Targeted Gene Therapy and Nanomedicine Approaches to Treat Corneal Diseases

描述(由申请人提供)： 摘要眼睛的损伤和感染会造成角膜疤痕，每年影响超过150万美国人。尽管使用了战眼保护，我们在阿富汗和伊拉克的伤员中仍有16%的人眼睛受伤。目前治疗角膜疤痕或新生血管的方法只能提供短期缓解，会引起严重的副作用，而且往往无效。美国每年超过5万例角膜移植手术，以及在最近的战争中受伤的20%的美军士兵的眼睛被摘除，都突显了当前治疗的无能为力。角膜是基因治疗的理想器官，因为它的可及性和免疫特权状态。最近，我们确定了针对正常和病变兔眼的组织靶向GNPs或AAV5载体的基因治疗方法，并表明治疗性基因可以有效地导入靶向角膜细胞来治疗角膜瘢痕形成和血管生成。我们最近的研究表明，金纳米颗粒(GNPs)对角膜是安全的，因此可以用来在体内运送角膜中的基因。我们的中心假设是，组织选择性治疗性基因在角膜中的传递可以治愈角膜疾病，而不会引起明显的副作用。本研究项目将验证以下假设：稳定在阿拉伯树胶或聚乙烯亚胺中的GNPs是无毒、无免疫原性和高效的载体，可以将治疗性基因转移到体内的兔角膜基质细胞中，可以提供安全和新颖的基于纳米技术的非病毒基因治疗方法来治疗角膜疾病，并且靶向传递基因使转化生长因子-2(TGF2)功能失效的基因将在体内阻止角膜瘢痕形成。研究表明，TGF2基因通过促进角膜基质细胞向肌成纤维细胞转化，在体内引起角膜瘢痕形成。我们将确定用阿拉伯树胶或聚乙烯亚胺(AIM 1)稳定的GNPs在体内将治疗性基因导入兔角膜基质细胞的条件，并通过GNPs或AAV5载体在体内将选定的基因导入兔角膜基质细胞，测试可溶性II型TGF2受体(AIM 2)和骨形态发生蛋白7(AIM 3)基因控制角膜瘢痕形成的效果。我们的初步研究表明，被测试的GNPs和AAV5载体在体内可以高效地选择性地将基因导入兔角膜基质细胞。使用兔角膜瘢痕模型、组织靶向基因治疗方法(本实验室最近优化)、生物显微镜、免疫细胞化学、细胞和分子检测将完成对这些目的的检测。拟议研究的成功完成有可能扩展一种有效的新方法来治疗角膜疤痕和其他流行的角膜疾病。我们对GNPs的研究将极大地推动眼部纳米医学领域的研究工作。 公共卫生相关性： 项目简介：与退伍军人健康的相关性以临床为导向的基础科学研究是重要的，并实现了退伍军人健康管理局的目标，即为美国退伍军人实现卓越的医疗保健。这一建议为利用纳米金治疗角膜瘢痕形成的组织选择性基因治疗方法的发展提供了动力，从而提供了长期的好处。现有的治疗方法只提供短期的好处，而且往往无效。拟议的临床前研究为亟需的针对性、有效和长期治疗方法的开发奠定了基础，以治愈角膜疤痕。众所周知，眼睛的损伤和感染会导致角膜疤痕。我们在阿富汗和伊拉克战争中受伤的士兵中，大约16%的人眼睛受伤，尽管他们戴着战眼保护。眼外伤导致的视力丧失是我们军队的一个主要问题，目前还没有药物可以在不引起严重副作用的情况下治愈角膜疤痕。

DESCRIPTION (provided by applicant): Abstract Injury and infection to the eye cause corneal scarring and affect over 1.5 million Americans each year. Despite the use of combat eye protection, eye injuries occurred in 16% of our wounded troops in Afghanistan and Iraq. Current therapies for corneal scarring or angiogenesis provide only short-term relief, cause serious side effects and are often ineffective. Over fifty thousand corneal transplantations in Americans each year and removal of eyes in 20% of the U.S. troops injured in recent wars highlight the incompetence of current therapy. The cornea is an ideal organ for gene therapy because of its accessibility and immune-privilege status. Recently we defined tissue-targeted GNPs or AAV5 vector gene therapy approaches for the normal and diseased rabbit eye, and showed that therapeutic genes can be delivered into target corneal cells efficiently to treat corneal scarring and angiogenesis. Our recent studies suggest that gold nanoparticles (GNPs) are safe for the cornea and thus can be used to deliver genes in the cornea in vivo. Our central hypothesis is that tissue-selective therapeutic gene delivery in the cornea can cure corneal disorders without causing significant side effects. This research project will test the hypotheses that GNPs stabilized in gum arabic or polyethyleneimine are nontoxic, non-immunogenic and efficient vehicles for transporting therapeutic genes into keratocytes of the rabbit cornea in vivo, can provide safe and novel nanotechnology-based nonviral gene therapy modalities to treat corneal diseases, and targeted delivery genes disabling transforming growth factor-2 (TGF2) function will impede corneal scarring in vivo. TGF2 gene has been shown to cause scarring in the cornea in vivo by promoting keratocyte transformation to myofibroblasts. We will define conditions for delivering therapeutic genes into rabbit keratocytes in vivo using GNPs stabilized in gum arabic or polyethyleneimine (Aim 1), test the efficacy of soluble type II TGF2 receptor (Aim 2) and bone morphogenic protein 7 (Aim 3) genes to control corneal scarring by delivering selected genes into rabbit keratocytes in vivo with GNPs or AAV5 vector. Our preliminary studies show that tested GNPs and AAV5 vectors are highly efficient in introducing genes selectively into rabbit keratocyte in vivo. Using rabbit corneal scarring model, tissue-targeted gene therapy approaches (recently optimized in our laboratory), bio-microscopy, immunocytochemistry, cellular and molecular assays testing of these aims will be accomplished. Successful completion of proposed research has the potential to extend an efficient new method to treat corneal scarring as well as other prevalent corneal diseases. Our studies on GNPs will substantially advance the research efforts in the field of ocular nanomedicine. PUBLIC HEALTH RELEVANCE: Project Narrative: Relevance to Veterans Health Clinically-oriented basic science research is important and fulfills the objective of the Veterans Health Administration objective to achieve excellence in health care for American veterans. This proposal provides an impetus toward the development of tissue-selective gene therapy methods using gold nanoparticles to treat corneal scarring which provide long-term benefits. Existing therapies provide only short-term benefits and are often ineffective. The proposed preclinical research lays the groundwork for the much-needed development of targeted, effective and long-term therapies to cure corneal scarring. Injury and infections to eye is known to cause corneal scarring. About 16% of our wounded troops in Afghanistan and Iraq wars sustained eye injuries despite wearing combat eye protection. Vision loss from eye trauma is a major problem among our military and no drugs are currently available to cure corneal scarring without causing serious side effects.