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）是影响5700多万人的不可逆失明的主要原因 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变性是 青光眼的病理特征由于小梁功能障碍导致的眼内压（IOP）升高 网状结构（TM）是青光眼最重要的和唯一已知的可改变的危险因素。了解 脑昏迷TM功能障碍和神经退行性变的病理机制由于缺乏 模拟TM功能障碍和昏迷性神经变性的稳健且忠实的小鼠模型。 建立一个已知遗传原因的POAG小鼠模型是了解POAG发病机制的理想策略。 POAG的病理生理学myocilin（MYOC）基因突变是POAG最常见的遗传病因。 利用TARGATT位点特异性敲入策略，我们建立了Cre诱导的转基因小鼠， DsRed标记的人肌球蛋白的Y 437 H突变体（Tg. Cre-MYOCY 437 H）。该技术利用丝氨酸整合酶， PhiC 31（ΦC31）将任何感兴趣的基因（单拷贝）插入到预先选择的基因间和转录上。 活性基因组位点（Hipp 11），其已被工程化为具有对接位点。这使得稳定和网站- 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细胞， 表达Cre的腺病毒（HAd）5在小鼠TM中选择性地诱导人突变型肌球蛋白。 重要的是，Ad5.Cre注射导致Tg. Cre-MYOCY 437 H小鼠中显著且持续的IOP升高。我们 假设突变型肌球蛋白TM特异性表达导致显著和明显的IOP升高， Tg. Cre-MYOCY 437 H小鼠中的神经变性。本应用程序的主要目标是诱导 使用HAd 5-cre注射在TM中表达突变myocilin并表征青光眼表型 Tg. Cre-MYOCY 437 H小鼠。在目的1中，我们将确定HAd 5介导的Cre是否诱导突变型肌细胞素 Cre-MYOCY 437 H小鼠中的IOP升高。在目标2中，我们将确定HAd 5-Cre- 诱导的IOP升高导致Tg. Cre-MYOCY 437 H小鼠中的昏迷性神经变性。我们的建议将 采用高度创新的方法。这些包括使用高效且位点特异性的基因敲入策略， 转基因小鼠的产生，流出途径、RGC功能和结构的全面研究 视力丧失、视神经损伤和大脑视觉中枢损伤。我们提出的研究将大大发展 我们需要POAG小鼠模型来忠实地复制青光眼的所有特征。