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.

原发性开角型青光眼 (POAG) 是导致不可逆性失明的主要原因，影响超过 5700 万人 世界各地的人们。视网膜神经节细胞（RGC）的进行性丧失和视神经轴突的退化是 青光眼的病理特征。由于小梁功能障碍导致眼压（IOP）升高 meshwork (TM) 是青光眼最重要且唯一已知的可改变危险因素。的理解 青光眼 TM 功能障碍和神经变性的病理机制由于缺乏 模拟 TM 功能障碍和青光眼神经变性的稳健且忠实的小鼠模型。 开发已知 POAG 遗传原因的小鼠模型是了解 POAG 的理想策略 POAG 的病理生理学。肌纤蛋白 (MYOC) 基因突变是 POAG 最常见的遗传原因。 利用 TARGATT 位点特异性敲入策略，我们开发了一种 Cre 诱导型转基因小鼠，其表达 带 DsRed 标记的 Y437H 人肌纤蛋白突变体 (Tg.Cre-MYOCY437H)。该技术利用丝氨酸整合酶， PhiC31 (ΦC31) 将任何感兴趣的基因（单个拷贝）插入预选的基因间和转录中 活性基因组位点（Hipp11），已设计有对接位点。这允许稳定和现场 特异性转基因整合。在我们的初步研究中，我们观察到单次玻璃体内注射辅助剂 表达 Cre 的腺病毒 (HAd) 5 在小鼠 TM 中选择性诱导人类突变肌纤蛋白。 重要的是，Ad5.Cre 注射导致 Tg.Cre-MYOCY437H 小鼠的 IOP 显着且持续升高。我们 假设突变肌纤蛋白的 TM 特异性表达导致显着且显着的 IOP 升高，并且 Tg.Cre-MYOCY437H 小鼠中的青光眼神经变性。该应用程序的主要目标是诱导 使用 HAd5-cre 注射在 TM 中突变肌纤蛋白表达并表征青光眼表型 Tg.Cre-MYOCY437H 小鼠。在目标 1 中，我们将确定 HAd5 介导的 Cre 是否诱导突变肌纤蛋白 TM 中的表达并提高 Tg.Cre-MYOCY437H 小鼠的 IOP。在目标 2 中，我们将确定 HAd5-Cre- 是否 诱导的 IOP 升高会导致 Tg.Cre-MYOCY437H 小鼠青光眼神经变性。我们的建议将 利用高度创新的方法。其中包括使用有效的位点特异性基因敲入策略 转基因小鼠的产生，流出途径、RGC功能和结构的全面研究 损失、视神经损伤和大脑视觉中枢损伤。我们提出的研究将取得很大进展 需要能够忠实复制青光眼所有特征的 POAG 小鼠模型。