Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6.
Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6.
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DOI：
10.1128/msphere.00466-18
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发表时间：
2018-10-24
期刊：
影响因子：
4.8
通讯作者：
Pellett S
中科院分区：
文献类型：
--
作者：
Moritz MS;Tepp WH;Bradshaw M;Johnson EA;Pellett S
肉毒杆菌神经毒素（BoNT）已被证明是治疗大量神经病的有效方法。 BoNT 包含一个锌金属蛋白酶大家族，但 BoNT/A1 几乎专门用于制药目的。天然菌株中第二个 BoNT 基因的基因失活使得能够从培养物中表达和分离单个 BoNT/A6。其表征表明，BoNT/A 亚型 A6 的作用持续时间与 A1 相当，同时它能更快、更有效地进入神经元，并且在肌内注射后保持更局部化。 BoNT/A6 的这些特性对于 BoNT/A6 作为一种新型的基于 BoNT 的治疗剂的潜在用途很有意义，该治疗剂有效、起效快、安全性提高、作用持续时间长。与目前使用的治疗方法相比，使用 BoNT/A6 作为药物还有可能揭示新的治疗主题。肉毒杆菌神经毒素 (BoNT) 是人类已知的最有效的毒素，也是肉毒中毒的病原体，通过进入运动神经元并裂解和灭活 SNARE 蛋白来发挥作用，而 SNARE 蛋白对于神经递质的释放至关重要。 BoNT 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。 BoNT 包含 7 种血清型和 40 多种异构体（亚型）。 BoNT/A1 因其高效力和作用持续时间长而成为临床上唯一使用的 A 亚型。虽然其他 BoNT/A 亚型已被纯化和描述，但只有 BoNT/A2 正在研究作为 BoNT/A1 的替代品。在这里，我们描述了与 BoNT/A1 相比具有改善的药理学特性的 BoNT/A6 亚型。它是从肉毒梭菌 CDC41370 中分离出来的，后者产生 BoNT/B2 和 BoNT/A6。编码 BoNT/B2 的基因被基因灭活，A6 的分离纯度高于 95%。 A6 在培养的原代啮齿动物神经元培养物和人类诱导多能干细胞衍生的神经元中非常有效，与 A1 相比，引起 50% SNAP-25 裂解所需的毒素少 20 倍。其次，A6 比 A1 更快、更有效地进入 hiPSC，但具有与 BoNT/A1 类似的较长作用持续时间。第三，小鼠腹腔注射后，BoNT/A6与BoNT/A1具有相似的LD50；然而，局部肌肉注射比 BoNT/A1 具有更低的全身毒性和更高的 (i.m.) LD50，表明其作为更安全药物的潜力。这些数据表明 BoNT/A6 的新颖特性及其作为改进药物的潜力，因为神经细胞进入更有效、在注射部位保持局部能力更强以及持续时间长。重要性 肉毒杆菌神经毒素 (BoNT) 已被证明是治疗大量神经病的有效方法。 BoNT 包含一个锌金属蛋白酶大家族，但 BoNT/A1 几乎专门用于制药目的。天然菌株中第二个 BoNT 基因的基因失活使得能够从培养物中表达和分离单个 BoNT/A6。其表征表明，BoNT/A 亚型 A6 的作用持续时间与 A1 相当，同时它能更快、更有效地进入神经元，并且在肌内注射后保持更局部化。 BoNT/A6 的这些特性对于 BoNT/A6 作为一种新型的基于 BoNT 的治疗剂的潜在用途很有意义，该治疗剂有效、起效快、安全性提高、作用持续时间长。与目前使用的治疗方法相比，使用 BoNT/A6 作为药物还有可能揭示新的治疗主题。
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. 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 Clostridium botulinum 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 LD50 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.) LD50, 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. IMPORTANCE 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.