Novel Glaucoma Treatment Using Genome Editing

描述(由申请人提供)：原发性开角型青光眼(POAG)是导致不可逆性视力丧失和失明的主要原因。目前POAG的治疗旨在降低眼压，眼压是POAG发生和发展的最重要的危险因素。然而，目前的青光眼治疗方法并没有解决潜在的疾病机制。肌红素基因(MYOC)突变是青光眼最常见的遗传原因。三项进展为我们提供了一个机会来研究针对青光眼潜在疾病机制的新的治疗方法：1.我们实验室建立的POAG小鼠模型(TG-MYOCY437H)表达由MYOC基因编码的突变的myoclin；2.利用TG-MYOCY437H小鼠模型在体内证明突变的myoclin积聚在内质网(ER)并诱导作为导致青光眼的关键机制组件的小梁网(TM)的ER应激；以及3.基因组编辑核酸酶作为基因治疗应用的有价值的工具的最新发展。此外，肌球蛋白相关的POAG是由于功能突变的显性获得，以及内质网应激诱导TM中的细胞凋亡导致POAG的事实，为通过体内消除突变的MYOC和/或凋亡途径基因(如CHOP)来探索新的青光眼治疗方法提供了机会。我们建议利用TG-MYOCY437H小鼠青光眼模型进一步探讨内质网应激在POAG中的作用，并使用一种新的基因组编辑方法(CRISPR)来探索新的POAG治疗方法，特别是从TG-MYOCY437H小鼠的TM中消除突变的myoclin，以及从TM细胞中消除ER应激基因。簇状规则间隔短回文重复序列(CRISPR)是一个内源核酸酶系统，它使用RNA序列来指导CRISPR相关核酸酶Cas9对DNA的切割。此外，我们还将探讨内质网应激在激素性青光眼中的作用，以确定青光眼治疗的基因组编辑策略是否适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流器官培养系统中的应用，将我们的工作扩展到人类。我们相信Cas9可以用来去除突变的MYOC基因，为MYOC突变引起的青光眼提供第一个有效的治疗方法。此外，眼睛是确定Cas9技术治疗潜力的理想试验点，这项工作将为Cas9应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.