Lacritin Regulation of Homeostasis and Ocular Surface Health

泪泌素对体内平衡和眼表健康的调节

基本信息

批准号：
10280641
负责人：
Sarah Monica Knox
金额：
$ 51.52万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10280641
关键词：
Acceleration Address Afferent Neurons Autophagocytosis Binding Binding Sites Biochemical C-terminal CRISPR/Cas technology Calcineurin Calcium California Cell surface Cellular Stress Cessation of life Chronic Complement Complementary DNA Complex Controlled Study Cornea Cryoelectron Microscopy Cytoplasmic Tail Data Distal Dose Dry Eye Syndromes Elements Epithelial Extracellular Domain Eye Development FOXO1A gene FOXO3A gene G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Gene Targeting Genes Goals Health Homeostasis Human Inflammation Interferon Type II Ion Channel Label Ligation Lipids Lysophosphatidic Acid Receptors Masks Mass Spectrum Analysis Mediator of activation protein Mitochondria Modeling Mus Mutation Oxidative Phosphorylation Peroxidases Pertussis Toxin Phase II Clinical Trials Placebos Proteoglycan Proteome Randomized Receptor Signaling Regulation Replacement Therapy Risk Factors Role San Francisco Sensory Signal Transduction Signs and Symptoms Sjogren&apos s Syndrome System T-Lymphocyte TNF gene Therapeutic Universities Virginia Work base corneal epithelium design effective therapy epithelial repair evaporation experimental study eye dryness genome-wide in vitro Model induced pluripotent stem cell knock-down lacrimal lysophosphatidic acid mTOR Signaling Pathway mouse model mutant nerve supply ocular surface phase II trial receptor relating to nervous system restoration small hairpin RNA syndecan tear proteins

Acceleration Address Afferent Neurons Autophagocytosis Binding Binding Sites Biochemical C-terminal CRISPR/Cas technology Calcineurin Calcium California Cell surface Cellular Stress Cessation of life Chronic Complement Complementary DNA Complex Controlled Study Cornea Cryoelectron Microscopy Cytoplasmic Tail Data Distal Dose Dry Eye Syndromes Elements Epithelial Extracellular Domain Eye Development FOXO1A gene FOXO3A gene G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Gene Targeting Genes Goals Health Homeostasis Human Inflammation Interferon Type II Ion Channel Label Ligation Lipids Lysophosphatidic Acid Receptors Masks Mass Spectrum Analysis Mediator of activation protein Mitochondria Modeling Mus Mutation Oxidative Phosphorylation Peroxidases Pertussis Toxin Phase II Clinical Trials Placebos Proteoglycan Proteome Randomized Receptor Signaling Regulation Replacement Therapy Risk Factors Role San Francisco Sensory Signal Transduction Signs and Symptoms Sjogren&apos s Syndrome System T-Lymphocyte TNF gene Therapeutic Universities Virginia Work base corneal epithelium design effective therapy epithelial repair evaporation experimental study eye dryness genome-wide in vitro Model induced pluripotent stem cell knock-down lacrimal lysophosphatidic acid mTOR Signaling Pathway mouse model mutant nerve supply ocular surface phase II trial receptor relating to nervous system restoration small hairpin RNA syndecan tear proteins

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项目摘要

Dry eye is a chronic disruption of ocular surface homeostasis. Evidence is accumulating for tear protein 'lacritin' and its natural C-terminal proteoforms 'N-94' and 'N-94/C-6' as master regulators of ocular surface homeostasis. Accordingly, their selective deficiency or absence in dry eye may be viewed as a major risk factor or even cause of dry eye for which replacement therapy may be a logical treatment approach. Indeed, in NCT03226444, a recent large multi-center, randomized, placebo-controlled, double masked phase 2 clinical trial, topical N-94/C- 6 significantly restored homeostasis within two weeks in Primary Sjögren's Syndrome dry eye - the most severe dry eye group. Although we understand in general how lacritin works, many details are missing - most notably the identity of the signaling receptor, and gaps in proximal and distal signaling. We recently performed unbiased, genome-wide CRISPR/Cas9 death screens. Our death screens identified genes that when disrupted by sgRNA/Cas9 editing abrogated the capacity of N-94 to restore homeostasis of cultured human corneal (HCE-T) cells stressed with lethal doses of IFNγ and TNF in an in vitro model of dry eye. Out of 19,114 genes targeted by 76,441 sgRNA's, with 1,000 controls, GPR87 was the top receptor hit as validated by targeted CRISPR/Cas 9 editing, shRNA knockdown without or with GPR87 cDNA complementation, and syndecan-1 pulldown. GPR87 is expressed in the cornea, shares lacritin signaling mediators (pertussis toxin sensitive G-protein, IP3, calcium, NFAT), and although considered by some to be deorphanized as a lysophosphatidic acid (LPA) receptor, a recent well-controlled study by others failed to detect specific LPA binding in competition nor functional experiments. Nor does LPA rescue HCE-T cells. Our working hypothesis is that GPR87 with syndecan-1 are essential elements of the lacrimal - ocular surface axis. Our immediate goal is to elucidate how GPR87 interacts with syndecan-1 and both N-94 and N-94/C-6, and explore CRISPR/Cas9 mediator hits to expand our understanding of lacritin signaling mechanisms. Our long-term goal is to harness this information towards the effective and lasting treatment of dry eye disease. University of Virginia Charlottesville Virginia University of California San Francisco San Francisco California

干眼症是眼表稳态的慢性破坏。证据正在积累泪蛋白“乳泪蛋白” 及其天然C末端蛋白基础“ N-94”和“ N-94/C-6”作为眼表稳态的主要调节剂。彼此之间，他们的选择性缺陷或干眼症可能被视为主要危险因素，甚至可能导致干眼症的替代疗法可能是一种逻辑治疗方法。确实，在NCT03226444中最近的大型多中心，随机，安慰剂对照，双掩盖2期临床试验，局部n-94/c- 6在初级Sjögren综合征干眼中，在两周内重新恢复了体内平衡 - 最严重干眼组。尽管我们一般来说，我们总体上了解了乳葡萄蛋白的工作原理，但缺少许多细节 - 最值得注意的是信号受体的身份，以及近端和圆盘信号传导中的间隙。我们最近表演了公正，全基因组CRISPR/CAS9死亡屏幕。我们的死亡屏幕确定了被破坏的基因 SGRNA/CAS9编辑的N-94能力恢复了培养的人角膜（HCE-T）的能力在干眼的体外模型中，细胞用致命剂量的IFNγ和TNF强调。在目标的1914个基因中由76,441个SGRNA的控制，GPR87是由目标CRISPR/CAS验证的最高受体。 9编辑，shRNA敲低无或完成GPR87 cDNA的完成，以及Syndecan-1下拉。 GPR87 在角膜中表达，共享流蛋白信号介质（百日咳毒素敏感的G蛋白，IP3，钙， NFAT），尽管有些人认为被视为脱氧磷脂酸（LPA）受体A，但其他人最近进行的良好控制的研究未能检测到竞争中特定的LPA结合或功能实验。 LPA也不营救HCE-T细胞。我们的工作假设是，使用Syndecan-1的GPR87是泪 - 眼表轴的基本要素。我们的直接目标是阐明GPR87如何相互作用使用Syndecan-1以及N-94和N-94/C-6，并探索CRISPR/CAS9介质命中以扩展我们了解透质蛋白信号传导机制。我们的长期目标是利用这些信息有效且持久的干眼病。弗吉尼亚大学夏洛茨维尔弗吉尼亚大学加利福尼亚大学旧金山旧金山加利福尼亚大学