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

CCR 4配体CCL 17和CCL 22代表了众所周知的特应性疾病的生物标志物。使用小鼠模型，我们证明了CCL 17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的主要作用，而CCR 4缺陷小鼠产生了增强的CHS反应。迄今为止，CCL 17/CCL 22-CCR 4轴的药理学靶向主要针对CCR 4，主要导致调节性T细胞的抑制。值得注意的是，接受CCR 4中和抗体治疗的患者经常出现皮疹（Stevens-Johnson综合征）。我们的假设是CCL 17和CCL 22通过CCR 4或其他受体发挥不同的功能。因此，我们的目标是产生适体，选择性地抑制这些趋化因子。适体相对于中和抗体的优势在于它们的小尺寸、高亲和力以及它们可以化学合成的事实。到目前为止，我们使用指数富集系统进化（SELEX）鉴定了对鼠CCL 17特异的2 '脱氧-2'氟RNA（2 '-fRNA）适体，其有效地抑制体内CHS。我们现在打算选择针对人和鼠CCL 17以及针对鼠CCL 22的其他RNA和DNA适体，或者可替代地点击体。对于DNA适体，我们将进行手动SELEX，对于2 '-fRNA适体，将使用自动化Biomek NX平台。将通过下一代测序监测核酸文库的富集，并检测单个序列的亲和力和结合特异性。适体对人CCL 17的抑制能力将在使用CCR 4+淋巴瘤系Mac-1以及原代人T细胞的迁移测定中测定。同时，我们将优化适体在CHS模型中的治疗应用条件，并探索表皮应用的可能性。为了阐明CCL 22的功能，我们使用设计者核酸酶产生了新的CCL 22缺陷型和CCL 17/CCL 22双缺陷型小鼠品系。我们将评估CHS的发展和T细胞迁移在这些小鼠相比，CCL 17和CCR 4缺陷小鼠。此外，我们还将研究CCL 17和CCL 22对调节性T细胞选择的影响。

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.