Gene Therapy in the Cornea

描述（由申请人提供）：角膜疾病是全球失明的主要原因，每年影响150万美国人。角膜的组织靶向和局部基因治疗有望治愈、减少和/或预防由创伤、损伤、感染或遗传性营养不良引起的角膜失明。我们研究的长期目标是确定临床上适用的，简单的，微创的靶向基因治疗角膜盲。最近，我们证明了靶向腺相关病毒（AAV）-核心蛋白聚糖基因治疗有效地抑制兔角膜纤维化和新生血管在体内。它没有 显示任何短期毒性。我们的中心假设是核心蛋白聚糖是角膜愈合的主要调节剂，并且作为信号分子，它与许多细胞因子和/或其受体相互作用，并防止角膜过度愈合和疾病。我们发现角膜中的核心蛋白聚糖与转化生长因子相互作用？(TGF??),表皮生长因子受体（EGFR）和血管内皮生长因子（VEGF）。我们的RNA敲除研究表明，TGF？通过Smad信号介导角膜纤维化，Smad 7是阻断TGF β 1的有吸引力的靶点。病理活动该项目的目标是：i）测试有效的AAV-核心蛋白聚糖基因疗法是否对角膜具有任何长期毒性，ii）收集角膜特异性数据并理解角膜中核心蛋白聚糖功能的分子机制，以及iii）鉴定用于角膜失明的另外的基于基因的疗法。由于医学状况、对药物的敏感性、耐受性水平等的变化，一种治疗方法并不适用于所有患者。具体目的是：（1）测试以下假设：在体内将核心蛋白聚糖靶向递送到兔角膜细胞中不会改变基质中的原纤维结构或特征组织，调节角膜细胞分化、迁移和增殖，并调节角膜愈合。(2)验证靶向AAV-Smad 7基因治疗是破坏TGF？信号传导并防止体内角膜中的肌成纤维细胞形成和纤维化。(3)检验组织选择性AAV-PEDF基因治疗在体内递送到兔角膜细胞中抑制兔角膜新生血管化而没有显著不良反应的假设。这些目标将使用定义明确的兔疾病模型、最近定义的靶向AAV治疗方法、已建立的方案和技术（包括裂隙灯、立体显微镜和光学相干断层扫描、免疫组织化学、免疫印迹、定量PCR、Southern印迹、透射电子和共聚焦显微镜等）来测试。所提出的实验将为AAV介导的核心蛋白聚糖如何在细胞内表达提供重要答案。Smad 7和PEDF基因治疗可以单独或联合使用，以恢复视力或预防角膜瘢痕或新血管形成导致的失明。他们还将提供有关角膜愈合的许多基本问题的答案，以及角膜细胞和细胞因子在角膜疾病和疾病中的作用。 公共卫生相关性：创伤、损伤或感染后的角膜瘢痕形成和新生血管形成可导致失明。通过简单的局部技术递送至角膜的靶向基因疗法提供了通过调节导致这些致盲角膜病症的过度愈合机制来治愈、治疗和/或预防视力丧失的希望。

DESCRIPTION (provided by applicant): Corneal disorders are the leading cause of global blindness and affect 1.5 million Americans each year. Tissue-targeted and localized gene therapy for the cornea offers promise to cure, reduce and/or prevent corneal blindness caused by trauma, injury, infection or inherited dystrophies. The long-range goal of our research is to define clinically applicable, simple, minimally invasive targeted gene-based therapies for corneal blindness. Recently, we demonstrated that targeted adeno-associated virus (AAV)-decorin gene therapy efficiently inhibits corneal fibrosis and neovascularization in vivo in rabbits. It did not show any short-term toxicity. Our central hypothesis is that decorin is a master regulator of corneal healing and, acting as a signaling molecule, it interacts with many cytokines and/or their receptors and prevents excessive healing and disease in the cornea. We found decorin in the cornea interacts with transforming growth factor ? (TGF??), epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF). Our RNA knockdown studies revealed that TGF?? mediates corneal fibrosis via Smad signaling, and Smad7 is an attractive target to block TGF?? pathologic activity. The goals of this project are: i) test whether efficacious AAV-decorin gene therapy has any long-term toxicity for the cornea, ii) collect cornea-specific data and an understanding of the molecular mechanism of decorin functions in the cornea, and iii) identify additional gene-based therapy for corneal blindness. One treatment does not work for all patients due to variations in medical conditions, susceptibility to medication, tolerance level etc. The Specific Aims are: (1) Test the hypothesis that targeted decorin delivery into rabbit keratocytes in vivo does not alter fibril structure or characteristic organization in the stroma, modulates keratocyte differentiation, migration and proliferation, and regulates corneal healing. (2) Test the hypothesis that targeted AAV-Smad7 gene therapy is a novel approach to disrupt TGF? signaling and prevent myofibroblast formation and fibrosis in the cornea in vivo. (3) Test the hypothesis that tissue-selective AAV-PEDF gene therapy delivered into rabbit keratocytes in vivo inhibits corneal neovascularization in rabbits without significant adverse effects. These aims will be tested using well-defined rabbit disease models, recently defined targeted AAV therapy approaches, established protocols and techniques, including slit-lamp, stereomicroscope and optical coherence tomography, immunohistochemistry, immunoblotting, quantitative PCR, southern blot, transmission electron and confocal microscopy, etc. The proposed experiments will provide important answers to how AAV- mediated decorin, Smad7 and PEDF gene therapy may be used, alone and in combination, to restore vision or prevent blindness from corneal scarring or neovascularization. They also will provide answers to many fundamental questions about corneal healing, and the role of keratocyte and cytokines in corneal disorders and diseases. PUBLIC HEALTH RELEVANCE: Corneal scarring and neovascularization following trauma, injury or infection cause blindness. Targeted gene therapy delivered to the cornea with a simple topical technique offers promise to cure, treat and/or prevent vision loss by modulating excessive healing mechanisms that cause these blinding corneal disorders.