Motor neuron-targeted adenovirus antidotes for botulism

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。BONT是已知的最有效的毒素，可以相对容易地生产出来。如果大量的人接触到这种毒素，即使是很小的剂量，他们也会瘫痪，需要辅助呼吸，这很容易使有限的呼吸器供应不堪重负。我们已经开发了小蛋白制剂，当在中毒的神经元中表达时，促进BONT蛋白水解酶的快速降解，并提高中毒的恢复率。我们将这些生物分子制剂称为靶向F-盒(TFBs)，因为它们由一个15 kDa的F-box结构域与一个14 kDa的骆驼VHH结构域融合而成，与BONT蛋白酶具有结合特异性。在这里，我们建议开发以腺病毒为基础的肉毒杆菌中毒解毒剂，这种解毒剂将专门针对运动神经元，并导致TFB制剂在中毒神经元内的胞浆表达。由于几个原因，腺病毒(Ads)是将TFBs运送到运动神经元的理想载体。首先，Ad不会将它们的基因组整合到受感染的细胞中，并且已经产生了非复制的、用于治疗的安全的Ad载体。其次，可以对Ad进行修饰，以显著降低正常嗜性(肝脏)，同时增加选定细胞群体的感染。第三，可以对Ad载体进行修饰，以产生仅持续几周的瞬时转基因表达，之后受感染的细胞恢复正常。最后，已发现ADS可以感染运动神经元，导致转基因表达，令人惊讶的是，BONT中毒的运动神经元比正常神经元更有效地感染。在这个拟议项目的R21阶段，我们将开发一种适配器蛋白，通过使用来自BONT的神经元特异性受体结合域(RBD)来阻止正常的Ad趋向性，同时向运动神经元传递趋向性。重组Ad将被设计成表达TFB试剂，该TFB试剂针对来自ONT血清A型(BONT/A)的蛋白酶进行降解，并将用神经元靶向适配器进行预处理。然后，这种改良的Ad将作为肉毒杆菌中毒的解毒剂进行测试，方法是将其局部注射到小鼠的BoNT/A中毒肌肉中。如果成功，在R33阶段，我们将对Ad载体进行额外的修饰，以进一步提高运动神经元的特异性，并消除对适配蛋白的需求。我们还将设计一种针对另一种BONT血清型BONT/B的Ad解毒剂。这些Ad载体将在系统给药后测试加速小鼠从肉毒杆菌中毒瘫痪中恢复的能力。如果成功，通过简单地将TFBs的VHH结构域替换为对不同BONT蛋白酶具有特异性的其他结构域，就可以容易地针对所有七个已知的BONT血清型开发类似的试剂。此外，我们开发的神经元靶向Ad载体可能在其他重要的神经元病理学中也有应用。

DESCRIPTION (provided by applicant): Botulism is caused by Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote exists. BoNT is the most potent toxin known and can be produced with relative ease. If large numbers of people were exposed to even a small dose of this toxin, they would become paralyzed and require assisted breathing which could easily overwhelm the limited supplies of respirators. We have developed small protein agents that, when expressed within intoxicated neurons, promote rapid degradation of BoNT proteases and improve rates of recovery from intoxication. We call these biomolecule agents "targeted F-boxes" (TFBs) because they consist of a 15 kDa F-box domain fused to a 14 kDa camelid VHH domain with binding specificity for a BoNT protease. Here we propose to develop adenovirus-based botulism antidotes that will specifically target motor neurons and lead to cytosolic expression of a TFB agent within intoxicated neurons. Adenoviruses (Ads) are ideal vehicles for delivery of TFBs to motor neurons for several reasons. First, Ad does not integrate their genome into infected cells and Ad vectors have been produced that are non-replicating and safe for therapeutic use. Second, Ad can be modified to dramatically reduce the normal tropism (liver) while increasing infection of a selected cell population. Thirdly, Ad vectors can be modified to produce a transient burst of transgene expression lasting only a few weeks after which the infected cells revert to normal. Finally, Ads have been found to infect motor neurons leading to transgene expression and, surprisingly, BoNT intoxicated motor neurons are much more efficiently infected than normal neurons. In the R21 phase of this proposed project, we will develop an adapter protein that will block normal Ad tropism while imparting tropism for motor neurons by employing a neuron-specific receptor binding domain (RBD) that derives from BoNT. Recombinant Ad will be engineered to express a TFB agent that targets the protease from BoNT serotype A (BoNT/A) for degradation and will be pre-treated with the neuron-targeting adapter. This modified Ad will then be tested as a botulism antidote by local injection into BoNT/A intoxicated muscles in mice. If successful, in the R33 phase we will make additional modifications to the Ad vector to further improve specificity for motor neurons and obviate the need for an adapter protein. We will also engineer an Ad antidote for another BoNT serotype, BoNT/B. These Ad vectors will be tested for the ability to accelerate recovery from botulism paralysis in mice following a systemic administration of the therapeutic agent. If successful, similar agents can be readily developed for all seven known BoNT serotypes by simply replacing the VHH domain of the TFBs with others having specificity for different BoNT proteases. In addition, the neuron-targeted Ad vehicles we develop may have applications in other important neuronal pathologies.