Sanofi, Biologics Research, Industriepark Höchst, 65926 Frankfurt am Main, Germany.
Ablynx, a Sanofi Company, Technologiepark 21, 9052 Zwijnaarde, Belgium.
Mol Pharm. 2021 Mar 1;18(3):1048-1060. doi: 10.1021/acs.molpharmaceut.0c01001. Epub 2021 Jan 14.
Targeted extrahepatic delivery of siRNA remains a challenging task in the field of nucleic acid therapeutics. An ideal delivery tool must internalize siRNA exclusively into the cells of interest without affecting the silencing activity of siRNA. Here, we report the use of anti-EGFR Nanobodies (trademark of Ablynx N.V.) as tools for targeted siRNA delivery. A straightforward procedure for site-specific conjugation of siRNA to an engineered -terminal cysteine residue on the Nanobody (trademark of Ablynx N.V.) is described. We show that siRNA-conjugated Nanobodies (Nb-siRNA) retain their binding to EGFR and enter EGFR-positive cells via receptor-mediated endocytosis. The activity of Nb-siRNAs was assessed by measuring the knockdown of a housekeeping gene (AHSA1) in EGFR-positive and EGFR-negative cells. We demonstrate that Nb-siRNAs are active in vitro and induce mRNA cleavage in the targeted cell line. In addition, we discuss the silencing activity of siRNA conjugated to fused Nbs with various combinations of EGFR-binding building blocks. Finally, we compare the performance of Nb-siRNA joined by four different linkers and discuss the advantages and limitations of using cleavable and noncleavable linkers in the context of Nanobody-mediated siRNA delivery.
在核酸治疗领域，靶向肝外递送达 RNA 仍是一项具有挑战性的任务。理想的递药工具必须将 siRNA 特异性内化到靶细胞中，而不影响 siRNA 的沉默活性。在这里，我们报告了使用抗 EGFR Nanobody（Ablynx N.V. 的商标）作为靶向 siRNA 递药的工具。本文描述了一种用于将 siRNA 定点偶联到 Nanobody（Ablynx N.V. 的商标）末端半胱氨酸残基上的简单方法。我们表明，siRNA 偶联的 Nanobody（Nb-siRNA）保留了与 EGFR 的结合，并通过受体介导的内吞作用进入 EGFR 阳性细胞。通过测量 EGFR 阳性和 EGFR 阴性细胞中管家基因（AHSA1）的敲低来评估 Nb-siRNA 的活性。我们证明 Nb-siRNA 在体外具有活性，并在靶细胞系中诱导 mRNA 切割。此外，我们还讨论了与各种 EGFR 结合构建块融合的 Nb 偶联 siRNA 的沉默活性。最后，我们比较了通过四种不同接头连接的 Nb-siRNA 的性能，并讨论了在 Nanobody 介导的 siRNA 递药中使用可切割和不可切割接头的优缺点。
