Hypoxia Regulated Gene Therapy for Neovascularization

描述(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，以阻止新的血管生长和相关的血管通透性。这种方法可能从根本上改变糖尿病视网膜病变和AMD的临床治疗。我们建议的创新方面包括：a)开发一种新的基因治疗策略，使用带有缺氧反应元件(HRE)的重组AAV(RAAV)载体将抗血管生成分子特异性地输送到视网膜缺氧区内的RPE细胞或Muller细胞，在那里新的血管开始生长；b)评估Muller细胞或RPE细胞中的基因表达是视网膜前新生血管的更合适靶点，以及哪种细胞类型是脉络膜NV的更合适靶点；以及c)测试激光激活基因表达的新想法，以便精确输送到特定的病理。 为实现这些目标，我们将： 1)构建AAV-HRE-载体，并对其体外和体内的特异性和氧反应进行鉴定。 2)利用小鼠ROP模型，确定血管抑素、内皮抑素或tubedown-1(一种新的血管生长调节因子)在Muller细胞特异性载体(带有GFAP启动子)中提供最好的抑制作用。 3)使用ROP模型，确定NV的抑制是否依赖于表达治疗性基因的细胞位置(RPE或Muller细胞)。 4)类似地，使用CNV模型，确定NV的抑制是否依赖于表达基因的细胞。(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了Muller细胞特异性载体(GFAP启动子)(rAAV-GFAP-GFP)处理的小鼠的报告基因(GFP)的表达。

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).