Novel therapeutic approaches to treatment of botulinum neurotoxin poisoning.

治疗肉毒杆菌神经毒素中毒的新治疗方法。

• 批准号: 9976431
• 负责人: Konstantin Ichtchenko
• 金额: $ 112.74万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9976431
• 关键词: Affect; Affinity; Animals; Antibodies; Antidotes; Benefits and Risks; Binding; Bioterrorism; Bontoxilysin; Botulinum Antitoxin; Botulinum Toxin Type A; Botulism; Budgets; Bypass; Caring; Cavia; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chimeric Proteins; Clinical; Collaborations; Communicable Diseases; Complex; Cytoplasm; Cytosol; Data; Development; Disease; Dose; Effectiveness; Engineering; Epithelial; Epithelium; Epitopes; Equipment; Event; Failure; Food Contamination; Formulation; Funding; Generations; Goals; Hospitals; Hour; Human; Human Resources; IgE; Immune; Immunization; Immunoglobulin Therapy; In Vitro; Ingestion; Inhalation; Institution; Intestines; Intoxication; Laboratories; Lead; Libraries; Light; Mass Immunization; Mechanical ventilation; Metalloproteases; Modality; Molecular; Motor Neurons; Mus; National Security; Neurodegenerative Disorders; Neurons; Pathogenicity; Patients; Pattern; Peptide Hydrolases; Physiological; Poisoning; Process; Production; Recombinants; Recording of previous events; Recovery; Research; Research Personnel; Respiratory Muscles; Respiratory Signs and Symptoms; Safety; Scientist; Septic Toxemia; Serotyping; Specificity; Structure; Suggestion; Symptoms; Synapses; System; Systems Development; Targeted Toxins; Testing; Therapeutic; Therapeutic Uses; Time; Toxic effect; Toxin; Translating; Treatment Efficacy; Viral; Viral Vector; Wound Infection; acute symptom; animal rule; antitoxin; base; biodefense; botulinum; botulinum toxin type B; botulinum toxin type E; cytotoxic; cytotoxicity; experience; high risk; improved; in vitro Model; in vivo Model; in vivo evaluation; innovation; lead candidate; molecular trojan horse; mouse model; nanobodies; neurotropic; novel; novel therapeutic intervention; passive antibodies; pre-clinical; presynaptic; receptor; response; safety testing; side effect; small molecule inhibitor; small molecule therapeutics; success; therapeutic target; trafficking; vector; weapons

PROJECT SUMMARY/ABSTRACT: Human botulism can result from the ingestion of food contaminated with botulinum neurotoxin (BoNT), from the infection of wounds or intestine, or from direct inhalation of the toxin, which can be transcytosed across epithelial barriers as a physiologically active molecule. Intoxication, if untreated, may lead to death due to respiratory muscle failure, while recovery from botulism is a prolonged process (up to several months) and BoNTs are classified as one of the six highest‐risk threat agents for bioterrorism by the CDC. Developing may require intensive and expensive hospital care. Due to their extreme toxicity and ease of production, antidotes to botulinum neurotoxins is a priority goal of the Biodefense Research Agenda. During the previous funding period, we developed a molecular engineering platform that takes advantage of structural features and trafficking of BoNTs to enable the delivery within the presynaptic compartment of intoxicated neurons of single domain camelid antibodies (sdAbs) that bind and inactivate the wt light chain (LC) of BoNT metalloproteases. We developed a post-exposure anti-botulinum therapeutic (PEABT) targeting wt BoNT/A1, which is the first compound ever to show reversal of respiratory symptoms in mice at times post-intoxication when standard antibodies are ineffective. Preliminary data also demonstrated the effectiveness of a second lead candidate against wt BoNT/B, and attest to the generalizability of the platform. We succeeded in developing a novel principle for delivering single domain antibodies to the presynaptic compartment of neurons without a viral vector. In addition, we developed a murine model of botulism that is specifically appropriate to test botulism antidotes with an intra-neuronal mode of action. The murine model is currently being translated to a guinea pig, in response to an FDA suggestion that the guinea pig was the species to use for an Animal Rule pivotal study. Our objective is to expand the therapeutic range of this platform to two other known human pathogenic serotypes – wt BoNT/B and wt BoNT/E, and to further increase the potency and therapeutic window of the wt BoNT/A1 countermeasure. We will identify sdAbs that bind and broadly neutralize BoNT/A, /B, and /E LC subtypes, and we will pursue strategies to increase the potency of PEABTs by increasing the affinity of sdAbs for the wt LC target and by improving therapeutic efficacy. The team of scientists assembled for this project includes researchers with unique expertise, who are well-qualified for the task at hand, including a long and successful history in generating BoNT-neutralizing sdAbs, extensive expertise in expressing neuron-targeted physiologically active fusion proteins that use metalloprotease-inactivated BoNT/C1 as a molecular vehicle for delivery of sdAbs, expertise in test systems for development of compounds promoting accelerated degradation of intracellular targets through the endogenous proteasomal system, and extensive experience in developing in vitro and in vivo models that can be used to test novel botulism antidotes.

