Intracellular targeting Hsp70 for pulmonary cytoprotection after toxin inhalation

描述(由申请人提供)：2003年，美国环保局确定了全国123家化工厂，这些工厂的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，细胞死亡的一个主要原因就是氧化应激。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，需要将过多的物质输送到肺部，可能会造成危险的副作用。我们开发了穿透细胞的抗体mAb3E10的Fv片段，作为细胞内转运蛋白，将热休克蛋白70(Fv-Hsp70)运送到细胞内，并在体内外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气雾剂配方的临床有效性，这种气雾剂是便携的，如果怀疑接触有毒吸入剂，可以自行给药。追求这一目标的目的是建立对抗光气暴露的有效性的原则证明，鉴于光气在化学工业中的普遍存在，光气暴露是一种未得到满足的医疗需求。我们的中心假设是，FV-HSP70在预防暴露于光气中造成的肺损伤方面将具有治疗效果。这项拟议研究的基本原理是，穿透细胞的抗体3E10是一种细胞内转运蛋白， 可以将Hsp70直接输送到细胞内，从而将氧化应激引起的蛋白质变性和聚集降至最低。该抗体结合细胞外DNA和核苷，在有受损细胞的地方很容易接触到这些靶标，并通过平衡的核苷挽救途径穿透仍然活着的细胞。3E10的独特之处在于它可以穿透细胞，没有明显的伤害，并且已经在没有毒性证据的情况下用于人体。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，当给中风后的大鼠注射时，将大脑组织中的脑梗塞体积最小化68%。我们申请资金以实现以下特定目标：1)作为基于细胞的效力测试的一部分，评估FV-Hsp70对三光气(一种光气模拟物)暴露的肺细胞在体外存活和屏障完整性的影响。2)建立FV-Hsp70气雾剂的产品稳定性图谱，并测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。单剂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。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、不依赖能量的细胞内蛋白质疗法递送系统。在体内诱导热休克蛋白的产生需要时间，而我们产品的影响是将Hsp70快速输送到受损细胞中，以防止细胞死亡。

DESCRIPTION (provided by applicant): 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 effectivenes 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% of 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.