Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Center, Nijmegen, The Netherlands.
Methods Mol Biol. 2022;2434:129-141. doi: 10.1007/978-1-0716-2010-6_8.
Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1-254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.
磷酰二胺吗啉寡聚物 (PMO) 因其高稳定性和与靶序列的亲和力，在翻译阻断或剪接调节方面具有很大的应用潜力。然而，尽管与天然寡核苷酸或硫代磷酸酯类似物相比，它们带中性电荷，但它们仍然对细胞膜的通透性很小，这突出了需要有效的细胞质递送策略。此外，实施有效的细胞靶向策略对于最大限度地减少副作用和使药物在作用部位的剂量最大化非常重要。炭疽毒素是一种由三种蛋白质组成的毒素，其中形成孔的蛋白质炭疽保护抗原 (PA) 可以被重新导向到所需的受体，而两种底物蛋白之一的致死因子 (LF) 可以与各种货物结合，以实现有效的细胞质货物递送。在本方案中，我们描述了通过炭疽保护抗原生成的阳离子选择性孔来实现 PMO 细胞质递送所需的蛋白质和蛋白质缀合物的生产步骤。该方法依赖于在截断 LF (aa 1-254) 的 C 末端引入一个独特的半胱氨酸，在大肠杆菌中高效表达（截断）毒素蛋白，用马来酰亚胺基团功能化 PMO，并通过马来酰亚胺-巯基缀合化学将马来酰亚胺功能化的 PMO 偶联到 LF 上的独特半胱氨酸上。通过与 PA 共同给予 LF-PMO 缀合物，可以实现 PMO 的有效细胞质递送。
