Hypoxia Regulated Gene Therapy for Neovascularization

描述（由申请人提供）：我们的假设是，引起新生血管（NV）的缺氧形成了一种新的治疗方法的机制基础，以阻止新血管的生长和相关的血管通透性。这种方法可以从根本上改变糖尿病视网膜病变和AMD的临床管理。我们的建议的创新方面是：)发展的一个新的基因治疗策略使用与hypoxia-responsive重组AAV (rAAV)向量元素()一定是专门提供抗血管生成分子穆勒RPE细胞或细胞的缺氧区域内视网膜血管开始生长,b)的评估是否Muller细胞或RPE细胞中基因表达的更合适的目标前新血管形成,和细胞类型是脉络膜的NV的更合适的目标;c)测试激光激活基因表达的新想法，以精确传递到特定的病理。

DESCRIPTION (provided by applicant): Our hypothesis is that the hypoxia that elicits neovascularization (NV) forms a mechanistic basis for a novel therapeutic approach to arrest new vessel growth and associated vascular permeability. This approach could radically alter the clinical management of diabetic retinopathy and AMD. The innovative aspects of our proposal are: a) the development of a new gene therapy stategy using recombinant AAV (rAAV) vectors with hypoxia-responsive elements (HRE) to deliver anti-angiogenic molecules exclusively to either RPE cells or Muller cells within the hypoxic regions of retina where new vessels begin to grow, b) the evaluation of whether gene expression in the Muller cells or RPE cells is the more appropriate target for preretinal neovascularization, and which cell type is the more appropriate target for choroidal NV; and c) test a novel idea for laser-activated gene expression for precise delivery to specific pathologies. To accomplish these goals we will: 1 ) Construct AAV-HRE-vectors and characterize their specificity and oxygen response in vitro and in vivo. 2) Using the murine ROP model, identify whether angiostatin, endostatin, or tubedown-1 (a novel regulator of blood vessel growth) offers the best inhibiton in Muller-cell specific vectors (with a GFAP promoter). 3) Using the ROP model, determine whether the inhibition of NV is dependent on the cell location (RPE or Muller cell) in which the therapeutic gene (identified in 2) is expressed. 4) Similarly, using the CNV model, determine whether inhibition of NV is dependent on the cell in which the gene is expressed. (The location of gene expression is controlled by cell-specific promoters.) 5) Since laser treatment upregulates GFAP, we will determine whether it also activates expression of a reporter gene (GFP) in mice treated with Muller-cell specific vectors (GFAP promoters) (rAAV-GFAP-GFP).