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“设计者E3-连接酶”，其靶向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，用于加速泛素介导的降解.我们的试剂由TrCP的F-box结构域与对BoNT蛋白酶具有结合特异性的callid VHH结构域融合组成。这些多肽剂，具有小于30 kD的大小，导致快速，神经元内破坏的中毒BoNT蛋白酶。在这里，我们建议开发一种基于艰难梭菌毒素B（TcdB）的载体，用于将BoNT设计者E3-连接酶递送到中毒神经元的胞质溶胶中。我们建议测试工程化的TcdB试剂在培养的神经元内以及随后在小鼠中逆转肉毒杆菌中毒症状，可能使用单剂量疗法。 TcdB具有高度进化的特征，这使得其对于将BoNT定向的设计者E3连接酶递送至患者体内肉毒中毒神经元的胞质溶胶是理想的。TcdB天然地结合于在细胞中广泛表达的表面受体，然后通过胞吞作用内化并递送使Rho GTP酶失活的葡糖基转移酶（GT），从而导致细胞死亡。该毒素还含有蛋白酶活性，其将其酶促“货物”从“递送载体”切割，并将其释放到细胞溶质中。当GST与毒素GT结构域的氨基末端融合时，TcdB已显示出向细胞胞质溶胶递送显著量的功能性谷胱甘肽-S-转移酶（GST）。我们已经成功地表达了大量的全尺寸，生物活性，重组TcdB，和无毒的变体TcdB，在微生物宿主细胞。在这个拟议的项目中，我们将开发和测试神经元特异性TcdB，用来自BoNT/A的等效RBD结构域替换TcdB受体结合结构域（RBD）。第二;我们将测试通过用BoNT/A设计者E3-连接酶与无毒性TcdB的融合物处理是否可以在中毒的神经元内加速BoNT/A蛋白酶周转。最后，将在小鼠后肢轻瘫模型中测试将BoNT设计者连接酶靶向神经元的工程化TcdB逆转肉毒杆菌毒素诱导的麻痹症状的能力。 如果成功的话，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那些，类似的TcdB试剂应该是容易可能的并且快速可用于所有肉毒杆菌血清型。其次，我们将证明TcdB是用于将生物分子递送至细胞的胞质溶胶的通用载体-由RBD的特异性靶向。最后，我们递送到这些细胞的生物分子货物，设计者E3-连接酶，是骆驼VHH靶向结构域和TrCP F-盒之间的简单融合物，并且预计可以开发类似的试剂以靶向几乎任何细胞溶质蛋白的加速周转，用于研究或治疗应用。 公共卫生相关性：通过这项提案，我们直接寻求开发一种单剂量肉毒杆菌中毒治疗方法，肉毒杆菌中毒是由几种CDC A类肉毒杆菌毒素引起的，目前没有解毒剂。如果成功的话，类似的药剂可以迅速开发出用于所有肉毒杆菌血清型。相同的一般策略可用于将治疗剂递送至特定细胞以靶向其他致病性胞质蛋白，从而快速破坏蛋白酶体。

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.