Immune cell skewing with RNA target site oligonucleotides to promote vascular smooth muscle cell homeostasis

慢性同种异体排斥反应仍然是实体器官移植长期成功的主要障碍，尽管 免疫抑制的改进和共刺激阻断的进展。在免疫损伤之后， 血管平滑肌细胞(VSMCs)从分化的收缩表型转变为合成表型， 增殖，导致内膜扩张和血管受损，经常导致缺血性器官衰竭。 同种异体移植物特异性调节性T细胞(Treg)可以在没有免疫抑制的情况下抑制免疫细胞，但它们直接 对病理性VSMC表型转换的影响研究还不够深入。此外，Treg的异质性 以及与microRNAs对基因表达的复杂转录后调控相关的可塑性 代表了需要扩大体外Treg的细胞治疗的挑战。我们假设mrna- 翻译的特异性增强可以使同种抗原特异性树的分泌体偏向于促进 VSMC动态平衡。这个探索性、开发性(R21)项目将设计和评估一种新的方法 在同种异体血管移植免疫反应的背景下促进血管平滑肌细胞动态平衡 通过调节Treg分泌组。在目标1中，我们将识别富含Treg的分泌产品，以促进 VSMC动态平衡。我们将扩展同种异体抗原特异性Treg并识别具有秘密因子的克隆 对VSMC表型的有利影响。使用两个互补的分泌组分析(多分析物 Isoplexis和脉冲SILAC的免疫分析，然后LC/MS-MS)，我们将选择2-3个Treg富集物 调制的因素。在目标2中，我们将设计和评估锁定核酸(Lna)修饰的mrna靶点。 位点阻滞剂(TSB)，特异性解除miRNA介导的翻译抑制并增加 IL-10，作为我们的模型候选，和新发现的富含Treg的靶点，促进VSMC动态平衡。这个 TSB对Treg和VSMC表型的影响将在体外进行评估，然后在体内进行验证 模型：(1)因子对次级淋巴器官抗原激活的Treg的增强作用；(2)血管生成 次要抗原(HY)不相合的同种异体主动脉的稳定作用。这项探索性研究的结果将全新 揭示Treg-VSMC相互作用的特定调控网络，并为一种新类别提供概念证明 旨在改善长期血管化同种异体移植结果的治疗方法。

Chronic allograft rejection remains a major obstacle to long-term success of solid organ transplantation despite improvements in immunosuppression and advances in costimulation blockade. Following immune injury, vascular smooth muscle cells (VSMCs) switch from a differentiated contractile to a synthetic phenotype and proliferate, resulting in intimal expansion and vascular compromise, often causing ischemic organ failure. Allograft-specific regulatory T cells (Treg) may inhibit immune cells without immunosuppression but their direct effects on the pathologic VSMC phenotype switch have been insufficiently studied. Moreover, Treg heterogeneity and plasticity associated with complex post-transcriptional regulation of gene expression by microRNAs represent challenges for cellular therapies requiring expansion of Treg ex vivo. We hypothesize that mRNA- specific enhancement of translation can skew the secretome of the alloantigen-specific Tregs toward promoting VSMC homeostasis. This exploratory, developmental (R21) project will design and evaluate a novel approach to promoting vascular smooth muscle cell homeostasis in the context of immune responses to vascular allografts through modulation of the Treg secretome. In Aim 1, we will identify Treg-enriched secreted products promoting VSMC homeostasis. We will expand alloantigen-specific Treg and identify clones that secret factors with favorable effects on VSMC phenotype. Using two complementary secretome analyses (multianalyte immunoassays from Isoplexis and pulsed SILAC followed by LC/MS-MS), we will select 2-3 Treg-enriched factors for modulation. In Aim 2, we will design and evaluate locked nucleic acid (LNA)-modified mRNA target site blockers (TSB) that specifically relieve miRNA-mediated translational repression and enhance production of IL-10, as our model candidate, and newly identified Treg-enriched targets, to promote VSMC homeostasis. The effects of the TSB on Treg and VSMC phenotype will be evaluated in vitro, followed by validation in 2 in vivo models: (1) factor-enhancing effect on secondary lymphoid organ antigen-activated Treg, and (2) vascular- stabilizing effect on minor antigen (HY) mismatch aortic allografts. The results of this exploratory study will newly reveal specific regulatory networks of Treg-VSMC interactions and provide a proof of concept for a novel class of therapeutics directed at improving long-term vascularized allograft outcomes.