Development and characterization of an inducible model for myocilin POAG

原发性开角型青光眼(POAG)是影响5700多万人不可逆性失明的主要原因 世界各地的人们。进行性视网膜神经节细胞(RGC)丢失和视神经轴突变性 青光眼的病理特征。小梁功能障碍引起的高眼压 NetHWork(TM)是青光眼最重要也是唯一已知的可改变的危险因素。了解 青光眼TM功能障碍和神经退行性变的病理机制因缺乏 健壮而忠实的小鼠模型，模拟TM功能障碍和青光眼神经退行性变。 开发POAG已知遗传原因的小鼠模型是了解POAG的理想策略 开角型青光眼的病理生理。霉菌素(myocin，MYOC)基因突变是引起POAG的最常见的遗传原因。 利用TARGATT定点敲打策略，我们开发了一种Cre诱导的转基因小鼠，其表达 DsRed标记的人肌红素Y437H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)插入到预先选择的基因间和转录上 活性基因组位点(Hipp11)，它已经被设计成有一个对接位点。这允许稳定和现场- 特定的转基因整合。在我们的初步研究中，我们观察到一次玻璃体内注射助手 表达Cre的腺病毒(HAD)5在小鼠TM中选择性诱导人突变型Myoclin蛋白。 重要的是，注射Ad5.Cre后，Tg.Cre-MYOCY437H小鼠的眼压持续显著升高。我们 假设突变体myoclin的TM特异性表达导致显著和显著的眼压升高和 Tg.Cre-MYOCY437H小鼠的青光眼神经变性。此应用程序的主要目标是诱导 用HAd5-cre注射在TM中表达突变的myocin并鉴定青光眼的表型 Tg.Cre-MYOCY437H小鼠。在目标1中，我们将确定HAd5介导的Cre是否诱导突变的肌球蛋白 Tg.Cre-MYOCY437H小鼠TM的表达并升高眼压。在目标2中，我们将确定HAd5-Cre- 诱导的眼压升高导致Tg.Cre-MYOCY437H小鼠的青光眼神经变性。我们的建议将 利用高度创新的方法。这些措施包括使用高效和特定部位的基因敲击策略 转基因小鼠的产生、外流途径、RGC功能和结构的综合研究 丧失、视神经损伤和大脑视觉中心的损伤。我们提议的研究将会有很大的发展 需要能够忠实复制青光眼所有特征的POAG小鼠模型。

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.