Plant-Made Nanobodies for Botulism Treatment

描述（由申请人提供）：本申请中描述的研究的最终目标是开发一种经济、安全、有效的治疗方法，可以逆转肉毒杆菌中毒的麻痹症状。成功实现这一目标可以在发生肉毒杆菌毒素生物恐怖袭击时挽救无数人的生命。注射抗毒素抗体的目的是迅速彻底地将毒素从体内清除。目前可用的肉毒杆菌中毒免疫疗法（马抗毒素、人肉毒杆菌免疫球蛋白、单克隆抗体）只能去除尚未到达运动神经元的毒素分子。一旦毒素的催化轻链到达运动神经元的细胞质，在那里它会切割参与神经传递的蛋白质，正常抗体就无法进入。这就是为什么肉毒杆菌神经毒素中毒的受害者可能需要数周的呼吸机支持治疗。我们建议使用一种新的单链可变片段，即纳米体，来对抗毒素轻链的催化位点，同时使用允许蛋白质渗透到运动神经元的细胞质中的技术，来逆转由毒素引起的瘫痪。我们将从一个单域可变片段开始，从我们的合作者詹姆斯·马克斯博士（UCSF）实验室的美洲驼抗体库中分离出来，它抑制毒素轻链切割蛋白质底物的能力。我们将使用植物表达系统将这种纳米体融合到各种细胞穿透肽中。然后，我们将测试这些纳米体融合物进入原代大鼠神经元细胞并抑制毒素活性的能力。最后，我们将在小鼠身上测试细胞穿透纳米体的毒性。

DESCRIPTION (provided by applicant): The ultimate goal of the research described in this application is to develop an economical, safe and effective therapeutic that can reverse the paralytic symptoms of botulism. Success in achieving this goal could save countless lives in the event of a bioterrorist attack by botulinum toxin. The goal of administering an anti-toxin antibody is rapid and complete removal of the toxin from the body. Currently available immunotherapies for botulism (equine antitoxin, human botulism immune globulin, monoclonal antibodies) can remove only toxin molecules that have not yet reached motor neurons. Once the catalytic light chain of the toxin reaches the cytosol of motor neurons, where it cleaves proteins involved in neurotransmission, it is inaccessible to normal antibodies. This is why victims of botulinum neurotoxin poisoning may need supportive care on a respirator for many weeks. We propose using a new single-chain variable fragment, a nanobody, against the catalytic site of the toxin light chain, along with technology that allows proteins to penetrate into the cytosol of motor neurons, to reverse the paralysis caused by the toxin. We will start with a single-domain variable fragment, isolated from a llama antibody library in the laboratory of our collaborator, Dr James Marks (UCSF), that inhibits the ability of the toxin light chain to cleave it's protein substrate. We will express fusions of this nanobody to various cell-penetrating peptides, using a plant expression system. We will then test the ability of these nanobody fusions to enter primary rat neuron cells and inhibit toxin activity. Finally, we will test the toxicity of the cell-penetrating nanobody in mice. PUBLIC HEALTH RELEVANCE: Botulinum toxin is the deadliest biological substance known, and would be easy for a terrorist to produce and use to cause large numbers of casualties. This makes developing an effective and economical countermeasure to protect the public a public health priority. The research supported by this grant will lay the groundwork for using plants to produce a highly protective, safe and economical antitoxin therapy.