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

RNA靶位点寡核苷酸倾斜免疫细胞促进血管平滑肌细胞稳态

基本信息

批准号：
10593490
负责人：
JEFFREY R. BENDER
金额：
$ 25.13万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-11-10 至 2024-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10593490
关键词：
3&apos Untranslated Regions Adjuvant Alloantigen Allograft Tolerance Allografting Antigens Aorta Arteries Binding Blood Vessels CTLA4 gene Cell Communication Cell Therapy Cell secretion Cells Chronic Clonal Expansion Clone Cells Complex Computer Analysis Conditioned Culture Media Data Development Endogenous Factors Environment FOXP3 gene Female Gene Expression Gene Expression Regulation Generations Goals H-Y Antigen Heterogeneity Homeostasis Immune Immune response Immunoassay Immunosuppression In Vitro Inflammation Influentials Injury Interleukin-10 Interleukin-2 Internal Ribosome Entry Site Intervention Ischemia Maintenance Mediating Messenger RNA Metabolism MicroRNAs Minor Mitotic Modeling Molecular Mus Oligonucleotides Organ Transplantation Organ failure Outcome Pathologic Peptides Phenotype Physiologic pulse Post-Transcriptional Regulation Production Proliferating Proteins Proteomics RNA RNA-Binding Proteins Regulation Regulatory T-Lymphocyte Reporter Research Design Signal Transduction Site Small RNA Smooth Muscle Myocytes Solid Sorting Specificity T cell differentiation T-Cell Activation T-Lymphocyte Testing Therapeutic Translational Repression Translations Validation Vascular Smooth Muscle Vascular remodeling Vascularization adverse outcome allograft rejection blood vessel transplantation candidate identification candidate selection cell dedifferentiation cytokine design enhancing factor immunoregulation improved in vivo in vivo Model liquid chromatography mass spectrometry locked nucleic acid mRNA Stability mRNA Translation male novel novel strategies novel therapeutic intervention organ transplant rejection pharmacologic posttranscriptional pre-clinical preservation prevent release factor response secondary lymphoid organ success transplant model vascular smooth muscle cell proliferation

项目摘要

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.

尽管慢性同种异体移植排斥仍然是实体器官移植长期成功的主要障碍免疫抑制的改善和共刺激阻断的进展。免疫损伤后，血管平滑肌细胞（VSMC）从分化的收缩型转变为合成表型，增殖，导致内膜扩张和血管受损，常常引起缺血性器官衰竭。同种异体移植物特异性调节性 T 细胞 (Treg) 可能会在不产生免疫抑制的情况下抑制免疫细胞，但它们的直接作用对病理性 VSMC 表型转换的影响尚未得到充分研究。此外，Treg异质性和与 microRNA 对基因表达的复杂转录后调控相关的可塑性代表了需要离体扩增 Treg 的细胞疗法的挑战。我们假设 mRNA- 翻译的特异性增强可以使同种异体抗原特异性 Tregs 的分泌组偏向于促进 VSMC 稳态。这个探索性、开发性 (R21) 项目将设计和评估一种新颖的方法在血管同种异体移植免疫反应的背景下促进血管平滑肌细胞稳态通过调节 Treg 分泌蛋白组。在目标 1 中，我们将鉴定富含 Treg 的分泌产物，促进 VSMC 稳态。我们将扩展同种异体抗原特异性 Treg 并鉴定分泌因子的克隆对 VSMC 表型有有利的影响。使用两种互补的分泌组分析（多分析物 Isoplexis 和脉冲 SILAC 免疫测定，然后进行 LC/MS-MS），我们将选择 2-3 个 Treg 富集调制因素。在目标 2 中，我们将设计和评估锁核酸 (LNA) 修饰的 mRNA 靶标位点阻断剂 (TSB)，可特异性缓解 miRNA 介导的翻译抑制并增强 IL-10 作为我们的候选模型，以及新发现的富含 Treg 的靶标，可促进 VSMC 稳态。这 TSB 对 Treg 和 VSMC 表型的影响将在体外进行评估，然后在 2 体内进行验证模型：(1) 对次级淋巴器官抗原激活的 Treg 的因子增强作用，以及 (2) 血管- 对次要抗原（HY）不匹配的同种异体主动脉移植物具有稳定作用。这项探索性研究的结果将新揭示 Treg-VSMC 相互作用的特定调控网络，并为新型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。