Aptamer-based inhibition of the chemokines CCL17 and CCL22 in allergic and inflammatory reactions

基于适体的趋化因子 CCL17 和 CCL22 抑制过敏和炎症反应

• 批准号: 418270278
• 负责人: Professorin Dr. Irmgard Förster
• 金额: --
• 依托单位: Abteilung Immunologie und Umwelt
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/418270278?language=en
• 关键词: Aptamer; based; inhibition; chemokines; CCL17

The CCR4 ligands CCL17 and CCL22 represent well-known biomarkers for atopic diseases. Using mouse models, we demonstrated a major role of CCL17 in the pathogenesis of contact hypersensitivity (CHS), atopic dermatitis, and other inflammatory diseases, whereas CCR4-deficient mice developed enhanced CHS responses. Pharmacological targeting of the CCL17/CCL22-CCR4 axis has been mainly directed towards CCR4 so far, primarily leading to inhibition of regulatory T cells. Remarkably, patients treated with CCR4 neutralizing antibodies often develop skin rashes (Stevens-Johnson Syndrome). Our hypothesis is that CCL17 and CCL22 exert differential functions via CCR4 or other receptors. Therefore, we aim to generate aptamers, which selectively inhibit either of these chemokines. The advantage of aptamers over neutralizing antibodies is their small size, high affinity and the fact that they can be chemically synthesized. So far, we identified a 2’deoxy-2’fluoro RNA (2’-fRNA) aptamer specific for murine CCL17, which efficiently inhibits CHS in vivo, using Systematic Evolution by Exponential Enrichment (SELEX). We now intend to select further RNA- and DNA aptamers, or alternatively clickmers, directed against human and murine CCL17, and against murine CCL22. For DNA aptamers we will perform manual SELEX and for 2’-fRNA aptamers an automated Biomek NX platform will be used. The enrichment of the nucleic acid libraries will be monitored by next-generation sequencing, and individual sequences will be tested for affinity and binding specificity. The inhibitory capacity of aptamers against human CCL17 will be determined in migration assays using the CCR4+ lymphoma line Mac-1 as well as primary human T cells. In parallel, we will optimize the conditions for therapeutic application of aptamers in the CHS model and explore the possibility of epicutaneous application. To unravel the function of CCL22, we generated novel CCL22-deficient and CCL17/CCL22-double deficient mouse strains using designer nucleases. We will assess the development of CHS and T cell migration in these mice in comparison to CCL17- and CCR4-deficient mice. In addition, we will also study the influence of CCL17 and CCL22 on the selection of regulatory T cells.

CCR4配体CCL17和CCL22是特应性疾病的著名生物标志物。利用小鼠模型，我们证明了CCL17在接触性超敏反应(CHS)、特应性皮炎和其他炎症性疾病的发病机制中起主要作用，而CCR4缺陷小鼠则表现出增强的CHS反应。到目前为止，针对CCL17/CCL22-CCR4轴的药物靶向主要针对CCR4，主要导致对调节性T细胞的抑制。值得注意的是，接受CCR4中和抗体治疗的患者经常出现皮疹(史蒂文斯-约翰逊综合征)。我们的假设是CCL17和CCL22通过CCR4或其他受体发挥不同的功能。因此，我们的目标是产生适体，选择性地抑制这两种趋化因子中的任何一种。与中和抗体相比，适配子的优势在于它们体积小，亲和力高，而且可以化学合成。到目前为止，我们利用系统进化指数富集法(SELEX)鉴定出了小鼠CCL17特异性的2‘脱氧-2’-FRNA(2‘-FRNA)适配子，它能在体内有效地抑制CHS。我们现在打算选择更多针对人和小鼠CCL17以及针对小鼠CCL22的RNA和DNA适配子，或者替代点击分子。对于DNA适配子，我们将执行手动SELEX，而对于2‘-FRNA适配子，将使用自动化的Biomek NX平台。核酸库的丰富将通过下一代测序进行监测，并将测试单个序列的亲和力和结合特异性。适体对人CCL17的抑制能力将在使用CCR4+淋巴瘤株Mac-1和原代人类T细胞的迁移试验中确定。同时，我们将优化适体在CHS模型中的治疗应用条件，并探索皮肤表面应用的可能性。为了揭示CCL22的功能，我们利用设计的核酸酶获得了新的CCL22缺陷和CCL17/CCL22双缺陷小鼠品系。我们将评估这些小鼠与CCL17和CCR4缺陷小鼠的CHS和T细胞迁移的发展。此外，我们还将研究CCL17和CCL22对调节性T细胞选择的影响。