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

描述（由申请人提供）：包括肉毒杆菌中毒在内的神经退行性疾病的治疗发展面临的一个主要挑战是现有治疗方法的低效率和低特异性，这些治疗方法选择性地靶向治疗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输送到神经元细胞中，现在我们准备将这种原型货物输送载体转化为与肉毒杆菌中毒有关的医学上的抑制货物输送载体。我们在此提出了两种抗毒素方法来对抗肉毒中毒引起的瘫痪，这两种方法都基于我们的BoNT/A-HC平台。为了证明我们基于BoNT/A-HC的治疗性生物分子载体平台的可行性和多功能性，我们建议将我们的原型GFP-BoNT/A-HC构建中的GFP载体与两种不同类型的载体部分交换，这将包括本提案的两个目标：目标1。基于肽/蛋白的抑制性货物递送载体，以及Aim 2。生物素链亲和素连接的抑制性货物运载工具。拟议的研究将分三个阶段进行：第一阶段（R21组件，1-2年）-基于BoNT/ a - hc的货物运输车辆的构建，纯化，表征和优化，以及货物运输车辆在培养的神经元细胞中神经元特异性细胞摄取功能的初步测试。第二阶段（R33组分，3-4年）-进一步优化运载工具的稳定性、表达量和细胞内货物释放。在培养的神经元细胞中测试货物-BoNT/A-HC递送载体在神经元特异性细胞摄取中的功能（货物递送，抑制BoNT/ a介导的SNAP25切割）；离体腓神经- edl肌制剂；小鼠体内注射后。第三阶段（R33组成部分，4-5年）-测试货物-BoNT/A- hc运载工具在神经保护活性方面的功能和有效性，例如BoNT/A攻击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。

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.