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: 10666529
• 负责人: Sarah Monica Knox
• 金额: $ 49.98万
• 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/10666529
• 关键词: Acceleration; Address; Afferent Neurons; Autophagocytosis; Binding; Binding Sites; Biochemical; C-terminal; CRISPR/Cas technology; Calcineurin; Calcium; California; Cell surface; Cells; Cellular Stress; Cessation of life; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complementary DNA; Complex; Controlled Study; Cornea; Cryoelectron Microscopy; Cytoplasm; Cytoplasmic Tail; Development; Distal; Dose; Dry Eye Syndromes; Elements; Epithelium; Extracellular Domain; FOXO1A gene; FOXO3A gene; FRAP1 gene; G-Protein-Coupled Receptors; 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; 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 Pathway; Signal Transduction; Signs and Symptoms; Sjogren' s Syndrome; System; TNF gene; Therapeutic; Universities; Virginia; Work; corneal epithelium; data integration; design; effective therapy; epithelial repair; evaporation; experimental study; eye dryness; genome-wide; improved; in vitro Model; induced pluripotent stem cell; knock-down; lacrimal; lysophosphatidic acid; mouse model; mutant; nerve supply; neural; nuclear factors of activated T-cells; ocular surface; phase II trial; receptor; 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“泪分泌素”的证据正在积累 及其天然 C 端蛋白“N-94”和“N-94/C-6”作为眼表稳态的主要调节因子。 因此，干眼症中它们的选择性缺乏或缺失可能被视为主要危险因素，甚至是导致干眼症的原因。 干眼症的替代治疗可能是一种合理的治疗方法。事实上，在 NCT03226444 中， 最近大型多中心、随机、安慰剂对照、双盲2期临床试验，局部N-94/C- 6 原发性干燥综合症干眼症（最严重的）在两周内体内平衡显着恢复 干眼症组。尽管我们大体了解催乳素的工作原理，但仍缺少许多细节 - 最值得注意的是 信号传导受体的身份以及近端和远端信号传导的间隙。我们最近表现得不偏不倚， 全基因组 CRISPR/Cas9 死亡筛选。我们的死亡筛选发现了当被破坏时的基因 sgRNA/Cas9 编辑消除了 N-94 恢复培养人角膜 (HCE-T) 稳态的能力 在体外干眼模型中，细胞受到致死剂量的 IFNγ 和 TNF 的应激。 19,114 个目标基因中 由 76,441 个 sgRNA 和 1,000 个对照组成，GPR87 是经靶向 CRISPR/Cas 验证的最佳受体命中 9 编辑、无或有 GPR87 cDNA 互补的 shRNA 敲低以及 syndecan-1 下拉。探地雷达87 在角膜中表达，共享乳泌素信号传导介质（百日咳毒素敏感 G 蛋白、IP3、钙、 NFAT），尽管有些人认为它是一种溶血磷脂酸（LPA）受体，但 其他人最近进行的良好对照研究未能检测到竞争或功能中的特定 LPA 结合 实验。 LPA 也不能拯救 HCE-T 细胞。我们的工作假设是 GPR87 与 syndecan-1 是 泪腺-眼表面轴的基本要素。我们的近期目标是阐明 GPR87 如何相互作用 与 syndecan-1 以及 N-94 和 N-94/C-6 一起，并探索 CRISPR/Cas9 介体命中以扩展我们的研究 了解泪素信号传导机制。我们的长期目标是利用这些信息来实现 有效且持久地治疗干眼症。 弗吉尼亚大学弗吉尼亚州夏洛茨维尔分校 加州大学旧金山分校 旧金山 加利福尼亚州

项目成果

A synthetic tear protein resolves dry eye through promoting corneal nerve regeneration.

• DOI: 10.1016/j.celrep.2022.111307
• 发表时间: 2022-08-30
• 期刊: CELL REPORTS
• 影响因子: 8.8
• 作者: Efraim, Yael;Chen, Feeling Yu Ting;Cheong, Ka Neng;Gaylord, Eliza A.;McNamara, Nancy A.;Knox, Sarah M.
• 通讯作者: Knox, Sarah M.

Lacripep for the Treatment of Primary Sjögren-Associated Ocular Surface Disease: Results of the First-In-Human Study.

• DOI: 10.1097/ico.0000000000003091
• 发表时间: 2023-07-01
• 期刊: Cornea
• 影响因子: 2.8

Autophagy in the normal and diseased cornea.

• DOI: 10.1016/j.exer.2022.109274
• 发表时间: 2022-12
• 期刊: EXPERIMENTAL EYE RESEARCH
• 影响因子: 3.4
• 作者: Dias-Teixeira, Karina Luiza;Sharifian, Gh. Mohammad;Romano, Jeff;Norouzi, Fatemeh;Laurie, Gordon W.
• 通讯作者: Laurie, Gordon W.

Guidelines for an optimized differential centrifugation of cells.

• DOI: 10.1016/j.bbrep.2023.101585
• 发表时间: 2023-12
• 期刊: Biochemistry and biophysics reports
• 影响因子: 2.7