative research, and has developed RNA-based therapies in the past.