Novel Glaucoma Treatment Using Genome Editing

描述（由申请人提供）：原发性开角型青光眼（POAG）是不可逆视力丧失和失明的主要原因。目前POAG的治疗旨在降低眼内压（IOP），这是该疾病发展和进展的最重要风险因素。然而，目前的青光眼治疗没有解决潜在的疾病机制。Myocilin基因（MYOC）突变是青光眼最常见的已知遗传原因。三个发展为我们提供了一个机会，研究针对潜在的疾病机制的青光眼新的治疗方法：1。在我们实验室中开发了POAG小鼠模型（Tg-MYOCY 437 H），其表达由MYOC基因编码的突变型肌球蛋白; 2.使用Tg-MYOCY 437 H小鼠模型在体内证明突变型肌球蛋白在内质网（ER）中积累并诱导眼小梁网（TM）中的ER应激作为导致青光眼的关键机制组分;和3.基因组编辑核酸酶作为基因治疗应用的有价值工具的最新发展。此外，肌球蛋白相关的POAG由功能突变的显性获得引起的事实和TM中ER应激诱导的细胞凋亡导致POAG的事实提供了通过体内消除突变MYOC和/或细胞凋亡途径基因（例如Chop）来探索新的青光眼治疗的机会。我们建议利用Tg-MYOCY 437 H小鼠青光眼模型进一步探索ER应激在POAG中的作用，并使用新的基因组编辑方法（CRISPR）探索新的POAG治疗方法，特别是从Tg-MYOCY 437 H小鼠的TM中消除突变型肌球蛋白，以及从TM细胞中消除ER应激基因。规则间隔短回文重复序列（CRISPR）是一种内源性核酸酶系统，它使用RNA序列来引导CRISPR相关核酸酶Cas9切割DNA。此外，我们将探索ER应激在类固醇诱导的青光眼中的作用，以确定青光眼治疗的基因组编辑策略是否可以应用于这种形式的青光眼。重要的是，我们将通过探索基因组编辑在人眼灌注器官培养系统中的效用，将我们的工作扩展到人类。我们相信Cas9可以用于去除突变的MYOC基因，并为MYOC突变诱导的青光眼提供第一个有效的治疗方法。此外，眼睛是建立Cas9技术治疗潜力的理想试验场所，这项工作将为Cas9用于治疗各种遗传性眼部疾病以及影响其他器官系统的疾病铺平道路。

DESCRIPTION (provided by applicant): Primary open angle glaucoma (POAG) is a leading cause of irreversible vision loss and blindness. Current treatments of POAG are aimed at reducing intraocular pressure (IOP), the most important risk factor for the development and progression of the disorder. However, current glaucoma treatments do not address the underlying disease mechanisms. Mutations in the myocilin gene (MYOC) are the most common known genetic cause of glaucoma. Three developments provide us with an opportunity to investigate novel treatments of glaucoma directed at the underlying disease mechanisms: 1. The development of a POAG mouse model (Tg- MYOCY437H) in our laboratory that expresses mutant myocilin encoded by the MYOC gene; 2. The use of the Tg-MYOCY437H mouse model to demonstrate in vivo that mutant myocilin accumulates in the endoplasmic reticulum (ER) and induces ER stress in the trabecular meshwork (TM) of the eye as a key mechanistic component leading to glaucoma; and 3. The recent development of genome editing nucleases as a valuable tool for gene therapy applications. Furthermore, the fact that myocilin-associated POAG results from dominant gain of function mutations and the fact that ER stress induced apoptosis in the TM leads to POAG provide an opportunity to explore novel glaucoma treatments by in vivo elimination of mutant MYOC and/or apoptosis pathway genes (e.g. Chop). We propose to utilize the Tg-MYOCY437H mouse model of glaucoma to further explore the role of ER stress in POAG and to use a novel genome editing method (CRISPR) to explore novel POAG treatments, specifically the elimination of mutant myocilin from the TM of Tg-MYOCY437H mice, as well as the elimination of ER stress genes from TM cells. Clustered regularly interspaced short palindromic repeats (CRISPR) is an endogenous nuclease system, which uses RNA sequences to guide the cleavage of DNA by the CRISPR-associated nuclease Cas9. In addition, we will explore the role of ER stress in steroid induced glaucoma for the purpose of determining whether the genome editing strategy of glaucoma treatment can be applied to this form of glaucoma. Importantly, we will extend our work to humans by exploring the utility of genome editing in a human eye perfusion organ culture system. We believe that Cas9 can be used to remove the mutant MYOC gene and provide the first effective cure for MYOC mutation-induced glaucoma. In addition, the eye is an ideal test site for establishing the therapeutic potential of the Cas9 technology, and this work will pave the path for the application of Cas9 for treating a variety of genetic eye disorders, as well disorders affecting other organ systems.