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: 8471649
• 负责人: Brenda A. Wilson
• 金额: $ 19.84万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8471649
• 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; 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

DESCRIPTION (provided by applicant): 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 w 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.

描述（由申请人提供）：开发神经退行性疾病（包括肉毒杆菌中毒）治疗方法的主要挑战是现有治疗方法的低效率和低特异性，这些治疗方法将治疗剂选择性靶向神经细胞，而不涉及次要作用部位。我们已经证明，我们可以在体外、离体和体内特异性地将原型货物GFP递送到神经元细胞中，并且我们现在准备将这种原型货物递送载体转化为针对肉毒杆菌中毒的医学相关的抑制性货物递送载体。我们在此提出了两种对抗肉毒杆菌中毒麻痹的抗毒素方法，两者都基于我们的BoNT/A-HC平台。为了证明我们的基于BoNT/A-HC的平台用于靶向神经元特异性递送的治疗性生物分子货物的可行性和多功能性，我们提出用两种不同类型的货物部分交换我们的原型GFP-BoNT/A-HC构建体中的GFP货物，这将包括该提议的两个目的：目的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-运载工具。针对每个目标的每个构建体，拟议的研究将分3个阶段进行：第一阶段（R21部分，第1-2年）-基于BoNT/A-HC的货物运输工具的构建、纯化、表征和优化，以及货物运输工具的初步测试在培养的神经元细胞中神经元特异性细胞摄取的功能。阶段II（R33组分，3-4年）-进一步优化货物递送载体的稳定性、表达产率和细胞内货物释放。测试货物-BoNT/A-HC递送媒介物在培养的神经元细胞中的神经元特异性细胞摄取中的功能性（货物的递送，BoNT/A介导的SNAP 25切割的抑制）;在腓神经-EDL肌肉制备物中的离体;以及在小鼠中注射后的体内。III期（R33部分，第4-5年）-测试货物-BoNT/A-HC递送载体在神经保护活性方面的功能性和有效性，例如BoNT/A激发后神经递质释放的保护或恢复，检测毒素中和scFv或骆驼抗体或DNA载体编码的SNAPI蛋白的表达增加。

项目成果

Global biosecurity in a complex, dynamic world.

复杂、动态的世界中的全球生物安全。

• DOI: 10.1002/cplx.20246
• 发表时间: 2008
• 期刊: Complexity
• 影响因子: 2.3
• 作者: Wilson,BrendaA