Targeted Gene Therapy and Nanomedicine Approaches to Treat Corneal Diseases

描述（由申请人提供）： 由于创伤或受伤造成的角膜瘢痕是我们退伍军人和部队失明的主要原因。它每年影响150万美国人，是全球失明的第三大原因。尽管使用了战斗护目镜，但在阿富汗和伊拉克战斗中受伤的士兵中有16%的人眼睛受伤。由于缺乏有效的治疗方法，每年有20%的受伤士兵被摘除眼睛，超过45，000名美国人接受角膜移植。该项目的重点是开发基于纳米技术的基因治疗方法，提供长期的缓解而没有显着的副作用，这是一种治疗角膜失明的新方法。是在过去一 在资助期内，我们在这方面取得了显著进展，这从我们的出版物和奖项中可以看出。我们成功地鉴定了一种有效的纳米颗粒载体、递送技术和靶向基因治疗方法，并使用兔模型测试了抗转化生长因子-β（TGF β）基因在体内治疗角膜盲的潜力。我们的直接目标是测试4个新的假设，这些假设被制定为4个特定的目标：1）测试聚乙烯亚胺缀合的金纳米颗粒（PEI 2-GNP）在眼睛中的驻留不影响体内角膜中的角膜基质细胞和内皮密度或原纤维均匀性，2）测试 通过PEI 2-GNP在角膜中的骨形态发生蛋白-7（BMP 7）递送通过减轻纤维化Smad信号传导、增加分化蛋白的抑制剂和阻断角膜角膜细胞分化成肌成纤维细胞来调节角膜愈合，肌成纤维细胞是体内瘢痕形成的机制，3）检测由肝细胞生长因子（HGF）和BMP 7或可溶性TGF β受体II组成的双基因联合治疗，（sTGF β RII）和BMP 7基因是一种创新的方法，可以在体内最大限度地消除固定和愈合的角膜瘢痕，以及4）测试通过在眼睛上局部施用伏立诺他（每天两次，持续3天）来广泛地表观遗传敲除TGF β诱导的促纤维化基因是一种治疗体内角膜瘢痕的新方法。 没有严重的副作用我们已发表和正在进行的研究强烈支持这些假设。对于成人眼睛的角膜透明度和正常功能，22，000 -24000个角膜细胞/mm 3、2400-3200个内皮细胞/mm 2和特征性胶原纤维组织是必需的。我们正在进行的研究表明，PEI 2-GNP是一种安全有效的眼部药物递送载体，sTGF β RII基因转移抑制TGF β，BMP 7调节Smad通路，HGF选择性诱导角膜肌成纤维细胞凋亡。我们的实验室拥有新颖的体内兔和体外人角膜纤维化模型，并在动物研究、临床眼部成像、纳米颗粒表征、组织学、电子显微镜、蛋白质印迹和分子生物学技术方面拥有丰富的经验。拟议研究的成功完成将显着提高我们对角膜伤口愈合的分子机制的理解，继续/推进新型眼部给药系统和角膜失明的新疗法的开发，并推进视觉研究领域。

DESCRIPTION (provided by applicant): Corneal scarring due to trauma or injury is a major cause of blindness in our veterans and troops. It affects 1.5 million Americans each year and is the 3rd leading cause of global blindness. Despite using combat goggles, eye injuries occurred in 16% of wounded troops fighting in Afghanistan and Iraq. A lack of efficient therapies caused eye removal in 20% of injured troops and corneal transplantation in >45,000 American each year. This project focuses on developing nanotechnology-based gene therapy approaches that offer long-term relief without significant side effects, which is a novel modality to treat corneal blindness. During last funding period, we made remarkable progress to this end as evident from our publications and awards. We successfully identified a potent nanoparticle vector, delivery techniques, and targeted gene therapy approaches, and we tested the potential of anti-transforming growth factor-� (TGF�) genes to treat corneal blindness in vivo using a rabbit model. Our immediate goal is to test 4 novel hypotheses formulated into 4 specific aims: 1) test that the residence of polyethyleneimine conjugated gold nanoparticles (PEI2-GNPs) in the eye does not affect corneal keratocyte and endothelial density or fibril uniformity in the cornea in vivo, 2) test that bone morphogenic protein-7 (BMP7) delivery in the cornea via PEI2-GNP regulates corneal healing by mitigating fibrotic Smad signaling, increasing inhibitors of differentiation proteins, ad blocking corneal keratocyte differentiation to myofibroblasts, a mechanism of scar development in vivo, 3) test that 2-gene combination therapy consisting of hepatocyte growth factor (HGF) and BMP7 or soluble TGF� receptor II (sTGF�RII) and BMP7 genes is an innovative approach to achieve maximum resolution of fixed- and healing- corneal scars in vivo, and 4) test that a broad-based epigenetic knockdown of TGF�-induced profibrotic genes by a topical vorinostat (twice a day for 3 days) application on the eye is a novel method to treat corneal scarring in vivo without major side effects. Our published and ongoing studies strongly support these hypotheses. For corneal clarity and normal function in the adult human eye, 22,000-24000 keratocytes/mm3, 2400-3200 endothelial/mm2 and characteristic collagen fibril organization are necessary. Our ongoing studies suggest that PEI2-GNPs are a safe and potent vector for ocular drug delivery, sTGF�RII gene transfer inhibits TGF�, BMP7 modulates the Smad pathway, and HGF selectively induces apoptosis in corneal myofibroblasts. Our lab has novel in vivo rabbit and in vitro human corneal fibrosis models and substantial experience in performing animal studies, clinical eye imaging, nanoparticle characterization, histology, electron microscopy, western blotting and molecular biology techniques. Successful completion of the proposed research will significantly improve our understanding of the molecular mechanisms of corneal wound healing, continue/progress the development of a novel ocular delivery system and newer therapies for corneal blindness, and advance the vision research field.