项目摘要/摘要： 人类肉毒杆菌可能是由于摄入被肉毒神经毒素（BONT）污染的食物而引起的， 从奖金或肠道感染，或直接吸入毒素，可以转换 跨上皮屏障作为身体活跃的分子。中毒，如果未经治疗，可能会导致死亡 呼吸道肌肉衰竭，而从肉毒杆菌中恢复是一个延长的过程（长达几个月），而 BONT被归类为CDC六种生物恐怖主义的最高风险威胁代理之一。发展 可能需要密集且昂贵的医院护理。由于它们的极端毒性和易于生产， 肉毒神经毒素对肉毒杆菌毒素的解毒剂是生物反应研究议程的优先目标。在上一个 资助期，我们开发了一个分子工程平台，该平台利用结构特征和 贩运BONT，以使单个单个神经元的突触前室内递送 结合并使BONT金属蛋白酶的WT轻型链（LC）结合并失活的结构域骆驼抗体（SDABS）。 我们开发了一种靶向WT BONT/A1的暴露后抗骨治疗（PEABT），这是第一个 当标准化时，有时会在小鼠中表现出小鼠呼吸道症状的逆转 抗体无效。初步数据还证明了第二个铅候选人的有效性 反对WT BONT/B，并证明平台的普遍性。我们成功地发展了一本小说 用于在没有病毒的神经元的突触前室中输送单个域抗体的原理 向量。此外，我们开发了一种鼠类肉毒杆菌的模型，该模型特别适合于测试肉毒杆菌 具有神经内神经元模式的解毒剂。鼠模型目前正在转化为豚鼠， 为了回应FDA的建议，即豚鼠是用于动物规则关键研究的物种。 我们的目标是将该平台的治疗范围扩展到另外两个已知的人类病原体 血清型 - WT BONT/B和WT BONT/E，并进一步增加wt的效力和治疗窗口 BONT/A1对策。我们将确定粘合并广泛中和BONT /A， /B和 /E LC的SDABS 亚型，我们将通过增加SDAB的亲和力来采取策略来提高PEABT的效力 用于WT LC目标并提高治疗效率。科学家团队为该项目组成了 包括具有独特专业知识的研究人员，他们在手头的任务中符合资格，包括长期和 成功产生散发性SDAB的成功历史，表达神经元靶向的广泛专业知识 使用金属蛋白酶灭活的BONT/C1作为分子媒介物的物理活性融合蛋白 交付SDAB，用于开发化合物的测试系统专业知识，促进加速降解 通过内源性蛋白酶体系统的细胞内靶标的以及在发展中的丰富经验 体外和体内模型，可用于测试新型肉毒杆菌抗原。