Novel Genome Editing for the Treatment of Glaucoma

摘要 青光眼是一组异质性的视神经疾病，是不可逆转的视力丧失和 世界范围内的失明。原发性开角型青光眼(POAG)是最常见的青光眼形式，通常 眼压升高是导致青光眼的主要危险因素。高眼压 是由于小梁网络(TM)受损，增加了对房水外流的阻力。当前 青光眼的治疗没有解决潜在的主要病理生理机制和疾病 尽管接受了治疗，但仍能继续进展。为了真正治愈青光眼，迫切需要永久抑制 或者逆转青光眼对TM的损害，恢复TM细胞的正常功能。新技术， 特别是CRISPR/Cas9基因组编辑，使研究人员能够直接修改与 致病损害。我们最近证明了以突变的myoclin基因为靶点的可行性 CRISPR-Cas9基因组编辑预防和逆转小鼠和小鼠肌球蛋白相关POAG的青光眼 人类捐献的眼睛。我们的目标是开发针对TM的基因组编辑方法，用于治疗 霉菌素相关的POAG，以及重要的POAG的一般治疗。我们有一个独特的机会来 通过靶向常见病理途径抑制正在进行的疾病病理治疗青光眼 与普瓦格将军有关。我们先前已经将内质网(ER)的病理作用联系起来。 小鼠高眼压模型与人青光眼视网膜损害及眼压升高的应激反应 捐献的眼睛。具体地说，我们已经证明了内质网应激诱导的ATF4和CHOP与 青光眼的TM损伤和ATF4和CHOP的独立抑制可能是一种新的治疗策略 普瓦格将军。因此，我们建议使用CRISPR-CAS9将这一共同的基础机制作为目标 系统。我们的总体目标是设计针对MYOC、CHOP和ATF4的编码CRISPR/Cas9的AAV载体 在小鼠高眼压模型中选择性地敲除TM和降低眼压中的这些基因，以及 在体外培养的人体灌流器官前段。我们假设基因组编辑敲除 使用AAV.CRISPR/Cas9载体靶向TM的MYOC、CHOP和ATF4将防止和逆转升高 青光眼的眼压。我们将使用AAV载体，因为这些试剂已经用于患者的基因治疗 患有特定的眼疾。在这个方案中，我们将首先确定对TM趋向性最有效的AAV载体(S 并设计高效的CRISPR/Cas9结构，有效地击倒TM中的MYOC、CHOP和ATF4。 我们还将评估CRISPR-Cas9在人类供体眼睛中的非靶向效应。第二，我们确定了疗效 AAV.cr.MYOC，AAV.cr.Chop和AAV.cr.TM中的ATF4击倒以及这些击倒的能力 降低小鼠体内高眼压和人体灌流器官培养前节的体外高眼压。我们的 该方法将通过直接干预青光眼对TM和 提供一次性治疗，有效治疗高眼压引起的青光眼。

Abstract Glaucoma is a heterogenous group of optic neuropathies and a leading cause of irreversible vision loss and blindness worldwide. Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma and is often associated with elevated intraocular pressure (IOP), a leading causative risk factor for glaucoma. Elevated IOP is due to damage to the trabecular meshwork (TM) that increases resistance to aqueous humor outflow. Current glaucoma treatments do not address the underlying primary pathophysiological mechanisms and the disorder can continue to progress despite treatment. To truly cure glaucoma, there is critical need to permanently inhibit or reverse the glaucomatous damage to the TM and restore normal TM cell functions. New technologies, specifically CRISPR/Cas9 genome editing, allow investigators to directly modify the genes associated with pathogenic damage. We recently demonstrated the feasibility of targeting the mutant myocilin gene using CRISPR-Cas9 genome editing to prevent and reverse glaucoma in myocilin-associated POAG in mice and in human donor eyes. It is our goal to develop genome editing methods targeting the TM for the treatment of myocilin-associated POAG, and importantly the treatment of POAG in general. We have a unique opportunity to cure glaucoma by inhibiting the ongoing disease pathology by targeting common pathological pathways associated with general POAG. We have previously linked the pathological role of endoplasmic reticulum (ER) stress to the glaucomatous TM damage and IOP elevation in mouse models of ocular hypertension and in human donor eyes. Specifically, we have shown that ER-stress induced ATF4 and CHOP are associated with glaucomatous TM damage and independent inhibition of ATF4 and CHOP can be a novel treatment strategy for general POAG. Therefore, we propose to target this common underlying mechanism using the CRISPR-Cas9 system. It is our overall goal to design AAV vectors encoding CRISPR/Cas9 targeting MYOC, CHOP and ATF4 to selectively knockout these genes in the TM and lower IOP in mouse ocular hypertension models, as well as in ex vivo human perfusion organ cultured anterior segments. We hypothesize that genome editing knockout of MYOC, CHOP and ATF4 using AAV.CRISPR/Cas9 vectors targeting the TM will prevent and reverse elevated IOP in glaucoma. We will use AAV vectors because these agents have been used for gene therapy in patients with specific eye diseases. In this proposal, we will first determine the most efficient AAV vector(s) for TM tropism and design efficient CRISPR/Cas9 constructs that effectively knockdown MYOC, CHOP and ATF4 in the TM. We will also evaluate CRISPR-Cas9 off-target effects in human donor eyes. Second, we determine the efficacy of AAV.cr. MYOC, AAV.cr. CHOP and AAV.cr. ATF4 knockouts in the TM and the ability of these knockouts to reduce elevated IOP in vivo in mice and ex vivo in human perfusion organ cultured anterior segments. Our approach will revolutionize glaucoma therapy by directly interfering with glaucomatous damage to the TM and provide a one-time therapy to effectively treat glaucoma resulting from elevated IOP.