Development and characterization of an inducible model for myocilin POAG

肌纤蛋白 POAG 诱导模型的开发和表征

• 批准号: 10661911
• 负责人: Gulab Zode
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10661911
• 关键词: Adenoviruses; Affect; Antibodies; Aqueous Humor; Axon; Biochemical; Blindness; Brain; Chronic; Development; Docking; DsRed; Engineering; Exhibits; Functional disorder; Gene Mutation; Gene Silencing; Generations; Genes; Genetic; Genetic Transcription; Glaucoma; Goals; Human; Inflammation; Injections; Integrase; Investigation; Knock-in; Lead; Light; Mediating; Methylation; Modeling; Molecular; Morphology; Mus; Mutation; Nerve Degeneration; Neurotransmitters; Ocular Hypertension; Optic Nerve; Pathologic; Pathology; Pathway interactions; Patients; Pattern; Persons; Phenotype; Physiologic Intraocular Pressure; Primary Open Angle Glaucoma; Proteins; Resistance; Retina; Retinal Ganglion Cells; Risk Factors; Route; Serine; Site; Stains; Structure; Technology; Trabecular meshwork structure; Transgenes; Transgenic Mice; Transmission Electron Microscopy; Viral; Viral Packaging; Viral Vector; Virus Replication; Visual; Visual evoked cortical potential; Western Blotting; cis acting element; functional loss; genomic locus; immunogenicity; inducible Cre; innovation; insight; interest; intravitreal injection; modifiable risk; molecular sieving; mouse development; mouse model; mutant; myocilin; nerve damage; novel; promoter; retinal ganglion cell degeneration; treatment strategy

Primary Open Angle Glaucoma (POAG) is the leading cause of irreversible blindness affecting over 57 million people worldwide. Progressive loss of retinal ganglion cells (RGCs) and degeneration of optic nerve axons is the pathological hallmark of glaucoma. Elevated intraocular pressure (IOP) due to dysfunction of trabecular meshwork (TM) is the most significant and the only known modifiable risk factor for glaucoma. Understanding of the pathological mechanisms of glaucomatous TM dysfunction and neurodegeneration is limited due to lack of robust and faithful mouse model that mimics both TM dysfunction and glaucomatous neurodegeneration. Developing a mouse model of known genetic cause of POAG represents an ideal strategy to understand the pathophysiology of POAG. Mutations in myocilin (MYOC) gene are the most common genetic cause of POAG. Using TARGATT site-specific knockin strategy, we developed a Cre-inducible transgenic mice that expresses DsRed-tagged Y437H mutant of human myocilin (Tg.Cre-MYOCY437H). This technology utilizes serine integrase, PhiC31 (ΦC31) to insert any gene of interest (a single copy) into a preselected intergenic and transcriptionally active genomic locus (Hipp11), which has been engineered with a docking site. This allows stable and site- specific transgene integration. In our preliminary studies, we observed that a single intravitreal injection of helper adenovirus (HAd) 5 expressing Cre selectively induced human mutant myocilin protein in mouse TM. Importantly, Ad5.Cre injection resulted in significant and sustained IOP elevation in Tg.Cre-MYOCY437H mice. We hypothesize that TM-specific expression of mutant myocilin leads significant and pronounced IOP elevation and glaucomatous neurodegeneration in Tg.Cre-MYOCY437H mice. The major goals of this application are to induce mutant myocilin expression in TM using HAd5-cre injections and to characterize glaucoma phenotypes of Tg.Cre-MYOCY437H mice. In Aim 1, we will determine whether HAd5-mediated Cre induces mutant myocilin expression in TM and elevates IOP in Tg.Cre-MYOCY437H mice. In Aim 2, we will determine whether HAd5-Cre- induced IOP elevation leads to glaucomatous neurodegeneration in Tg.Cre-MYOCY437H mice. Our proposal will utilize highly innovative approaches. These include use of efficient and site-specific gene knockin strategy for generation of transgenic mice, a comprehensive investigation of outflow pathway, RGC functional and structural loss, optic nerve damage and damage to the visual centers of the brain. Our proposed studies will develop much needed mouse model of POAG that faithfully replicate all features of glaucoma.

初级开角色青光眼（POAG）是不可逆失明的主要原因，影响超过5700万 全球人。视网膜神经节细胞（RGC）的进行性丧失和视神经轴突的变性为 青光眼的病理标志。由于小梁的功能障碍，眼内压（IOP）升高 网格工作（TM）是青光眼最重要，唯一已知的可修改风险因素。理解 青光眼TM功能障碍和神经变性的病理机制由于缺乏而受到限制 模仿TM功能障碍和青光眼神经变性的强大而忠实的小鼠模型。 开发已知遗传原因的小鼠模型是理解理想的策略 POAG的病理生理学。肌动蛋白（MYOC）基因的突变是POAG的最常见遗传原因。 使用Targatt特定地点的敲蛋白策略，我们开发了一种Cre-CRE诱导的转基因小鼠，表达 人类肌动蛋白的DSRED标记的Y437H突变体（TG.CRE-Myocy437h）。该技术利用丝氨酸积分酶， pHIC31（φC31）将任何感兴趣的基因（单个副本）插入预览的基因间和转录 主动基因组基因座（HIPP11），该基因座已通过对接站点进行了设计。这允许稳定和站点 - 特定的转换整合。在我们的初步研究中，我们观察到玻璃体内注射助手 腺病毒（HAD）5表达CRE在小鼠TM中选择性诱导的人突变肌蛋白蛋白。 重要的是，AD5.CRE注射导致TG.CRE-Myocy437H小鼠的显着和持续的IOP升高。我们 假设突变体肌动蛋白的TM特异性表达会导致显着且明显的IOP升高和 TG.CRE-Myocy437h小鼠中的青光眼神经退行性。该应用的主要目标是诱导 使用5-CRE注射在TM中的突变肌动蛋白表达，并表征青光眼表型 TG.CRE-MYOCY437H小鼠。在AIM 1中，我们将确定是否有5个介导的CRE诱导突变体肌动蛋白 在TM中的表达并升高IOP在TG.CRE-Myocy437h小鼠中。在AIM 2中，我们将确定是否有5个cre- 诱导的IOP升高导致TG.CRE-Myocy437h小鼠中的青光眼神经退行性变化。我们的建议将 利用高度创新的方法。这些包括将高效和特定于位点的基因敲除策略用于 转基因小鼠的产生，这是对出口通路，RGC功能和结构的全面研究 损失，视神经损伤和对大脑视觉中心的损害。我们提出的研究将发展很大 需要忠实复制青光眼所有特征的POAG的小鼠模型。