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

描述(申请人提供)：开发包括肉毒杆菌中毒在内的神经退行性疾病的治疗方法的一个主要挑战是现有治疗方法的低效率和低特异性，用于选择性地将治疗剂靶向神经细胞，而不涉及第二作用部位。我们已经证明，我们可以在体外、体外和体内将原型货物GFP特异性地运送到神经细胞中，现在我们准备将这种原型货物运送载体转化为具有医学意义的抗肉毒杆菌中毒抑制性货物运送载体(S)。我们在此提出了两种对抗肉毒杆菌中毒瘫痪的抗毒素方法，两者都基于我们的BONT/A-HC平台。为了证明我们基于BONT/A-HC的平台用于针对神经元特异性递送的治疗性生物分子货物的可行性和通用性，我们建议用两种不同类型的货物部分来交换我们原型GFP-BONT/A-HC结构中的GFP货物，这将包括本提案的两个目标：目的1.基于肽/蛋白质的抑制性货物递送载体，以及目的2.生物素-链霉亲和素连接的抑制性货物递送载体。拟议的研究将针对每个目标的每个构造分三个阶段进行：第一阶段(R21组件，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，并初步测试货物运输工具在培养神经元细胞中神经元特异性细胞摄取的功能。第二阶段(R33组分，3年至4年)-进一步优化货物输送载体，以实现稳定性、表达产量和细胞内货物释放。测试Cargo-BONT/A-HC递送载体在培养神经细胞中神经细胞特异性细胞摄取方面的功能性(递送货物，抑制BONT/A介导的SNAP25切割)；体外测试腓肠神经-EDL肌肉制剂；以及体内注射后的小鼠。第三阶段(R33组件，4年至5年)-测试Cargo-BONT/A-HC递送工具在神经保护活性方面的功能和有效性，例如保护或恢复BONT/A挑战后神经递质的释放，检测由DNA载体编码的毒素中和单链抗体或骆驼抗体或SNAPI蛋白表达增加。 公共卫生相关性：迫切需要解毒剂来逆转肉毒杆菌神经毒素造成的有害瘫痪，特别是在毒素已内化到神经细胞的情况下。将治疗剂定向到正确的细胞不仅是成功治疗的关键，而且治疗不被传递到错误的细胞也是重要的。拟议的神经元特异性递送载体将提供一种神经元特异性递送各种治疗性生物分子货物的手段，包括中和神经毒素麻痹活性的抗体或基于多肽的抑制剂。

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. PUBLIC HEALTH RELEVANCE: Antidotes are urgently needed that can reverse the detrimental paralysis caused by botulinum neurotoxin, particularly once the toxin has been internalized into nerve cells. Not only is targeting the therapeutic agent to the correct cells critical for successful treatment, but it is also important that the therapy is not delivered to th wrong cells. The proposed neuron-specific delivery vehicle will provide a means for neuronal-specific delivery of a variety of therapeutic biomolecule cargos, including antibody or peptide-based inhibitors that neutralize the paralyzing activity of the neurotoxins.