Targeted Gene Therapy and Nanomedicine Approaches to Treat Corneal Diseases

摘要 角膜纤维化(疤痕形成)是导致退伍军人、军人和平民失明的第三大原因。 全球有2300万人一只眼有角膜混浊/纤维化，490万人双眼失明。 目前，有16.7万名退伍军人在法律上失明，每年有7000名退伍军人失明，200万名退伍军人失明 视力受损。我们研究的长期目标是确定有效和安全的治疗角膜的方法。 人类的纤维化症。在过去四年的资助中，我们取得了许多新发现，并成功地推进了 发展包括首次发现MyoD(成肌细胞决定蛋白1)、IL-37(白介素37)和 角膜自噬相关基因及其在角膜创伤修复和纤维化中的重要作用。利用 这些，我们建议检验三个新的假说，以推进角膜创伤愈合和纤维化领域 进一步治疗。我们的中心假设是，角膜基质中肌成纤维细胞的形成和消除 无瘢痕角膜在体修复的时间敏感性基本机制及其适时策略 RESOLUTION将提供非手术的视力恢复手段。目前，角膜移植的标准是 护理恢复视力和非特异性非手术治疗存在治愈角膜纤维化而无副作用的 人类。该提案测试三个独立的特定目标：AIM-1测试PEI2-GNP的假设 纳米颗粒介导的定位和组织靶向MyoD基因沉默治疗将去除已建立的角膜 通过促进角膜肌成纤维细胞(CMF)去分化为成纤维细胞来促进瘢痕/纤维化 在兔创伤愈合模型中ERK/MAPK信号转导。AIM-2验证了一个假设，即双向作用的IL-37 滴眼液通过限制Caspase-1的可获得性来抑制角膜炎症，并通过以下方式防止角膜混浊 在已建立的显示角膜炎症和混浊的兔CIT模型中与Smad3的体内结合 在病人身上看到的病理。AIM-3测试了一种假说，即开阔的冲击伤会扰乱角膜基质细胞 MTORC1介导的功能失调自噬在体内的动态平衡机制和伤口愈合特性 通过LC3I/II、Beclin、SQSTM1、LAMP1/2、TFEB和vATPase蛋白表达。建议的研究将会完成 利用已建立的体外人和体内临床前兔和啮齿动物角膜纤维化模型，实际 露天爆炸现场；临床眼科检查，多模式2D/3D眼科成像，组织学，分子和细胞分析， 和使用我们发表的方法的透射电子显微镜。项目顺利完成将取得进展 治疗角膜纤维化的新的非手术策略的发展，填补了许多知识空白，并满足了 退伍军人健康管理局的使命是为我们的退伍军人提供最好的医疗保健。

Abstract Corneal fibrosis (scarring) is a 3rd leading cause of blindness in veterans, military personnel, and civilians. Globally, 23 million people have corneal opacity/fibrosis in one eye and 4.9 million people are blind in both eyes. Currently, >167,000 veterans are legally blind, >7000 veterans develop blindness each year and 2 million have impaired vision. The long-term goal of our research is to define efficient and safe therapies to treat corneal fibrosis in humans. In last 4 years of funding, we made many new discoveries and successfully advanced development including first time finding of MyoD (myoblast determination protein 1), IL-37 (interleukin 37), and autophagy-related genes in cornea and their significant role in corneal wound repair and fibrosis. Capitalizing on these, we propose testing three new hypotheses to advance the field of corneal wound healing and fibrosis treatment further. Our central hypothesis is that formation and elimination of myofibroblast in corneal stroma is a time-sensitive essential mechanism for scarless corneal repair in vivo and strategies targeting its timely resolution will provide non-surgical means of vision restoration. Currently, corneal transplantation is standard of care to restore vision and no-specific non-surgical therapies exist to cure corneal fibrosis without side effects in humans. This proposal tests three independent specific aims: Aim-1 tests a hypothesis that PEI2-GNP nanoparticle mediated localize and tissue-targeted MyoD gene silencing therapy will remove established corneal scars/fibrosis by facilitating de-differentiation of corneal myofibroblasts (CMFs) into fibroblast cells via an ERK/MAPK signaling in vivo in a rabbit model of wound healing. Aim-2 tests a hypothesis that dual-action IL-37 eye drops will inhibit corneal inflammation by limiting the availability of Caspase-1 and prevent corneal haze by binding to SMAD3 in vivo in an established rabbit CIT model that shows corneal inflammation and haze pathologies seen in patients. Aim-3 tests a hypothesis that open-field blast injury upsets corneal keratocytes’ homeostatic mechanism and wound healing properties in vivo via mTORC1-mediated dysfunctional autophagy through LC3I/II, Beclin, SQSTM1, LAMP1/2, TFEB, & vATPase proteins. Proposed studies will be accomplished employing established in vitro human and in vivo pre-clinical rabbit and rodent models of corneal fibrosis, actual open field blast site; clinical eye exams, multimodal 2D/3D eye imaging, histology, molecular and cellular assays, and transmission electron microscopy using our published method. Successful completion of project will advance the development of novel non-surgical strategies to treat corneal fibrosis, fill many knowledge gaps, and meet the mission of the Veterans Health Administration to provide best health care to our veterans.