Lacritin Regulation of Homeostasis and Ocular Surface Health

泪泌素对体内平衡和眼表健康的调节

• 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
• 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' 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

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

干眼是眼表动态平衡的一种慢性破坏。越来越多的证据表明，眼泪蛋白是一种“催泪蛋白” 其天然的C末端蛋白形式‘N-94’和‘N-94/C-6’是眼表面动态平衡的主要调节因子。 因此，干眼的选择性缺陷或缺失可能被视为一个主要的危险因素甚至原因。 对于干眼症，替代疗法可能是一种合理的治疗方法。事实上，在NCT03226444中，一个 最近的大型多中心、随机、安慰剂对照、双掩蔽的2期临床试验，外用N-94/C- 6在两周内显著恢复动态平衡的原发性干燥综合征-最严重的干眼 干眼组。尽管我们大体上了解了lacritin是如何起作用的，但仍缺少许多细节--最值得注意的是 信号受体的特性，以及近端和远端信号的差距。我们最近不偏不倚地表演， 全基因组CRISPR/Cas9死亡筛查。我们的死亡筛选器识别出当被破坏时的基因 SgRNA/Cas9编辑使N-94恢复培养人角膜(HCE-T)动态平衡的能力丧失 在干眼的体外模型中，细胞受到致死剂量的干扰素、γ和肿瘤坏死因子的刺激。在19,114个目标基因中 在具有1,000个对照的76,441个sgRNA中，GPR87是靶向CRISPR/CA验证的最高受体 9编辑，不带或带GPR87 cDNA互补的shRNA敲除，以及syndecan-1下拉。GPR87 在角膜中表达，分享撕裂蛋白信号媒介(百日咳毒素敏感G蛋白，IP3，钙， NFAT)，尽管一些人认为它是一种溶血磷脂酸(LPA)受体，但a 最近由其他人进行的严格控制的研究未能检测到竞争或功能上的特定LPA结合 实验。LPA也不能拯救HCE-T细胞。我们的工作假设是带有syndecan-1的GPR87是 泪眼表面轴的基本要素。我们的直接目标是阐明GPR87如何相互作用 使用Syndecan-1和N-94和N-94/C-6，并探索CRISPR/Cas9介体命中，以扩展我们的 了解催乳素信号转导机制。我们的长期目标是利用这些信息 有效而持久地治疗干眼症。 弗吉尼亚大学夏洛茨维尔分校 加州大学旧金山分校旧金山加州