Reversing botulism with agents that accelerate intraneuronal toxin degradation

描述(申请人提供)：肉毒杆菌中毒是由肉毒杆菌神经毒素(BONT)引起的，这是一种CDC A类生物防御威胁剂，目前尚无解毒剂。我们已经开发了两种不同的“设计E3连接酶”，它们针对A型或B型肉毒神经毒素的细胞内酶(BONT/A，BONT/B)，以加速泛素介导的降解。我们的试剂由TrCP的F-box结构域与与BONT蛋白酶具有结合特异性的ComLID VHH结构域融合而成。这些多肽制剂的大小小于30kD，导致令人陶醉的BONT蛋白水解酶在神经细胞内快速破坏。在这里，我们建议开发一种基于艰难梭菌毒素B(TcdB)的载体，将BONT设计的E3-连接酶输送到中毒神经元的胞浆中。我们建议在培养的神经元中测试工程TcdB制剂逆转肉毒杆菌中毒症状，然后在小鼠身上进行试验，可能只需一次剂量治疗。TcdB具有高度进化的特征，使其成为将BONT导向的设计E3-连接酶输送到患者体内肉毒杆菌中毒神经元的细胞液中的理想选择。TcdB天然地与在细胞中广泛表达的表面受体结合，然后被内吞作用内化，并产生葡萄糖转移酶(GT)，使Rho GTP酶失活，导致细胞死亡。这种毒素还含有一种蛋白酶活性，可以将其酶“货物”从“运送载体”上分解出来，并将其释放到胞浆中。研究表明，当谷胱甘肽-S-转移酶(GST)与毒素GT结构域的氨基末端融合时，TcDB能将大量的GST递送到细胞质中。我们已经成功地在微生物宿主细胞中表达了大量全尺寸的、具有生物活性的重组TcdB和无毒突变体TcdB。在这个拟议的项目中，我们将通过用来自BONT/A的等效RBD结构域替换TcDB受体结合域(RBD)来开发和测试神经元特异性TcDB。其次，我们将测试通过将BONT/A设计者E3-连接酶融合到无毒性TcDB的处理是否可以加速中毒神经元中BONT/A蛋白酶的转换。最后，将在小鼠后肢瘫痪模型中测试针对BONT设计者连接酶的神经元的工程化TcdB是否有能力逆转肉毒杆菌毒素诱导的瘫痪症状。如果成功，通过简单地将VHH结构域切换到那些对其他BONT蛋白酶具有特异性的结构域，类似的TcdB试剂应该很容易并迅速地用于所有肉毒杆菌血清型。其次，我们将证明TcdB是一种通用的载体，可以将生物分子输送到细胞的胞浆中--靶向于RBD的特异性。最后，我们正在向这些细胞运送的生物分子货物，设计者E3-连接酶，是骆驼VHH靶向结构域和TrCP F-box之间的简单融合，预计可以开发类似的试剂来靶向几乎任何用于研究或治疗应用的胞浆蛋白的加速周转。 公共卫生相关性：通过这项提议，我们直接寻求开发一种治疗肉毒杆菌中毒的单剂治疗性疗法，肉毒杆菌中毒是由几种CDC A类肉毒杆菌毒素引起的，目前还没有解毒剂可供使用。如果成功，可以迅速开发出适用于所有肉毒杆菌血清型的类似试剂。同样的一般策略可以用来将治疗剂输送到特定的细胞，以靶向其他致病的胞浆蛋白，以快速破坏蛋白酶体。

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.