Local Angiogenic Gene Therapy/VEGF/Treat Diabetic Ulcers

说明(由申请人提供)本提案的目标是确定 如果局部血管生成基因治疗与Adv-VEGF联合应用加速了血管的愈合 实验性糖尿病小鼠溃疡。每年，超过90,000个肢体和手指 在美国，84%的糖尿病患者接受截肢手术 其中有一例是足部溃疡。与以下疾病相关的5年死亡率 截肢的比例在39%到68%之间。预计每年 90亿美元的费用不包括帕金森综合症、痛苦和发病率 糖尿病足溃疡。伤口血管生成减少，此外 神经病变和压力，是导致骨折延迟愈合的主要原因 这些伤口。目前，还没有直接的血管生成疗法可用于 治疗糖尿病足溃疡。血管内皮生长因子刺激血管生成的能力很强 已经成立了。由于血管生成在伤口愈合和修复中起着关键作用 糖尿病创面局部应用血管内皮生长因子实验降低 可提供有效的治疗，无论是单独治疗还是联合治疗的一部分 治疗糖尿病创面的患者。局部腺病毒介导的基因 治疗提供了持续给药的理论优势 毒性最小的分子。尽管VEGF165基因治疗已经成为 能有效逆转四肢外周缺血、心肌缺血， 糖尿病神经病变，尚未在神经病理性糖尿病患者中得到彻底测试 溃疡，这是已知的减少血管生成。我们假设 局部伤口注射Adv-VEGF将导致VEGF的持续释放， 从而刺激血管生成，促进实验性骨愈合 糖尿病小鼠伤口，同时表现出最小的全身副作用。我们 将Adv-VEGF用于6周龄小鼠的雌性糖尿病小鼠模型， 有可接受的代谢控制，以确定ADV-VEGF的最小剂量 这将导致统计上的时间显著减少到100% 实验性糖尿病溃疡的闭合。我们将衡量潜在的系统性方面 影响(例如，视网膜病变和肝脏毒性)，并确定是否增加 血清中存在一定水平的血管内皮生长因子。末梢器官毒性将由血清检测 化学检查，全血细胞计数，以及肠道组织学分析， 视网膜、肺、肾、脾、胸腺、脑、肝和心脏。我们还将 检查是否有局部伤口毒性的迹象。将使用聚合酶链式反应来评估 腺病毒载体的潜在全身分布。 血管生成反应与变化的特异性定量比较 在胶原沉积和伤口愈合方面，将对上皮化进行评估。 此外，我们将分析伤口液以确定颞叶 血管内皮生长因子在糖尿病溃疡中的表达

DESCRIPTION (provided by applicant) The goal of this proposal is to determine if local angiogenic gene therapy with ADV-VEGF accelerates healing in experimental diabetic mouse ulcers. Yearly, over 90,000 limb and digit amputations are performed on diabetic patients in the United States, 84 percent of these are proceeded by a foot ulcer. The 5-year mortality associated with these amputations is between 39 percent and 68 percent. The estimated annual cost of $9 billion doesn't include the pam, suffering, and morbidity of diabetic foot ulcers. Decreased angiogenesis in the wound, in addition to neuropathy and pressure, is a major causative factor for the delayed healing of these wounds. Currently, there is no directly angiogenic therapy available to treat diabetic foot ulcers. VEGF's ability to stimulate angiogenesis is well established. Since angiogenesis maintains a critical role in wound healing and VEGF is experimentally decreased in diabetic wounds, locally administered VEGF may provide an effective treatment, either alone or as part of combination therapy, for patients with diabetic wounds. Local adenoviral mediated gene therapy provides the theoretical advantage of sustained delivery of the target molecule with minimal toxicity. Although VEGF165 gene therapy has been effective in reversing peripheral ischemia of the limbs, cardiac ischemia, and diabetic neuropathy, it has not been thoroughly tested in neuropathic diabetic ulcers, which are known to have decreased angiogenesis. We hypothesize that local wound injection of ADV-VEGF will result in a sustained release of VEGF, thereby stimulating angiogenesis and accelerating healing in experimental diabetic mouse wounds, while demonstrating minimal systemic side effects. We will use ADV-VEGF in the female diabetic mouse model with 6-week mice, which have acceptable metabolic controls, to determine the minimal dose of ADV-VEGF that will result in a statistically significant decrease in time to 100 percent closure m experimental diabetic ulcers. We will measure potential systemic side effects (e.g., retinopathy and liver toxicity) and determine if increased levels of VEGF occur in the serum. End organ toxicity will be assayed by serum chemistries, complete blood count, and histologic analysis of the intestine, retina, lungs, kidney, spleen, thymus, brain, liver and heart. We will also check for signs of local wound toxicity. PCR will be used to assess the potential systemic distribution of the adenoviral vector. Specific quantification of the angiogenic response as compared to the changes in collagen deposition and wound healing epithelialization will be evaluated. In addition, we will analyze the wound fluid in order to determine the temporal expression of VEGF in a diabetic ulcer.