Novel Gene Therapy for Critical Limb Ischemia and Limb Salvage in Vascular Occlusive Disease

项目概要/摘要： 严重肢体缺血（CLI）是外周动脉疾病（PAD）的最晚期。它表现为 腿部疼痛、行走距离缩短、伤口不愈合和坏疽。大约1/3的CLI患者没有 对标准治疗有反应并最终接受大截肢。这一关键的未满足需求要求 一个解决方案CLI在美国每年导致>105例严重截肢。我们的首要目标是开发一个 新的保肢疗法基因治疗是诱导治疗性血管生成的一种有前途的选择， 修复缺血肢体的血流。这种方法为“没有选择”的CLI患者带来了很大的希望。 然而，先前的基因治疗方法使用编码血管生成因子的裸质粒，如 VEGF和FGF未能显示出实质性的临床益处。此外，早期腺病毒介导的基因传递 系统引发了对安全性的担忧。因此，需要更多的努力来确定替代有效的 转基因和安全/有效的递送载体至关重要。CLI基因治疗的成功需要激活 以及内源性愈合机制的参与，包括干细胞/祖细胞的募集/归巢， 缺血组织增强的募集/归巢依赖于“适宜的”组织微环境， 接受内源性再生细胞。我们之前的研究发现，在缺血时， 这些关键的修复细胞依赖于通过一组粘附分子，包括E- 选择素/配体对。缺血肢体组织中血管E-选择素水平下调。我们 假设E-选择素是募集干/祖细胞所必需的关键， 缺血组织中的新血管形成。E-选择素可以作为内源性干细胞/祖细胞的对接位点 细胞进行锚。由此产生的下游细胞-细胞信号级联可以增加血管生成和组织增生。 修复/再生。我们最近已经证明了E-选择素/AAV-1的可行性、安全性和上级功效。 用于改善肢体灌注、增加新血管形成、减少缺血、减少缺血/再灌注损伤、 坏疽，最大限度地减少组织损伤，增强缺血骨骼肌的再生，并延长 跑步能力，在小鼠模型中。在临床环境中，我们认为这种生物效应可以增加 功能性行走能力和防止肢体丧失。因此，我们建议诱导支持组织 微环境的影响，通过在毛细血管中的内皮细胞和其他缺血性肢体组织细胞中使用 粘附分子，E-选择素，使用安全有效的AAV 2载体。我们将使用临床相关的小鼠 CLI模型-足垫坏疽，以测试这种新的基因治疗。目标是验证和优化 未来大型动物临床前研究的转化方案，并进行必要的IND启用 GLP/GMP动物研究，用于后续的初步人体研究。这种新型基因治疗的发展 通过加速治疗性血管生成和组织再生， 再生，并有望彻底改变PAD/CLI患者的治疗。

Project Summary/Abstract: Critical limb ischemia (CLI) is the most advanced stage of peripheral arterial disease (PAD). It manifests with leg pain, decreased walking distance, non-healing wounds and gangrene. About 1/3 of CLI patients don't respond to standard therapy and ultimately undergo major limb amputation. This critical unmet need demands a solution. CLI causes >105 major limb amputations in the USA per year. Our overarching goal is to develop a novel limb-salvage therapy. Gene therapy is a promising option to induce therapeutic angiogenesis, increasing blood flow in ischemic limbs for repair. This approach has raised a great deal of hope for `no-option' CLI patients. However, previous gene therapy approaches using naked plasmids encoding angiogenic factors, such as VEGF and FGF, failed to show substantial clinical benefits. Moreover, early adenovirus-mediated gene delivery systems raised concerns over safety. Hence, more efforts for the identification of alternative effective transgenes and safe/efficient delivery vectors are crucial. Success of gene therapy for CLI requires activation and participation of endogenous healing mechanisms including recruitment/homing of stem/progenitor cells to ischemic tissue. Enhanced recruitment/homing depends upon a `hospitable' tissue microenvironment that is receptive of endogenous regenerative cells. Our previous research discovered that in ischemia, homing for these critical repair cells relies on specific cell-cell interactions via a panel of adhesion molecules, including E- selectin/ligand pairs. The levels of vascular E-selectin in the ischemic limb tissues is down-regulated. We hypothesize that E-selectin is pivotal in the recruitment of stem/progenitor cells necessary for neovascularization in ischemic tissues. E-selectin can serve as a docking site for endogenous stem/progenitor cells to anchor. The resulting downstream cell-cell signaling cascades can increase angiogenesis and tissue repair/regeneration. We have recently demonstrated feasibility, safety and superior efficacy of E-selectin/AAV- based gene therapy for improving limb perfusion, increasing neovascularization, decreasing ischemia, reducing gangrene, minimizing tissue damage, augmenting regeneration of the ischemic skeletal muscle, and extending running capacity, in murine models. In the clinical setting, we believe that such biologic effects can increase functional walking capacity and prevent limb loss. We thus propose to induce a supportive tissue microenvironment by priming endothelial cells in capillaries and other ischemic limb-tissue cells with the adhesion molecule, E-selectin, using a safe and efficient AAV2 vector. We will utilize a clinically relevant mouse model of CLI – the footpad gangrene, to test this novel gene therapy. The objectives are to validate and optimize the translational protocol for a future large-animal pre-clinical study and conduct the requisite IND-enabling GLP/GMP animal research for a subsequent pilot human study. The development of this novel gene therapy may ultimately eliminate the need for most amputations by accelerating therapeutic angiogenesis and tissue regeneration, and promises to revolutionize the treatment for PAD/CLI patients.