New Therapeutic Approach for Ricin-Induced Lung Injury

描述(申请人提供)：世界范围内日益严重的生物恐怖主义威胁加剧了对可用作生物武器的毒素疗法的开发需求。蓖麻毒素是蓖麻籽和一种B类毒素的天然产物。它是一种核糖体失活蛋白，有可能被用作生物武器，因为它具有热稳定性，可在全球范围内获得，易于生产，并可作为气雾剂传播，这可能是恐怖分子可能使用的路线。当作为可呼吸的气雾剂吸入时，它会在呼吸道引起不可逆转的病理变化，导致上皮坏死、肺水肿和肺泡积水，最终导致死亡。蓖麻毒素(RT)是一种由二硫键连接的两条多肽链组成的凝集素，需要进入细胞才能产生毒性。B链(RTB)促进毒素进入细胞，而A链(RTA)具有RNA N-糖苷酶活性，通过提纯28S真核核糖体RNA(RRNA)中的单个腺嘌呤来阻止翻译，从而诱导核毒性。在体外选择已经被用来产生催化RTA的特异性RNA配体(适配子)。这些RNA适配子可抑制RTA的脱嘌呤，并保护其免受蓖麻毒素的核毒作用。我们假设，暴露后的治疗可以通过在细胞内传递这些新型的抗RTA适配子来抑制RTA的活性和毒性来对抗蓖麻毒素的核毒性。为了验证我们的假设，我们提出了以下目标：(1)在细胞培养模型中测试聚阳离子聚乙烯亚胺(PEI)在促进细胞摄取适配子方面的有效性以及对抗蓖麻毒素细胞毒性的保护作用；(2)确定PEI-适配子在防止蓖麻毒素诱导的肺损伤和致命性方面的效力，并表征PEI-适配子在肺内的摄取情况；(3)在雾化蓖麻毒素小鼠模型中验证PEI-适配子的有效性。与公共卫生的相关性：由于目前还没有治疗蓖麻毒素毒性的方法，因此迫切需要开发有效的蓖麻毒素解毒剂。因为蓖麻毒素是一种重要的生物恐怖威胁，我们的长期目标是产生人类使用的抗毒素方案，以保护平民和军事人口免受在恐怖主义行为或战争中使用蓖麻毒素的影响。

DESCRIPTION (provided by applicant): The increasing threat of bioterrorism worldwide has escalated the demand for the development of therapeutics for toxins that can be used as biological weapons. Ricin is a natural product of the castor bean (Ricinus communis) and a Category B toxin. It is a ribosome-inactivating protein that has the potential of being used as a biological weapon because of its heat stability, worldwide availability, ease in production and can be disseminated as an aerosol, a likely route that terrorists may use. When inhaled as a respirable aerosol, it causes irreversible pathological changes in the respiratory tract resulting in epithelial necrosis, pulmonary edema and alveolar flooding, and eventual death. The ricin toxin (RT) is a lectin consisting of two polypeptide chains linked by a disulfide bridge and cellular entry is required for toxicity. The B-chain (RTB) facilitates entry of the toxin into the cell and the A-chain (RTA) possesses RNA N-glycosidase activity that disables translation by depurinating a single adenine in the 28S eukaryotic ribosomal RNA (rRNA), thereby inducing ribotoxicity. In vitro selection has been used to generate RNA ligands (aptamers) specific for the catalytic RTA. These RNA aptamers inhibited RTA depurination and protects against ricin ribotoxicity. We hypothesize that post-exposure treatment can be developed against ricin ribotoxicity by delivering these novel anti-RTA aptamers intracellularly to inhibit RTA activity and toxicity. To test our hypothesis, the following aims are proposed: (1) To test the efficacy of the polycation polyethylenimine (PEI) in facilitating cellular uptake of aptamers and protective effects against ricin cytotoxicity in cell culture models; (2) To ascertain the efficacy of PEI-aptamers in the protection against ricin-induced lung injury and lethality and characterize the uptake of PEI-aptamers in the lungs; and (3) To validate the efficacy of PEI-aptamers in an aerosolized ricin mouse model. Relevance to Public Health: Since there is currently no treatment for ricin toxicity, there is an urgent need to develop effective antidotes for ricin. Because ricin is an important bioterrorist threat, our long-term goal is generate anti-toxin regimen for human use to protect the civilian and military populations against the use of ricin in acts of terrorism or war.