A Bioengineering Approach to Gene Therapy for Peripheral Arterial Disease

描述(申请人提供)：外周动脉疾病(PAD)是由动脉粥样硬化引起的，导致通往下肢的动脉阻塞。PAD的患病率几乎与缺血性心脏病相当。在相当大一部分严重形式的PAD患者中，一条或多条主要动脉完全闭塞，因此流向下肢远端的血流取决于侧支血管的范围、数量和功能。对于患有PAD的患者，尚无药物治疗可以有利地调节侧支循环的数量或功能以及对下肢的血流灌注。刺激新血管生长(“治疗性血管生成”)以改善PAD患者肢体血流的概念现在是一个有20年历史的研究策略。绝大多数人体试验使用的是通过肌肉内(IM)载体或腺病毒载体进行的基因转移。在经历了数十次试验和数千个课题后，这一领域的成功微乎其微。人类研究未能令人信服地显示IM注射后的治疗性基因表达，这可能是治疗性血管生成临床试验失败的原因。 到目前为止进行的。腺相关病毒在为PAD的治疗性血管生成提供从小鼠到人的联系方面具有变革性。在全身(静脉)给药后，几种AAV血清型可以有效地转导骨骼肌。虽然PAD可能会限制AAV9进入缺血肌的途径，但我们发现全身给药后缺血肌与非缺血肌相比基因表达增强，我们研究了可能的机制。在此基础上，这项修订建议的中心假设是，生物工程AAV9载体是唯一适合通过使用隔离肢体灌流的局部递送方式在PAD中实现治疗性血管生成的载体。这三个互补但独立的目标是：特定目标1：作为临床应用的一个环节，在PAD的小动物和大动物模型中，使用全身和单独的肢体灌流，优化AAV9介导的缺血肌基因传递。我们将使用报告基因和基因表达的定量生物分布研究来比较小鼠和猪缺血肌肉相对于非靶标组织的全身和局部给药。此外，我们将纳入低氧反应元件，并在PAD的小鼠和猪后肢缺血模型中检测免疫反应。具体目标2：建立缺血诱导的终止，导致唾液酸化糖蛋白和终止糖蛋白比率的差异，是临床前和临床PAD受试者与非PAD对照组相比，缺血肌基因表达增强的机制。具体目的3：确定经隔离肢体灌流的AAV介导的基因治疗在小鼠和猪后肢缺血模型中的疗效。为此，我们将比较ADAM12和EcSOD基因治疗在小鼠后肢PAD模型中的作用，然后与耶鲁大学的Albert Sinusas博士合作，确认在猪PAD后肢模型中最有效的基因治疗的有效性。

DESCRIPTION (provided by applicant): Peripheral arterial disease (PAD) is caused by atherosclerosis that results in obstructions in the arteries to the lower extremities. The prevalence of PAD is nearly equal to that of ischemic heart disease. In a sizeable fraction of patients with severe forms of PAD, there is a complete occlusion in one or more major arteries, and thus blood flow to the distal lower limb becomes dependent on the extent, number and function of collateral blood vessels. There are no medical therapies available for patients with PAD that favorably modulate collateral number or function and perfusion to the lower extremity. The concept of stimulating new blood vessel growth ('therapeutic angiogenesis') to improve limb perfusion in patients with PAD is now a 20 year old investigational strategy. The vast majority of human trials use gene transfer by intra-muscular (IM) delivery with plasmid or adenoviral vectors. After dozens of trials and thousands of subjects, success in this field has been meager. Human studies have failed to convincingly show therapeutic gene expression following IM injection, and this may well account for the failure of clinical trials in therapeutic angiogenesis conducted to date. Adeno-associated virus can be transformational in providing the link from mouse to man for therapeutic angiogenesis in PAD. Following systemic (intravenous) delivery, several AAV serotypes efficiently transduce skeletal muscle. While PAD could potentially limit the access of AAV9 to ischemic muscle, we found enhanced gene expression in ischemic compared to non-ischemic muscle following systemic delivery and we have examined potential mechanisms. Building on this foundation, the central hypothesis of this revised proposal is that bioengineered AAV9 vectors are uniquely suited to accomplish therapeutic angiogenesis in PAD via local delivery using isolated limb perfusion. The three complementary but independent Aims are to: Specific Aim 1: Optimize AAV9-mediated gene delivery to ischemic muscle using systemic vs. isolated limb perfusion in small and large animal models of PAD as a link to clinical application. We will use reporter genes and quantitative biodistribution studies of gene expression to compare systemic versus local delivery in ischemic muscle relative to off-target tissues in mice and pigs. Further, we will incorporate hypoxia-response elements and examine immune responses in both murine and porcine hind-limb ischemia models of PAD. Specific Aim 2: Establish that ischemia-induced desialylation, which results in a difference in the ratio of sialylated to desialylated glycoproteins, is a mechanism for enhanced gene expression in ischemic muscle in pre-clinical and clinical PAD subjects vs. non-PAD controls. Specific Aim 3: Determine the efficacy of AAV-mediated gene therapy delivered via isolated limb perfusion in both mouse and porcine hind-limb ischemia models of PAD. In this aim, we will compare ADAM12 vs. EcSOD gene therapy in the mouse hind-limb ischemia model of PAD, then confirm the efficacy of the most effective gene therapy in the porcine hind-limb model of PAD in collaboration with Dr. Albert Sinusas (Yale).