Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA.
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA
mSphere. 2018 Oct 24;3(5):e00466-18. doi: 10.1128/mSphere.00466-18.
Botulinum neurotoxins (BoNTs), the most potent toxins known to humans and the causative agent of botulism, exert their effect by entering motor neurons and cleaving and inactivating SNARE proteins, which are essential for neurotransmitter release. BoNTs are proven, valuable pharmaceuticals used to treat more than 200 neuronal disorders. BoNTs comprise 7 serotypes and more than 40 isoforms (subtypes). BoNT/A1 is the only A-subtype used clinically due to its high potency and long duration of action. While other BoNT/A subtypes have been purified and described, only BoNT/A2 is being investigated as an alternative to BoNT/A1. Here we describe subtype BoNT/A6 with improved pharmacological properties compared to BoNT/A1. It was isolated from CDC41370, which produces both BoNT/B2 and BoNT/A6. The gene encoding BoNT/B2 was genetically inactivated, and A6 was isolated to greater than 95% purity. A6 was highly potent in cultured primary rodent neuronal cultures and in human induced pluripotent stem cell-derived neurons, requiring 20-fold less toxin to cause 50% SNAP-25 cleavage than A1. Second, A6 entered hiPSCs faster and more efficiently than A1 and yet had a long duration of action similar to BoNT/A1. Third, BoNT/A6 had similar LD as BoNT/A1 after intraperitoneal injection in mice; however, local intramuscular injection resulted in less systemic toxicity than BoNT/A1 and a higher (i.m.) LD, indicating its potential as a safer pharmaceutical. These data suggest novel characteristics of BoNT/A6 and its potential as an improved pharmaceutical due to more efficient neuronal cell entry, greater ability to remain localized at the injection site, and a long duration. Botulinum neurotoxins (BoNTs) have proved to be an effective treatment for a large number of neuropathic conditions. BoNTs comprise a large family of zinc metalloproteases, but BoNT/A1 is used nearly exclusively for pharmaceutical purposes. The genetic inactivation of a second BoNT gene in the native strain enabled expression and isolation of a single BoNT/A6 from cultures. Its characterization indicated that BoNT/A subtype A6 has a long duration of action comparable to A1, while it enters neurons faster and more efficiently and remains more localized after intramuscular injection. These characteristics of BoNT/A6 are of interest for potential use of BoNT/A6 as a novel BoNT-based therapeutic that is effective and has a fast onset, an improved safety profile, and a long duration of action. Use of BoNT/A6 as a pharmaceutical also has the potential to reveal novel treatment motifs compared to currently used treatments.
肉毒神经毒素（BoNTs）是人类已知的最有效毒素，也是肉毒中毒的致病因子，通过进入运动神经元并切割和失活 SNARE 蛋白来发挥作用，SNARE 蛋白对于神经递质的释放至关重要。BoNTs 是经过验证的、有价值的药物，可用于治疗 200 多种神经元疾病。BoNTs 由 7 种血清型和 40 多种同工型（亚型）组成。BoNT/A1 是唯一一种临床上使用的 A 亚型，因为它具有高效力和长时间的作用。虽然已经纯化和描述了其他 BoNT/A 亚型，但只有 BoNT/A2 被作为 BoNT/A1 的替代品进行研究。在这里，我们描述了一种与 BoNT/A1 相比具有改善的药理学特性的亚型 BoNT/A6。它是从同时产生 BoNT/B2 和 BoNT/A6 的 CDC41370 中分离出来的。BoNT/B2 的基因被遗传失活，A6 的纯度大于 95%。A6 在培养的原代啮齿动物神经元培养物和人诱导多能干细胞衍生的神经元中具有高度效力，导致 50%SNAP-25 切割所需的毒素比 A1 少 20 倍。其次，A6 进入 hiPSCs 的速度比 A1 更快、更有效，但其作用持续时间与 BoNT/A1 相似。第三，BoNT/A6 在小鼠中的腹腔注射后的 LD 与 BoNT/A1 相似；然而，局部肌肉内注射导致比 BoNT/A1 更低的全身毒性和更高的（i.m.）LD，表明其作为更安全药物的潜力。这些数据表明 BoNT/A6 具有新的特性，并且由于其具有更高的神经元细胞进入效率、更大的在注射部位局部化的能力和更长的作用持续时间，因此具有作为改良药物的潜力。肉毒神经毒素（BoNTs）已被证明是治疗大量神经病变的有效方法。BoNTs 由一大类锌金属蛋白酶组成，但 BoNT/A1 几乎专门用于制药用途。在天然菌株中遗传失活第二种 BoNT 基因，使单一 BoNT/A6 能够从培养物中表达和分离。其特征表明，BoNT/A 亚型 A6 的作用持续时间与 A1 相当，而 A6 进入神经元的速度更快、效率更高，并且在肌肉内注射后保持更局部化。BoNT/A6 的这些特性对于将 BoNT/A6 用作新型基于 BoNT 的治疗药物具有潜在的兴趣，该药物具有疗效、起效迅速、改善的安全性和较长的作用持续时间。BoNT/A6 作为药物的使用也有可能揭示与目前使用的治疗方法相比的新的治疗模式。
