Neuronal-specific cargo-delivery platforms as post-exposure botulism therapies

作为暴露后肉毒杆菌疗法的神经元特异性货物递送平台

• 批准号: 8851508
• 负责人: Brenda A. Wilson
• 金额: $ 39.17万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8851508
• 关键词: Affinity; Antibodies; Antidotes; Antitoxins; Binding; Biotin; Bontoxilysin; Botulism; Cell Culture Techniques; Cells; Clostridium; Coupled; Cytosol; DNA; Data; Detection; Development; Effectiveness; Escherichia coli; Green Fluorescent Proteins; In Vitro; Injection of therapeutic agent; Intoxication; Light; Link; Mediating; Membrane; Mus; Muscle; Nerve-Muscle Preparations; Neurodegenerative Disorders; Neuronal Dysfunction; Neurons; Neurotoxins; Paralysed; Peptide antibodies; Peptides; Pharmacotherapy; Phase; Preparation; Prophylactic treatment; Proteins; Recombinants; Recovery; Research Project Grants; Serotyping; Site; Solubility; Specificity; Streptavidin; Testing; Therapeutic; Therapeutic Agents; Toxin; Translating; Viral Vector; abstracting; base; biotin 2; combat; design; gene therapy; in vivo; inhibitor/antagonist; neurotransmission; neurotransmitter release; neutralizing antibody; peroneal nerve; phase 1 study; prototype; receptor binding; targeted delivery; therapy development; tissue culture; uptake; vector

Abstract Summary A major challenge in development of therapies for neurodegenerative diseases, including botulism, is the low efficiency and low specificity of existing treatments for targeting the therapeutic agents selectively to nerve cells without involvement of secondary sites of action. We have already demonstrated that we can specifically deliver a prototype cargo GFP into neuronal cells in vitro, ex vivo and in vivo, and we are now poised to translate this prototype cargo-delivery vehicle into a medically relevant inhibitory cargo-delivery vehicle(s) against botulism. We propose herein two antitoxin approaches toward combating paralysis from botulism, both based on our BoNT/A-HC platform. To demonstrate feasibility and versatility of our BoNT/A-HC-based platform for therapeutic biomolecule cargos targeted for neuronal- specific delivery, we propose to exchange the GFP cargo in our prototype GFP-BoNT/A-HC construct with two different types of cargo moieties, which will comprise the two aims of this proposal: Aim 1. Peptide/protein-based inhibitory cargo-delivery vehicles, and Aim 2. Biotin- streptavidin-linked inhibitory cargos-delivery vehicles. The proposed studies will be conducted in 3 phases for each construct of each aim: Phase I (R21 component, years 1-2) - Construction, purification, characterization and optimization of the BoNT/A-HC-based cargo-delivery vehicles and preliminary testing of the cargo-delivery vehicles for functionality in neuronal-specific cellular uptake in cultured neuronal cells. Phase II (R33 component, years 3-4) - Further optimization of cargo-delivery vehicle for stability, expression yield and intracellular cargo release. Testing the cargo-BoNT/A-HC delivery vehicle for functionality (delivery of cargo, inhibition of BoNT/A-mediated SNAP25 cleavage) in neuronal-specific cellular uptake in cultured neuronal cells; ex vivo in peroneal nerve-EDL muscle preparations; and in vivo after injection in mice. Phase III (R33 component, years 4-5) - Testing the cargo-BoNT/A-HC delivery vehicle for functionality and effectiveness in neuroprotective activity, such as protection from or recovery of neurotransmitter release after BoNT/A challenge, detection of increased expression of toxin-neutralizing scFv or camelid antibodies or SNAPI proteins encoded by DNA vectors.

抽象概括 开发神经退行性疾病治疗方法的主要挑战，包括 肉毒杆菌中毒，是现有治疗靶向的低效率和低特异性， 治疗剂选择性地作用于神经细胞而不涉及次级作用部位。 我们已经证明，我们可以特别地将原型货物GFP递送到 神经元细胞在体外，离体和体内，我们现在准备翻译这个原型， 将药物递送载体转化为医学相关的抑制性药物递送载体， 肉毒杆菌中毒。我们在此提出了两种抗毒素的方法来对抗瘫痪， 肉毒杆菌中毒，两者都基于我们的BoNT/A-HC平台。为了证明的可行性和多功能性， 我们基于BoNT/A-HC的治疗性生物分子货物平台，靶向神经元- 具体的交付，我们建议在我们的原型GFP-BoNT/A-HC中交换GFP货物 构建具有两种不同类型的货物部分，这将包括本发明的两个目的。 建议：目标1。基于肽/蛋白质的抑制性货物递送载体，和Aim 2。生物素- 链霉亲和素连接的抑制性货物-递送载体。拟议的研究将在 每个目标的每个结构有3个阶段：第一阶段（R21组件，1-2年）-建造， 基于BoNT/A-HC的运载工具的纯化、表征和优化 和初步测试的货物输送车辆的功能，在神经元特异性 在培养的神经元细胞中的细胞摄取。第二阶段（R33部分，3-4年）-进一步 针对稳定性、表达产率和细胞内货物优化货物递送载体 release.测试cargo-BoNT/A-HC运载工具的功能性（货物运输， 抑制BoNT/A介导SNAP 25切割）在培养的神经元特异性细胞摄取中的作用 神经元细胞;腓神经-EDL肌肉制备物中的离体;以及 小鼠第三阶段（R33组件，第4-5年）-测试Cargo-BoNT/A-HC运载工具 对于神经保护活性的功能性和有效性，例如保护免受或 BoNT/A攻击后神经递质释放的恢复，检测表达增加 毒素中和scFv或骆驼抗体或由DNA载体编码的SNAPI蛋白。