Characterization of Botulinum Neurotoxin A Subtypes

描述（由申请人提供）：肉毒神经毒素（BoNT）是已知毒性最强的毒素，被归类为A类特选制剂。BoNT是人类肉毒杆菌中毒的病原体，并且用作治疗无数神经元疾病的有效药物。BoNT通常细分为7种血清型（A-G）。近年来，基于核苷酸序列的差异，已经为大多数血清型鉴定了几种亚型BoNT。BoNT/A有五种亚型（A1 - A5）。除了序列研究和初步数据表明显着的功能差异，很少有研究已经进行了亚型BoNT的分子和细胞生物学。本研究项目的目标是在体外和体内表征和辨别BoNT/A1-A5的分子和细胞生物学特性，并将亚型特异性生物学特性与结构特性相关联。拟议的研究代表了三个实验室之间的长期和富有成效的合作伙伴关系，该应用的动力是基于这些实验室在亚型BoNT上的最新进展，包括亚型在分子特征，动力学和中毒机制以及结构特征方面的差异。该提案将测试五种BoNT/A亚型具有独特的体内毒性特征的总体假设。目的1将检查的假设，每个BoNT/A亚型具有不同的体外毒性特征，重组BoNT/A亚型可以产生作为源材料的遗传修饰，以定义功能特性。目的2将检验BoNT/A1-A5具有不同的细胞进入动力学和体内毒性特征的假设。目的3将检验以下假设：BoNT/A亚型在神经元细胞模型中具有独特的细胞运输机制，并且BoNT/A亚型之间的细胞运输和生物学特性的差异是由于毒素的特定结构差异。该项目产生的数据有可能改善基于BoNT的药物的临床治疗，因为具有靶向特征和较少副作用的新药物和改良药物将成为必要。此外，毒素特性的表征将促进靶向抗BoNT治疗剂的未来开发，其B对所有BoNT/A亚型有效。 公共卫生相关性：拟议的工作将检查和比较五种肉毒杆菌神经毒素（BoNT）A亚型的生物学特性，并确定哪些结构特征负责毒素的特定功能。从这项研究中获得的知识和为表达和操纵重组BoNT开发的技术系统将通过为潜在的改进和替代的基于BoNT的治疗提供基础，积极和显著地影响肉毒杆菌毒素研究领域以及医学领域，这可以显著增加BoNT治疗选择和适应症。

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNTs) are the most poisonous toxins known and are classified as Category A select agents. BoNTs are the causative agent of human botulism, and are used as efficacious pharmaceuticals for treatment of a myriad of neuronal diseases. BoNTs are conventionally subdivided into 7 serotypes (A - G). In recent years, several subtype BoNTs have been identified for most serotypes based on differences in nucleotide sequences. Five subtypes of BoNT/A (A1 - A5) are recognized. Apart from sequence studies and initial data indicating significant functional differences, little researh has been conducted on the molecular and cell biology of the subtype BoNTs. The goal of this research project is to characterize and discern the molecular and cell biology properties of the BoNT/A1-A5 in vitro and in vivo, and relate subtype specific biological characteristics to structural properties. The proposed studies represent a strong partnership among three laboratories with long term and productive collaborations, and the impetus for this application is based upon recent progress in these laboratories on the subtype BoNTs, including that the subtypes differ in molecular characteristics, kinetics and mechanisms of intoxication, and structural features. This proposal will test the overall hypothesis that the five BoNT/A subtypes possess unique in vivo toxicity profiles. Aim 1 will examine the hypothesis that each BoNT/A subtype possesses distinct in vitro toxicity characteristics, and that recombinant BoNT/A subtypes can be produced as source materials for genetic modifications to define functional properties. Aim 2 will test the hypothesis that BoNTs/A1-A5 have distinct cell entry kinetics and in vivo toxicity profiles. Aim 3 will examine the hypothesis that BoNT/A subtypes have unique cell trafficking mechanisms in neuronal cell models, and that the differences in cellular traffickig and biological properties between BoNT/A subtypes are due to specific structural differences of the toxins. The data resulting from this project have the potential to improve clinical treatments with BoNT based pharmaceuticals, as new and modified pharmaceuticals with targeted characteristics and fewer side effects will become necessary. In addition, the characterization of toxin properties will facilitate future developments of targeted anti-BoNT therapeutics that will b effective against all BoNT/A subtypes. PUBLIC HEALTH RELEVANCE: The proposed work will examine and compare the biological properties of the five botulinum neurotoxin (BoNT) A subtypes and determine which structural features are responsible for specific functions of the toxin. The knowledge gained from this study and the technical systems developed for expression and manipulation of recombinant BoNTs will positively and significantly impact the botulinum toxin research field as well as the medical field by providing the basis for potentially improved and alternative BoNT based therapeutics, which could significantly increase BoNT treatment options and indications.