Characterization of Botulinum Neurotoxin A Subtypes

说明(申请人提供)：肉毒杆菌神经毒素(BoNTs)是已知的最有毒的毒素，被归类为A类精选药物。BoNTs是人类肉毒杆菌中毒的病原体，是治疗多种神经性疾病的有效药物。传统上将BoNTs细分为7个血清型(A-G)。近年来，基于核苷酸序列的差异，大多数血清型的BoNTs已经被鉴定出几个亚型。识别出BoNT/A(A1-A5)的五种亚型。除了序列研究和初步数据显示出显著的功能差异外，对BoNTs亚型的分子和细胞生物学研究还很少。本研究的目的是鉴定和鉴定BoNT/A1-A5的体内外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联系起来。拟议的研究代表了三个实验室之间长期和富有成效的合作关系，这一应用的动力是基于这些实验室在BoNTs亚型方面的最新进展，包括亚型在分子特征、中毒动力学和中毒机制以及结构特征方面的不同。这项提议将检验五种BoNT/A亚型具有独特的体内毒性特征的总体假设。目的1将验证如下假设，即每个BONT/A亚型都具有不同的体外毒性特征，并且重组的BONT/A亚型可以作为基因修饰的来源材料来确定功能特性。目的2验证BoNTs/A1-A5具有不同的细胞进入动力学和体内毒性的假设。目的3验证以下假设：在神经细胞模型中，BONT/A亚型具有独特的细胞转运机制，并且BONT/A亚型之间的细胞转运和生物学特性的差异是由于毒素的特定结构差异所致。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副作用较少的新的和经修改的药物将成为必要的。此外，毒素特性的表征将促进未来靶向抗BONT疗法的发展，该疗法将对所有BONT/A亚型有效。

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.