Reversing botulism with agents that accelerate intraneuronal toxin degradation

描述（由申请人提供）：肉毒杆菌中毒是由肉毒梭菌神经毒素（BoNT）引起的，这是一种疾病预防控制中心的a类生物防御威胁剂，目前还没有解药。我们已经开发了两种不同的“设计e3连接酶”，它们针对肉毒杆菌神经毒素血清型A或B （BoNT/A, BoNT/B）的细胞内蛋白酶，加速泛素介导的降解。我们的试剂由TrCP的F-box结构域融合到一个具有BoNT蛋白酶结合特异性的盖子VHH结构域组成。这些多肽制剂的大小小于30kd，可导致令人陶醉的BoNT蛋白酶在神经元内的快速破坏。在这里，我们提出开发一种基于艰难梭菌毒素B （TcdB）的载体，用于将BoNT设计物e3连接酶递送到中毒神经元的胞质中。我们建议在培养的神经元中测试工程TcdB药物逆转肉毒中毒症状，然后在小鼠中测试，可能是单剂量治疗。TcdB具有高度进化的特性，使其非常适合将BoNT定向设计e3连接酶输送到患者体内肉毒中毒神经元的胞质中。TcdB自然结合在细胞中广泛表达的表面受体，然后通过内吞作用内化并传递葡萄糖基转移酶（GT），使Rho gtpase失活，导致细胞死亡。这种毒素还含有一种蛋白酶活性，可以从“运载工具”上切割酶“货物”，并将其释放到细胞质中。研究表明，当GST融合到毒素GT结构域的氨基端时，TcdB可将大量功能性谷胱甘肽- s转移酶（GST）传递到细胞质中。我们已经成功地在微生物宿主细胞中表达了大量的全尺寸、生物活性的重组TcdB和一种有毒的TcdB变体。在本项目中，我们将开发和测试神经元特异性TcdB，方法是用BoNT/ a的等效RBD结构域取代TcdB受体结合结构域（RBD）。其次;我们将测试BoNT/A设计物e3 -连接酶与毒性TcdB的融合处理是否可以加速中毒神经元内BoNT/A蛋白酶的周转。最后，一种靶向BoNT设计连接酶的工程TcdB将在小鼠后肢轻瘫模型中进行测试，以逆转肉毒杆菌毒素引起的瘫痪症状。如果成功，通过简单地将VHH结构域转换为对其他BoNT蛋白酶具有特异性的结构域，类似的TcdB药物应该很容易并且快速地用于所有肉毒杆菌血清型。其次，我们将证明TcdB是一种将生物分子递送到细胞细胞质的通用载体-由RBD的特异性靶向。最后，我们传递给这些细胞的生物分子货物，设计e3连接酶，是骆驼VHH靶向结构域和TrCP F-box之间的简单融合，预计类似的药物可以开发用于研究或治疗应用的几乎任何细胞质蛋白的加速周转。

DESCRIPTION (provided by applicant): Botulism is caused by Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote exists. We have developed two distinct "designer E3-ligases" that target either the intracellular proteases of botulinum neurotoxin serotype A or B (BoNT/A, BoNT/B) for accelerated ubiquitin- mediated degradation. Our agents consist of the F-box domain of TrCP fused to a came lid VHH domain with binding specificity for the BoNT protease. These polypeptide agents, with a size less than 30 kD, lead to the rapid, intraneuronal destruction of the intoxicating BoNT proteases. Here we propose to develop a Clostridium difficile toxin B (TcdB) based vehicle for delivery of the BoNT designer E3-ligases to the cytosol of intoxicated neurons. We propose to test the engineered TcdB agents for reversal of botulism symptoms within cultured neurons and then in mice, likely with a single dose therapy. TcdB has highly evolved features that make it ideal for delivery of BoNT directed designer E3-ligases to the cytosol of botulism intoxicated neurons within patients. TcdB naturally binds to a surface receptor that is broadly expressed in cells, then is internalized by endocytosis and delivers a glucosyltransferase (GT) that inactivates Rho GTPases leading to cell death. This toxin also contains a protease activity that cleaves its enzymatic "cargo" from the "delivery vehicle", and releases it into the cytosol. TcdB has been shown to deliver significant quantities of functional glutathione-S-transferase (GST) to the cell cytosol when the GST is fused to the amino terminus of the toxin GT domain. We have successfully expressed large amounts of full-size, bioactive, recombinant TcdB, and an atoxic variant TcdB, in microbial host cells. In this proposed project, we will develop and test a neuron-specific TcdB by replacing the TcdB receptor binding domain (RBD) with the equivalent RBD domain from BoNT/A. Secondly; we will test whether BoNT/A protease turnover can be accelerated within intoxicated neurons by treatment with a fusion of BoNT/A designer E3-ligase to atoxic TcdB. Finally, an engineered TcdB targeting the BoNT designer ligase to neurons will be tested in the mouse hind limb paresis model for the ability to reverse the symptoms of botulinum toxin induced paralysis. If successful, similar TcdB agents should be readily possible and rapidly available for all botulinum serotypes by simply switching the VHH domain to those having specificity for other BoNT proteases. Secondly, we will have demonstrated that TcdB is a general vector for the delivery of biomolecules to the cytosol of cells- targeted by the specificity of the RBD. Finally, the biomolecular cargo we are delivering to these cells, designer E3-ligases, are simple fusions between a camelid VHH targeting domain and the TrCP F-box, and it is expected that similar agents could be developed to target accelerated turnover of virtually any cytosolic protein for research or therapeutic applications. PUBLIC HEALTH RELEVANCE: Through this proposal, we are directly seeking to develop a single-dose therapeutic cure for botulism, which is caused by several CDC Category A Clostridium botulinum toxins for which no antidote is currently available. If successful, similar agents can be quickly developed for all botulinum serotypes. The same general strategy could be used to deliver therapeutic agents to specific cells to target other pathogenic cytosolic proteins for rapid proteasomal destruction.