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

外泌体是一种小的纳米级细胞膜囊泡，在多种疾病的细胞间通讯中发挥重要作用。肿瘤源性外泌体具有促肿瘤作用，导致肿瘤生长和存活、血管生成和转移。MicroRNAs （miRNAs）是一种与靶mrna特异性相互作用从而抑制基因表达的小RNA分子。许多mirna在癌症中表现出异常的高水平，从而参与肿瘤发生和转移的过程。因此，mirna可以很好地作为抗mirna分子治疗抑制的靶标，为此，已经引入了抗mirna寡核苷酸（antimir）。虽然探索了不同的抗药物体内治疗性递送技术，但需要解决几个问题，包括通常低疗效、非特异性递送、毒性或不良副作用。值得注意的是，mirna可以通过外泌体运输，并被其他细胞选择性地摄取。这使得外泌体作为小RNA分子（如anti - irs）的运载工具变得特别有趣。在这个项目中，我们现在打算通过外泌体递送首次建立一种治疗性的基于抗mir的miRNA抑制，并探索使用患者外泌体的优势。为此，我们将重点讨论结直肠癌（CRC）。外泌体也可以作为生物标志物，外泌体mirna的分析代表了一种新的诊断/预后工具。这种方法的优势在于外泌体中这些mirna的选择性富集，这可能与细胞-细胞通信特别相关。这是莱比锡大学与俄罗斯Petrov肿瘤研究所（Anastasia Malek博士）的德俄合作项目，旨在(i)详细分析CRC患者血液外泌体中的miRNA谱，以及结果与CRC各种癌症亚型、疾病分期、化疗反应等相关临床参数的相关性（俄罗斯项目部分）；（ii）开发新的基于外泌体的抗ir传递和miRNA抑制策略，用于选定的miRNA候选物（Leipzig）。德国项目部分是该申请的主题，并涵盖以下目标：目标1：建立创新的基于外泌体的抗ir输送系统（Exo-antimiRs）。目的2a：评估新开发的exo -anti - ir策略对俄罗斯筛选中发现的新型miRNA候选物的抑制作用。目的2b：利用结直肠癌患者的外泌体来产生定制的外泌体-anti - mir。目的3：探讨新开发的基于患者外泌体的抗ir策略（Exo-antimiRs）在临床前体外和体内的抗肿瘤和抗转移潜力。

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.