Analysis and therapeutic blocking of Exosome-miRNA tumor-derived signaling in colorectal carcinoma (Short title: Exo-antimiR)

外切体是一种小的、纳米级的细胞膜囊泡，在各种疾病的细胞间通讯中发挥着重要的作用。肿瘤来源的外切体可以产生促肿瘤作用，导致肿瘤的生长和存活、血管生成和转移。MicroRNAs(MiRNAs)是一种小的RNA分子，它与靶mRNAs特异性地相互作用，从而抑制基因的表达。许多miRNAs在癌症中表现出异常高的水平，从而参与了肿瘤的发生和转移过程。因此，miRNAs可以很好地作为抗miRNA分子治疗抑制的靶点，为此，已经引入了抗miRNA寡核苷酸(AntimiRs)。虽然人们探索了不同的体内治疗性给药技术，但有几个问题需要解决，包括通常效率低、非特异性给药、毒性或不想要的副作用。值得注意的是，miRNAs可以被外体运输，并被其他细胞选择性地摄取。这使得外体作为抗核糖核酸分子等小分子的运输工具特别有趣。在这个项目中，我们现在打算首次建立一种通过外切体递送的基于治疗作用的抗iR的miRNA抑制，并探索使用患者的外切体的优势。为此，我们将重点关注结直肠癌(CRC)。外体也可以作为生物标记物，外体miRNAs的图谱代表了一种新的诊断/预后工具。这种方法的优势在于选择性地在外体内富集这些miRNAs，这可能在细胞间通信方面特别相关。这是德国-俄罗斯莱比锡大学与俄罗斯彼得罗夫肿瘤学研究所(Anastasia Malek博士)合作的项目，旨在(I)详细分析来自CRC患者血液的exosome中的miRNA谱，并将结果与不同的CRC癌症亚型、疾病分期、化疗疗效和其他相关临床参数之间的相关性进行分析(项目部分俄罗斯)，以及(Ii)针对选定的miRNA候选对象开发基于exosome的新型抗iR传递和miRNA抑制策略(莱比锡)。德国的项目部分是本申请的主题，涵盖以下目标：目标1：建立创新的基于外显子的抗iR递送系统(Exo-antimiRs)。目的2a：评估新开发的抑制俄罗斯筛选中发现的新的miRNA候选的Exo-AntimiR策略。目的2b：利用结直肠癌患者的外显子产生量身定制的外源抗iRs。目的：探讨新开发的基于外显子体抗肿瘤转移策略(Exo-AntimiRs，Exo-AntimiRs)的体外和体内抗肿瘤转移潜能。

Exosomes are small, nanoscale cellular membrane vesicles which can play important roles in cell-cell communication in various diseases. Tumor-derived exosomes can confer pro-tumorigenic effects, leading to tumor growth and survival, angiogenesis and metastasis. MicroRNAs (miRNAs) are small RNA molecules which specifically interact with target mRNAs, thus inhibiting gene expression. Many miRNAs show aberrantly high levels in cancer and thus contribute to the process of tumorigenesis and metastasis. Thus, miRNAs may well serve as targets for their therapeutic inhibition by anti-miRNA molecules, and for this purpose, anti-miRNA oligonucleotides (antimiRs) have been introduced. While different technologies are explored for the therapeutic in vivo delivery of antimiRs, several issues need to be addressed, including often low efficacies, non-specific delivery, toxicity or unwanted side effects. Notably, miRNAs can be transported by exosomes, and selectively taken up by other cells. This makes exosomes particularly interesting as delivery vehicles for small RNA molecules like antimiRs. Within this project, we now intend to establish for the first time a therapeutic antimiR-based miRNA inhibition through exosome delivery, and to explore the advantages of using exosomes from patients. To this end, we will focus on colorectal carcinoma (CRC). Exosomes can also serve as biomarkers, with the profiling of exosomal miRNAs representing a new diagnostic/prognostic tool. The advantage of this approach lies in the selective enrichment of these miRNAs within the exosome that may be particularly relevant in terms of cell-cell communication. This is a German-Russian collaboration project of the University of Leipzig with the Petrov Institute of Oncology, Russia (Dr. Anastasia Malek), aiming at (i) the detailed analysis of miRNA profiles in exosomes from CRC patients blood, and the correlation of the results with various CRC cancer subtypes, disease stages, chemotherapy response and other relevant clinical parameters (project part Russia), and (ii) the development of novel exosome-based antimiR delivery and miRNA inhibition strategies for selected miRNA candidates (Leipzig). The German project parts are subject of this application and cover the following aims: Aim 1: To establish innovative exosome-based antimiR delivery systems (Exo-antimiRs). Aim 2a: To assess the newly developed Exo-antimiR strategy for the inhibition of novel miRNA candidates identified from the Russian screening. Aim 2b: To employ exosomes from CRC patients for the generation of tailor-made Exo-antimiRs. Aim 3: To explore the anti-tumor and anti-metastatic potential of the newly developed patients exosome-based antimiR strategy (Exo-antimiRs) preclinically in vitro and in vivo.