Motor neuron-targeted adenovirus antidotes for botulism

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。BoNT是已知的最有效的毒素，可以相对容易地生产。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，这很容易超过有限的呼吸器供应。我们已经开发了小蛋白剂，当在中毒的神经元内表达时，促进BoNT蛋白酶的快速降解并提高从中毒中恢复的速率。我们将这些生物分子试剂称为"靶向F盒"（TFB），因为它们由融合至14kDa骆驼科VHH结构域的15kDa F盒结构域组成，所述14kDa骆驼科VHH结构域对BoNT蛋白酶具有结合特异性。在这里，我们建议开发基于腺病毒的肉毒中毒解毒剂，将专门针对运动神经元，并导致中毒神经元内的TFB剂的胞质表达。腺病毒（Ads）是将TFB递送至运动神经元的理想载体，原因有几个。首先，Ad不将其基因组整合到受感染的细胞中，并且已经产生了非复制型且对于治疗用途安全的Ad载体。第二，Ad可以被修饰以显著降低正常向性（肝脏），同时增加所选细胞群的感染。第三，Ad载体可以被修饰以产生仅持续几周的转基因表达的瞬时爆发，之后感染的细胞恢复正常。最后，已经发现Ad感染运动神经元，导致转基因表达，并且令人惊讶的是，BoNT中毒的运动神经元比正常神经元更有效地被感染。在这个项目的R21阶段，我们将开发一种衔接蛋白，它将阻止正常的Ad向性，同时通过采用来自BoNT的神经元特异性受体结合域（RBD）来赋予运动神经元向性。重组Ad将被工程化以表达靶向来自BoNT血清型A（BoNT/A）的蛋白酶用于降解的TFB试剂，并且将用神经元靶向衔接子预处理。然后通过局部注射到小鼠中BoNT/A中毒的肌肉中来测试这种修饰的Ad作为肉毒中毒解毒剂。如果成功，在R33阶段，我们将对Ad载体进行额外的修饰，以进一步提高对运动神经元的特异性，并消除对衔接蛋白的需求。我们还将设计另一种BoNT血清型BoNT/B的Ad解毒剂。将测试这些Ad载体在全身施用治疗剂后加速从小鼠中肉毒杆菌中毒麻痹恢复的能力。如果成功的话，通过简单地用对不同BoNT蛋白酶具有特异性的其他结构域替换TFB的VHH结构域，可以容易地开发用于所有七种已知BoNT血清型的类似试剂。此外，我们开发的靶向神经元的Ad载体可能在其他重要的神经元病理学中具有应用。 公共卫生相关性：肉毒杆菌神经毒素是一种极其危险的CDC A类生物防御威胁，广泛存在，易于生产，毒性极强，并且没有解毒剂。此外，至少存在七种不同的毒素类型， 每一种都需要使用常规方法的不同的、具有挑战性的、昂贵的和长期的解毒剂开发计划。相比之下，我们建议开发的创新的基于病毒的策略应该很快导致所有BoNT血清型的安全，经济和商业上可行的解毒剂。

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. PUBLIC HEALTH RELEVANCE: Botulinum neurotoxin is an extremely dangerous, CDC Category A biodefense threat that is widely available, easily produced, exceedingly toxic and for which no antidote is available. Furthermore, at least seven different toxin types exist, that would each require distinct, challenging, expensive and prolonged antidote development programs using conventional approaches. In contrast, the innovative, virus-based strategy we propose to develop should quickly lead to safe, economical and commercially viable antidotes for all BoNT serotypes.