Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin
Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin
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DOI：
10.1111/j.1742-4658.2012.08638.x
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发表时间：
2012-07-01
期刊：
影响因子：
5.4
通讯作者：
Dolly, J. Oliver
中科院分区：
文献类型：
--
作者：
Edupuganti, Om P.;Ovsepian, Saak V.;Dolly, J. Oliver
本发明描述了一种靶向药物载体（TDC），其用于将功能性蛋白质或肽转移到运动神经末梢中，运动神经末梢是治疗神经肌肉病症的治疗剂的关键位点。它利用了肉毒杆菌神经毒素B型（BoNT/B）与这些胆碱能神经末梢上的受体相互作用并内化的显著选择性。将编码无害的BoNT/B蛋白酶失活突变体（BoTIM）的基因与在大肠杆菌中表达的核心链霉亲和素的基因融合，并将纯化的蛋白质缀合至表面生物素化的脂质体。这种装饰的脂质体，载有荧光素作为可追踪的货物，获得了显着的特异性运动神经末梢在离体小鼠hemidiaphragms和促进神经元内转移的荧光，共聚焦显微镜显示。通过该TDC递送A型肉毒杆菌神经毒素（BoNT/A）的蛋白酶轻链加速神经肌肉麻痹的发作，表明该酶向突触前胞质溶胶的易位得到改善，随后分子量为25 kDa的突触体相关蛋白（SNAP-25）被蛋白水解失活，一种神经递质释放所必需的胞吐可溶性N-乙基-马来酰亚胺敏感因子附着蛋白受体（SNARE）。BoTIM偶联的脂质体，装载有蛋白酶的肽抑制剂，产生了相当大的衰减的神经麻痹作用的BoNT/A或BoNT/F作为其胞质转移的结果，第一次在原位证明设计师的抗蛋白酶抑制肉毒杆菌中毒的症状离体的能力。通过脂质体靶向全长BoTIM递送BoNT/A抑制剂证明比通过其C-末端神经受体结合结构域介导的更有效。这表明TDC的非病毒货物转移到胆碱能神经末梢的多功能性，揭示了其直接递送功能性靶标到运动神经末梢的潜力。
A targeted drug carrier (TDC) is described for transferring functional proteins or peptides into motor nerve terminals, a pivotal locus for therapeutics to treat neuromuscular disorders. It exploits the pronounced selectivity of botulinum neurotoxin type B (BoNT/B) for interacting with acceptors on these cholinergic nerve endings and becoming internalized. The gene encoding an innocuous BoNT/B protease-inactive mutant (BoTIM) was fused to that for core streptavidin, expressed in Escherichia coli and the purified protein was conjugated to surface-biotinylated liposomes. Such decorated liposomes, loaded with fluorescein as traceable cargo, acquired pronounced specificity for motor nerve terminals in isolated mouse hemidiaphragms and facilitated the intraneuronal transfer of the fluor, as revealed by confocal microscopy. Delivery of the protease light chain of botulinum neurotoxin type A (BoNT/A) via this TDC accelerated the onset of neuromuscular paralysis, indicative of improved translocation of this enzyme into the presynaptic cytosol with subsequent proteolytic inactivation of synaptosomal-associated protein of molecular mass 25 kDa (SNAP-25), an exocytotic soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) essential for neurotransmitter release. BoTIM-coupled liposomes, loaded with peptide inhibitors of proteases, yielded considerable attenuation of the neuroparalytic effects of BoNT/A or BoNT/F as a result of their cytosolic transfer, the first in situ demonstration of the ability of designer antiproteases to suppress the symptoms of botulism ex vivo. Delivery of the BoNT/A inhibitor by liposomes targeted with the full-length BoTIM proved more effective than that mediated by its C-terminal neuroacceptor-binding domain. This demonstrated versatility of TDC for nonviral cargo transfer into cholinergic nerve endings has unveiled its potential for direct delivery of functional targets into motor nerve endings.