Intracellular targeting Hsp70 for pulmonary cytoprotection after toxin inhalation

项目摘要 2003年，环保局确定了美国123家化工厂，这些工厂可能发生恐怖袭击或事故。 可能会使100多万人暴露在有毒气体云中。一旦接触到某些化学物质， 细胞死亡的主要原因是氧化应激。目前预防氧化应激的策略包括使用 小分子试剂，但它们缺乏特异性，需要过多的物质输送到肺部 可能会造成危险的副作用。我们开发了一种穿透细胞的抗体mAb的Fv片段 3E10，作为细胞内转运体，将热休克蛋白70(FV-Hsp70)输送到细胞内，我们 在体外和体内证明了对氧化损伤的细胞保护作用。我们的长期目标是确定 FV-HSP70气雾剂的临床疗效 怀疑接触有毒吸入剂。在追求这一目标的过程中，我们的目标是建立证据- 防止光气暴露的有效性原则，这是一种未得到满足的医疗需求，因为 化学工业中的光气。我们的中心假设是FV-HSP70将在治疗上有效 防止暴露在光气中造成的肺损伤。建议的理由是 研究表明，穿透细胞的抗体3E10是一种细胞内转运蛋白，可以传递热休克蛋白70 直接进入细胞，最大限度地减少氧化应激引起的蛋白质变性和聚集。这个 抗体结合细胞外DNA和核苷，在受损的地方很容易接触到这些靶点 它通过平衡的核苷回收途径穿透仍然活着的细胞。3E10是独一无二的 它可以穿透细胞而没有明显的伤害，并且已经在没有证据的情况下用于人体。 毒性。FV-Hsp70已经被创造出来，并在体内被证明是一种有效的细胞保护剂，通过 给中风后的大鼠服用，可使脑组织中的脑梗塞体积增加68%。我们正在申请资金，以 实现以下特定目标：1)评价Fv-Hsp70对体外存活和屏障完整性的影响 暴露于三光气(一种光气模拟物)的肺细胞，作为基于细胞的效力测试的一部分。2)建立 FV-Hsp70气雾剂的产品稳定性概况，并确定大鼠的最大耐受量(MTD)。 3)体内雾化Fv-Hsp70对光气暴露大鼠的治疗作用。一张单人- 剂量的FV-Hsp70气雾剂将在暴露前、暴露后30分钟或暴露后60分钟提供 曝光。这项拟议的研究意义重大，因为它为未得到满足的需求开发了一种关键的治疗方法。 这项拟议的研究具有创新性，因为它利用了一种独特的抗体介导的、不依赖能量的 蛋白质疗法的细胞内递送系统。在体内诱导热休克蛋白的产生可以 时间，而我们的产品的影响是快速输送Hsp70到受损细胞，以防止细胞死亡。

Project Summary In 2003, the EPA identified 123 chemical plants in the nation where a terrorist attack or accident could potentially expose more than 1 million people to a cloud of toxic gas. Upon exposure to certain chemicals, a major cause of cell death is from oxidative stress. Current strategies to prevent oxidative stress include the use of small molecule reagents, but they lack specificity and require excessive material to be delivered to the lungs possibly causing dangerous side effects. We developed the Fv fragment of a cell-penetrating antibody, mAb 3E10, as an intracellular transporter to deliver heat-shock protein 70 (Fv-Hsp70) into cells, and we demonstrated cytoprotection against oxidative damage in vitro and in vivo. Our long-term goal is to determine the clinical effectiveness of an aerosol formulation of Fv-Hsp70 that is portable and can be self-administered if exposure to toxic inhalants is suspected. The objective here in the pursuit of this goal is to establish proof-of- principle for effectiveness against phosgene exposure, an unmet medical need given the prevalence of phosgene in the chemical industry. Our central hypothesis is that Fv-Hsp70 will be therapeutically effective in protecting against lung damage as a result of exposure to phosgene gas. The rationale for the proposed research is that the cell penetrating antibody, 3E10, is an intracellular transporter that can deliver Hsp70 directly into cells where it minimizes protein denaturation and aggregation caused by oxidative stress. The antibody binds extracellular DNA and nucleosides, targets that are quite accessible where there are damaged cells, and it penetrates still viable cells through an equilibrative nucleoside salvage pathway. 3E10 is unique in that it penetrates cells without apparent harm and has been administered to humans without evidence of toxicity. Fv-Hsp70 has already been created and shown to be an effective cytoprotectant in vivo, minimizing by 68% the infarct volume in brain tissue when administered to rats after a stroke. We are requesting funding to achieve the following specific aims: 1) Evaluate the effect of Fv-Hsp70 on survival and barrier integrity in vitro of lung cells exposed to triphosgene (a phosgene simulant) as part of a cell-based potency assay. 2) Establish a product stability profile for the Fv-Hsp70 aerosol and determine the maximum tolerated dose (MTD) in rats. 3) Evaluate the therapeutic efficacy of aerosolized Fv-Hsp70 in vivo with rats exposed to phosgene. A single- dose of Fv-Hsp70 aerosol will be provided either pre-exposure, 30 minutes post-exposure, or 60 minutes post- exposure. The proposed research is significant because it develops a critical therapeutic for an unmet need. The proposed research is innovative because it utilizes a unique antibody-mediated, energy-independent intracellular delivery system for protein therapeutics. Inducing heat shock protein production in vivo can take time, whereas the impact of our product is the rapid delivery of Hsp70 into damaged cells to prevent cell death.