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

摘要摘要 开发神经退行性疾病的治疗方法面临的主要挑战包括 肉毒杆菌中毒，是现有的靶向治疗方法的低效率和低特异性。 治疗药物选择性地作用于神经细胞，而不涉及次级作用部位。 我们已经演示了，我们可以专门将一个原型货运GFP交付到 体外、体外和体内的神经细胞，我们现在准备将这个原型 货物运输车辆进入医学上相关的禁止性货物运输车辆(S) 肉毒杆菌中毒。我们在此提出了两种对抗麻痹的抗毒素方法。 肉毒杆菌中毒，都是基于我们的BONT/A-HC平台。要展示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针对神经元- 具体交付，我们建议在我们的原型GFP-BONT/A-HC中交换GFP货物 用两种不同类型的货物部分建造，这将构成本项目的两个目标 建议：目标1.基于多肽/蛋白质的抑制性货物运输工具，以及目标2.生物素- 链霉亲和素链霉亲和素链霉亲和抑制货运车辆。建议的研究将在 每个目标的每个建设分三个阶段：第一阶段(R21组成部分，1-2年)--建设， 基于BONT/A-HC的货运车辆的提纯、表征和优化 以及对货运车辆神经元特异性功能的初步测试 培养的神经细胞的细胞摄取。第二阶段(R33部分，3年至4年)--进一步 基于稳定性、表达量和胞内载量的载货载体优化 放手。测试货物-BONT/A-HC递送车辆的功能(货物递送， 抑制ONT/A介导的SNAP25裂解)在培养的神经细胞特异性摄取中的作用 神经细胞；体外腓神经-EDL肌肉制剂；体内注射后 老鼠。第三阶段(R33组件，第4-5年)-测试货物-BONT/A-HC运送车辆 在神经保护活动中的功能性和有效性，如保护或 BONT/A刺激后神经递质释放的恢复，检测到表达增加 由DNA载体编码的毒素中和单链抗体或骆驼抗体或SNAPI蛋白。

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.