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: 10477335
• 负责人: Sarah Monica Knox
• 金额: $ 48.48万
• 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/10477335
• 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; Placebo Control; 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

干眼症是眼表稳态的慢性破坏。泪液蛋白“lacritin”的证据正在积累 以及其天然C-末端蛋白形式“N-94”和“N-94/C-6”作为眼表面稳态的主要调节剂。 因此，它们在干眼中的选择性缺乏或缺失可被视为主要的风险因素或甚至原因 替代疗法可能是一种合理的治疗方法。实际上，在NCT 03226444中， 最近进行的大型多中心、随机、安慰剂对照、双盲、II期临床试验，外用N-94/C- 6例在两周内显著恢复了原发性干燥综合征干眼的稳态-最严重的 干眼症组。尽管我们大体上了解了lacritin的工作原理，但仍缺少许多细节--最明显的是 信号受体的身份，以及近端和远端信号传导的缺口。我们最近进行了公正的， CRISPR/Cas9死亡筛查我们的死亡筛选鉴定出的基因， sgRNA/Cas9编辑消除了N-94恢复培养的人角膜（HCE-T）的稳态的能力 干眼体外模型中用致死剂量的IFNγ和TNF应激的细胞。在19，114个靶向基因中， 通过76，441个sgRNA，1，000个对照，GPR 87是靶向CRISPR/Cas验证的最高受体命中 9编辑、不具有或具有GPR 87 cDNA互补的shRNA敲低以及多配体蛋白聚糖-1下拉。GPR87 在角膜中表达，共享催乳素信号传导介质（百日咳毒素敏感性G蛋白，IP 3，钙， NFAT），并且尽管被一些人认为是作为溶血磷脂酸（LPA）受体去磷酸化的，但它仍然是一种非特异性受体。 其他人最近进行的一项对照良好的研究未能检测到LPA在竞争中的特异性结合， 实验LPA也不能拯救HCE-T细胞。我们的工作假设是，GPR 87与syndecan-1是相互作用的。 泪腺-眼表轴的基本要素。我们的直接目标是阐明GPR 87如何相互作用 与syndecan-1和N-94和N-94/C-6，并探索CRISPR/Cas9介导的命中，以扩大我们的研究。 了解lacritin信号机制。我们的长期目标是利用这些信息， 有效和持久的治疗干眼病。 University of Virginia弗吉尼亚大学 加州旧金山大学旧金山弗朗西斯科加州