Hypoxia Regulated Gene Therapy for Neovascularization

描述（由申请人提供）：我们的假设是，促进新生血管形成（NV）的缺氧形成了阻止新血管生长和相关血管通透性的新型治疗方法的机制基础。 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。 我们建议的创新之处在于：a）开发了一种新的基因治疗策略，其使用具有缺氧反应元件（HRE）的重组AAV（rAAV）载体将抗血管生成分子专门递送至视网膜缺氧区域内的RPE细胞或Muller细胞，在所述缺氧区域中新血管开始生长，B）评估Muller细胞或RPE细胞中的基因表达是否是视网膜前新生血管形成的更合适的靶标，以及哪种细胞类型是脉络膜NV的更合适的靶标;和c）测试激光激活基因表达的新想法，用于精确递送到特定病理。 为了实现这些目标，我们将： 1）构建AAV-HRE载体并在体外和体内表征其特异性和氧响应。 2)使用鼠ROP模型，确定血管抑制素、内皮抑制素或tubedown-1（一种新的血管生长调节剂）是否在Muller细胞特异性载体（具有GFAP启动子）中提供最佳载体。 3)使用ROP模型，确定NV的抑制是否依赖于表达治疗基因（在2中鉴定）的细胞位置（RPE或Muller细胞）。 4)类似地，使用CNV模型，确定NV的抑制是否依赖于基因表达的细胞。(The基因表达的位置由细胞特异性启动子控制。 5)由于激光治疗上调GFAP，我们将确定它是否也激活报告基因（GFP）的表达与穆勒细胞特异性载体（GFAP启动子）（rAAV-GFAP-GFP）治疗的小鼠。

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).