Botulinum Neurotoxin Derivatives for Targeted Neuronal Delivery

用于靶向神经元递送的肉毒杆菌神经毒素衍生物

• 批准号: 7446717
• 负责人: Konstantin Ichtchenko
• 金额: $ 24.1万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7446717
• 关键词: Amino Acid Sequence; Antidotes; Biological Testing; Bontoxilysin; Catalytic Domain; Chemicals; Class; Coupled; Cytosol; Data; Disulfides; Doctor of Philosophy; Dose; Ensure; Fluorescence Microscopy; Genes; Genetic; Genetic Engineering; Immunofluorescence Immunologic; Length; Light; Methods; Microscopy; Modification; Molecular; Motor Neurons; Mucous Membrane; Neurons; Object Attachment; Oral; Organism; Pathway interactions; Principal Investigator; Property; Protocols documentation; Recombinants; Residual state; Role; Series; Structure; Techniques; Testing; Therapeutic Agents; Toxic effect; Toxin; Work; base; biodefense; design; disulfide bond; interest; neurotoxicity; neurotropic; polypeptide; programs; prototype; research study; size; targeted delivery; tool; trafficking

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNT) are a high priority of the national biodefense initiative because of their ability to target the cytosol of motor neurons at extremely low doses, even when delivered via mucosa. Extensive work characterizing the molecular mechanism of BoNT action has created the potential to use atoxic derivatives of BoNT as neuron-targeting delivery vehicles for diverse therapeutic agents, including agents directed against neurotropic toxins and organisms, and antidotes to BoNT itself. We are therefore designing and testing a genetic engineering platform for producing atoxic BoNT derivatives that target the neuronal cytosol after oral or inhalational administration. Our genetic constructs facilitate manipulation and modification of BoNT gene segments, and enable expression of recombinant, full-length, atoxic BoNT derivatives that retain the native toxin's key structural features, disulfide bonding and targeting properties. We here propose expressing and purifying a prototype atoxic BoNT derivative in quantities, sufficient for biological testing, to ensure that any residual toxicity has been eliminated, and that the capacity to deliver atoxic light chains to the neuronal cytosol in reasonable quantities is retained. Subsequently, we will use this genetic engineering platform to produce a series of BoNT derivatives in which a fluorescent marker has been built directly into the derivative's amino acid sequence. These enable BoNT trafficking pathways to be evaluated using direct fluorescence microscopy, and will compliment data from indirect immunodetection techniques. Four fluorescent BoNT derivatives are proposed, differing in the size and structure of the flurophor, and the size of the LC polypeptide segment retained. Experiments comparing the intra-cellular trafficking of the fluorescent BoNT derivatives to each other, and to the prototype non-fluorescent BoNT derivative, will provide important information about the role of the LC catalytic domain in BoNT targeting, and establish limits to the size and structure of cargo molecules that can transported to the neuronal cytosol using a BoNT-based mechanism. One of the fluorescent BoNT derivatives is of particular interest because the fluorophor is enzymatically coupled to a short amino acid sequence inserted at the N-terminus of the LC construct, but which should be otherwise identical to native BoNT ad. It therefore provides a new tool to study the molecular mechanism of BoNT trafficking that resolves ongoing problems associated with the non- selective attachment of flurophors to BoNT using chemical methods. Project Narrative Botulinum neurotoxins (BoNT) are a high biodefense priority because they target motor neurons at extremely low doses, even when delivered via mucosa. We are therefore developing a genetic engineering platform that can take advantage of BoNT's neuron-targeting properties to deliver diverse therapeutic agents to the neuronal cytosol, including antidotes to BoNT itself. This proposal will evaluate the molecular features required for neuron targeting, and produce a new class of fluorescent BoNT derivatives that will be broadly useful to study the molecular mechanisms of BoNT trafficking.

描述（由申请人提供）：肉毒杆菌神经毒素（BONT）是国家生物诱变倡议的高优先级，因为即使在通过粘膜交付时，它们也能够以极低的剂量靶向运动神经元的细胞质。表征BONT作用的分子机制的广泛工作已经创造了将BONT的氧化衍生物用作神经元靶向的递送车的潜力，包括针对神经旋转的毒素和生物体和生物体的药物，以及解毒剂本身。因此，我们正在设计和测试一个基因工程平台，用于生产靶向口服或吸入施用后靶向神经元胞质的毒性骨衍生物。 我们的遗传构建体有助于操纵和修改BONT基因段，并能够表达重组，全长，毒性BONT衍生物，这些衍生物保留了天然毒素的关键结构特征，二硫键键合和靶向性质。我们在这里提出表达和净化原型的毒性BONT衍生物的数量，足以确保消除任何残留的毒性，并保留将毒性光链传递到合理量的神经元细胞质的能力。随后，我们将使用这个基因工程平台生成一系列的bont衍生物，其中将荧光标记物直接构建到衍生物的氨基酸序列中。 这些使得可以使用直接荧光显微镜评估BONT运输途径，并将补充来自间接免疫探测技术的数据。 提出了四个荧光骨衍生物，在氟载体的大小和结构上有所不同，并且保留了LC多肽段的大小。 比较荧光骨衍生物的细胞内运输以及与原型非荧光散发衍生物的实验，将提供有关LC催化域在BONT靶向中的作用的重要信息，并建立对使用基于神经元的机制的货物大小和结构的限制。荧光散发衍生物之一特别令人感兴趣，因为荧光摄影剂与插入LC构建体N末端的短氨基酸序列偶联，但否则应与本机Bont AD相同。因此，它提供了一种新的工具来研究BONT运输的分子机制，该工具可以解决与使用化学方法相关的flurophors非选择性附着与BONT相关的持续问题。 项目叙事肉毒杆菌神经毒素（BONT）是一个高生物反应的优先级，因为它们以极低剂量的运动神经元的靶向，即使通过粘膜递送也是如此。因此，我们正在开发一个基因工程平台，该平台可以利用BONT的神经元靶向特性，以将各种治疗剂传递到神经元的胞质醇，包括抗毒素以使其本身。 该提案将评估神经元靶向的分子特征，并产生一类新的荧光散发衍生物，这些荧光衍生物将对研究BONT运输的分子机制至关重要。