Department of Biomedicine, University Basel, 4031 Basel, Switzerland.
Department of Ophthalmology, Research Program for Ophthalmology and Glaucoma Research, Paracelsus University, 5020 Salzburg, Austria.
Mol Ther. 2022 Feb 2;30(2):855-867. doi: 10.1016/j.ymthe.2021.09.018. Epub 2021 Sep 20.
Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution in vivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.
细胞穿透肽 (CPP) 在治疗性蛋白的细胞内递送上具有巨大的应用潜力。然而，转导货物的内体捕获是阻碍其成功应用的主要瓶颈。在开发一种针对内皮素受体 A 表达的基于可转导锌指蛋白的人工转录因子时，我们发现 CPP 与内体膜的相互作用或内体纠缠是导致内体捕获的主要原因。为了实现内体松解，我们利用内体驻留蛋白酶，在人工转录因子进入内体时将其从 CPP 上切断。通过这种方法，我们极大地增强了正确亚细胞定位的解缠人工转录因子的生物活性和体内分布。通过对内切敏感性的合理设计，我们提出了一种新型可转导治疗性蛋白的设计原则，帮助 CPP 充分发挥其作为治疗性蛋白传递载体的潜力。
