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.

尽管免疫抑制的改善和共刺激封锁的进步。免疫损伤后，血管平滑肌细胞（VSMC）从分化的收缩物转换为合成表型和扩散，导致内膜扩张和血管损害，通常导致缺血器官衰竭。同种异体特异性调节T细胞（TREG）可能抑制免疫抑制的免疫细胞，但直接对病理VSMC表型开关的影响不足。此外，Treg异质性和与microRNA对基因表达的复杂转录后调节相关的可塑性代表了需要扩大TREG离体的细胞疗法的挑战。我们假设mRNA- 翻译的具体增强可能会使同种特异性特雷格的分泌偏向促进 VSMC稳态。这个探索性的发展（R21）项目将设计和评估一种新颖的方法在对血管同种异体的免疫反应的背景下促进血管平滑肌细胞体内平衡通过调节Treg Sextome。在AIM 1中，我们将确定促进富含Treg的分泌产品 VSMC稳态。我们将扩展同种抗原特异性的Treg，并确定具有秘密因素的克隆对VSMC表型的有利影响。使用两个互补的分泌组分析（多植物分析物来自等肌的免疫测定和脉冲Silac，然后是LC/MS-MS），我们将选择2-3个Treg富集调制因素。在AIM 2中，我们将设计和评估锁定的核酸（LNA）修饰的mRNA靶现场阻滞剂（TSB），专门缓解miRNA介导的翻译抑制并增强的产生 IL-10作为我们的模型候选人，以及新确定的Treg富含目标，以促进VSMC稳态。这 TSB对Treg和VSMC表型的影响将在体外评估，然后在2个体内进行验证模型：（1）增强因子增强作用对次级淋巴器官抗原激活的Treg的作用，以及（2）血管稳定对次要抗原（HY）不匹配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Treg-VSMC相互作用的特定调节网络，并为新班级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剂。