Aptamer therapeutic for safe and low-cost treatment of snake bite envenomation

蛇咬伤中毒在美国和世界范围内是一个严重的问题，可能会导致大量的发病率，甚至 死亡。蛇毒含有一种复杂的毒素混合物，包括神经毒素和肌肉毒素，以及 会扰乱正常止血，并可能导致致命出血。蛇毒还包括几类消化性和 一种蛋白水解酶，负责蛇咬造成的广泛的局部组织破坏，可能导致 永久性残疾或需要截肢。目前的抗蛇毒血清产品是由马饲养的抗体片段组成的 或绵羊，通常在稳定毒液患者方面是有效的，但它们有几个局限性 在减轻组织损伤方面，它们并不是对所有种类的北美毒蛇都有效，而且有不利的 如血清病、过敏和复发性凝血障碍等影响。此外，以抗体为基础的配方有 费用非常高，每名患者的治疗费用从2万美元到4万美元不等。 我们建议开发一种基于高亲和力适配子的新型抗蛇毒药物，该适配子由单链组成 形成茎环结构与其靶标结合的寡聚脱氧核苷酸。我们专有的X适配子选择 该技术利用基于珠基寡核苷酸组合文库，其中某些碱基包含蛋白质样侧 修改以增强结合亲和力。在这个项目中，我们将生成针对一组主要毒素类的X-适配子 在北美毒蛇中发现，以开发比目前更安全的新的抗蛇毒制剂 基于抗体的抗蛇毒血清。我们的初步研究表明，我们选择了能够中和的适体 南太平洋响尾蛇(C.O.helleri)在小鼠后肢瘫痪模型中的肌毒素。 该项目的目标是：1)产生针对北美五大类病毒的高亲和力寡核苷酸X-适配子 蛇毒毒素，并测定每个X-适配子及其靶毒素的解离常数；2)体内演示 X-适配子对抗蛇毒和粗蛇毒的有效性，采用小鼠出血性活动肌酸模型 激酶活性、后肢瘫痪和致死率，以确定每个适配子的有效剂量；3)进行剂量范围 以及使用混合适体抗蛇毒血清制剂对大鼠的毒理学研究。这些研究完成后， 展示一种基于适体的新配方的安全性和有效性，这将具有强大的影响力和市场潜力 在紧急救治毒蛇咬伤中毒中。

Snake bite envenomation is a significant problem in the US and worldwide that can result in substantial morbidity and even death. Snake venom contains a complex mixture of toxins including neurotoxins and myotoxins, as well as toxins that disrupt normal hemostasis and can produce fatal hemorrhage. Snake venoms also include several classes of digestive and proteolytic enzymes that are responsible for the extensive local tissue destruction from snake bites that can result in permanent disability or require limb amputation. Current antivenom products consist of antibody fragments raised in horses or sheep and are generally effective at stabilizing envenomation patients, however they come with several limitations in that they are not effective against all species of N. American venomous snakes in mitigating tissue damage, and there are adverse effects such as serum sickness, hypersensitivity, and recurrent coagulopathy. In addition, antibody-based formulations have an extremely high cost, with the cost of the drug ranging from $20,000 to $40,000 per patient treatment. We propose to develop a novel antivenom therapeutic based on high-affinity aptamers, which consist of single-stranded oligodeoxynucleotides that form stem-loop structures to bind to their targets. Our proprietary X-Aptamer selection technology utilizes bead-based oligo DNA combinatorial libraries in which certain bases contain protein-like side modifications to enhance binding affinity. In this project, we will generate X-Aptamers against a set of major toxin classes found in N. American venomous snakes, to develop novel antivenom formulation with a better safety profile than current antibody-based antivenoms. Our preliminary studies have demonstrated selection of aptamers capable of neutralizing myotoxin from Southern Pacific rattlesnakes (C. o. helleri) in a hind limb paralysis model in mice. The project aims are: 1) Generate high-affinity oligonucleotide X-Aptamers against the five major classes of N. American snake venom toxins and measure the dissociation constant for each X-Aptamer and its target toxin; 2) Demonstrate in vivo efficacy of X-Aptamers against snake venom toxins and crude venom, using mouse models of hemorrhagic activity, creatine kinase activity, hind-limb paralysis, and lethality, to determine the effective dose for each aptamer; 3) Perform dose-ranging and toxicology studies in rats with the blended aptamer antivenom formulation. The completion of these studies will demonstrate safety and efficacy of a novel aptamer-based formulation which will have a strong impact and market potential in the emergency treatment of snake bite envenomation.