Targeted Gene Therapy and Nanomedicine Approaches to Treat Corneal Diseases

摘要 角膜纤维化（瘢痕形成）是退伍军人、军事人员和平民失明的第三大原因。 在全球范围内，2300万人的一只眼睛患有角膜混浊/纤维化，490万人双眼失明。 目前，超过167，000名退伍军人在法律上是盲人，每年有超过7000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溃疡的方法。 人类的纤维化在过去4年的资助中，我们取得了许多新的发现，并成功地推进了 包括首次发现MyoD（成肌细胞决定蛋白1）、IL-37（白细胞介素37）和 角膜中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。充分利用 因此，我们提出测试三个新的假设，以推进角膜伤口愈合和纤维化的领域。 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是 体内无瘢痕角膜修复的时间敏感性基本机制及其及时靶向策略 分辨率将提供视力恢复的非手术手段。目前，角膜移植是 恢复视力的护理和无特异性的非手术疗法可治愈角膜纤维化而无副作用， 人类该提案测试了三个独立的具体目标：Aim-1测试了PEI 2-GNP 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， 在兔伤口愈合模型中的体内ERK/MAPK信号传导。Aim-2检验了双重作用IL-37 滴眼液将通过限制半胱天冬酶-1的可用性来抑制角膜炎症， 在显示角膜炎症和混浊的建立的兔CIT模型中与SMAD 3的体内结合 在病人身上看到的症状。目的-3检验一个假设，即开放场爆炸伤扰乱角膜基质细胞的功能。 通过mTORC 1介导的功能失调性自噬的体内稳态机制和伤口愈合特性 通过LC 3 I/II、Beclin、SQSTM 1、LAMP 1/2、TFEB和vATPase蛋白。将完成拟议的研究 使用已建立的角膜纤维化的体外人和体内临床前兔和啮齿动物模型， 开放视野爆炸部位;临床眼科检查，多模式2D/3D眼部成像，组织学，分子和细胞测定， 和透射电子显微镜使用我们公布的方法。项目顺利完成将推动 开发新的非手术策略来治疗角膜纤维化，填补许多知识空白，并满足 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.