Novel Genome Editing for the Treatment of Glaucoma

摘要 青光眼是一组异质性视神经病变，是不可逆视力丧失的主要原因， 全世界失明原发性开角型青光眼（POAG）是青光眼的最常见形式，并且通常是青光眼性的。 与升高的眼内压（IOP）相关，这是青光眼的主要致病风险因素。IOP升高 是由于小梁网（TM）的损伤增加了对房水流出的阻力。电流 青光眼治疗不能解决潜在的主要病理生理机制， 即使接受治疗也会继续恶化为了真正治愈青光眼，迫切需要永久抑制 或逆转对TM的昏迷性损害并恢复正常的TM细胞功能。新技术， 特别是CRISPR/Cas9基因组编辑，允许研究人员直接修改与以下相关的基因： 致病性损伤最近，我们证明了利用基因工程技术靶向突变型myocilin基因的可行性。 CRISPR-Cas9基因组编辑预防和逆转小鼠中肌青霉素相关POAG的青光眼 人类捐献的眼睛我们的目标是开发靶向TM的基因组编辑方法，用于治疗 肌球蛋白相关的开角型青光眼，以及重要的是开角型青光眼的一般治疗。我们有独特的机会 通过靶向常见病理途径抑制正在进行的疾病病理来治愈青光眼 与POAG有关。我们以前已经联系的病理作用，内质网（ER） 应激对高眼压小鼠模型和人类青光眼TM损伤和IOP升高的影响 捐献者的眼睛具体地说，我们已经表明，ER应激诱导的ATF 4和CHOP与 青光眼TM损伤和ATF 4和CHOP的独立抑制可能是一种新的治疗策略， 波格将军。因此，我们建议使用CRISPR-Cas9靶向这种常见的潜在机制， 系统我们的总体目标是设计编码靶向MYOC、CHOP和ATF 4的CRISPR/Cas9的AAV载体， 选择性地敲除TM中的这些基因并降低小鼠高眼压模型中的IOP，以及 在离体人灌注器官培养的眼前节中。我们假设基因组编辑敲除 靶向TM的CRISPR/Cas9载体将预防和逆转MYOC、CHOP和ATF 4的升高， 青光眼的IOP。我们将使用AAV载体，因为这些药物已用于患者的基因治疗 特定的眼部疾病。在这个提议中，我们将首先确定用于TM向性的最有效的AAV载体 设计有效的CRISPR/Cas9构建体，有效地敲除TM中的MYOC、CHOP和ATF 4。 我们还将评估CRISPR-Cas9在人类供体眼中的脱靶效应。其次，我们确定疗效 的AAV.cr. MYOC，AAV.cr. CHOP和AAV. cr. TM中的ATF 4敲除和这些敲除的能力， 降低小鼠体内和离体人灌注器官培养的前段中升高的IOP。我们 这种方法将通过直接干预青光眼对TM的损害， 提供一种有效治疗由IOP升高引起的青光眼的一次性疗法。

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.