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

• 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
• 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' 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

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.

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， 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）从分化的收缩细胞转变为合成表型， 增殖，导致内膜扩张和血管妥协，往往造成缺血性器官衰竭。 同种异体移植物特异性调节性T细胞（Treg）可以抑制免疫细胞而不具有免疫抑制作用，但它们的直接免疫抑制作用可能是免疫抑制性的。 对病理性VSMC表型转换的影响尚未充分研究。此外，Treg异质性 和可塑性与microRNA对基因表达的复杂转录后调控有关 代表需要离体扩增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TcR的分泌组偏向于促进TcR的表达。 VSMC稳态。这个探索性的发展（R21）项目将设计和评估一种新的方法， 在对血管同种异体移植物的免疫应答的背景下促进血管平滑肌细胞稳态 通过调节Treg分泌组。在目标1中，我们将鉴定Treg富集的分泌产物，其促进 VSMC稳态。我们将扩大同种异体抗原特异性Treg，并确定分泌因子的克隆， 对VSMC表型的有利影响。使用两种互补的分泌组分析（多分析物 来自Isoplexis和脉冲SILAC的免疫测定，随后LC/MS-MS），我们将选择2-3个Treg富集的 调制的因素。目的二是设计并评价锁核酸修饰的mRNA靶点 特异性缓解miRNA介导的翻译抑制并增强 IL-10作为我们的模型候选者，以及新鉴定的Treg富集靶点，以促进VSMC稳态。的 TSB对Treg和VSMC表型的影响将在体外进行评价，然后在2个体内进行验证 模型：（1）对次级淋巴器官抗原激活的Treg的因子增强作用，和（2）血管- 对次要抗原（HY）不匹配的主动脉同种异体移植物的稳定作用。这项探索性研究的结果将新 揭示了Treg-VSMC相互作用的特定调控网络，并为一类新的细胞提供了概念证明。 旨在改善长期血管化同种异体移植结果的治疗方法。