联合疗法在对抗癌症和相关耐药问题等复杂病理学方面发挥着关键作用。这在靶向治疗中尤其重要，其中药物靶标的抑制可以通过交叉激活互补途径来克服。不幸的是，迄今为止批准的药物组合（主要基于小分子）面临一些问题，例如毒性作用，这限制了它们的临床使用。为了解决这些问题，我们设计了一种新型的 RNase H 敏感结构 (3ASO)，它可以在进入细胞后在细胞内分解，从而同时释放三种不同的治疗性寡核苷酸 (ON)，从而解决每种寡核苷酸的 mRNA 问题。不同的蛋白质。在这里，我们使用大肠杆菌 RNase H1 作为模型来研究前所未有的识别和切割模式，这主要由我们基于 RNA·DNA 的杂合构建体的拓扑结构决定。作为我们技术的模型系统，我们创建了 3ASO 构建体，旨在特异性抑制 HER2 乳腺癌细胞中 HER2、Akt 和 Hsp27 的表达。这些三功能 ON 工具在 HER2 乳腺癌细胞中表现出非常低的毒性和良好的抗增殖活性。本研究在对抗涉及多个 mRNA 靶点的复杂病理学方面具有巨大潜力，因为所提出的可切割设计将允许同时有效地单剂量施用不同的 ON 药物。版权所有 © 2024 作者。由爱思唯尔公司出版。保留所有权利。

Combined therapies play a key role in the fight against complex pathologies, such as cancer and related drug-resistance issues. This is particularly relevant in targeted therapies where inhibition of the drug target can be overcome by cross-activating complementary pathways. Unfortunately, the drug combinations approved to date -mostly based on small molecules- face several problems such as toxicity effects, which limit their clinical use. To address these issues, we have designed a new class of RNase H-sensitive construct (3ASO) that can be disassembled intracellularly upon cell entry, leading to the simultaneous release of three different therapeutic oligonucleotides (ONs), tackling each of them the mRNA of a different protein. Here, we used Escherichia coli RNase H1 as a model to study an unprecedented mode of recognition and cleavage, that is mainly dictated by the topology of our RNA·DNA-based hybrid construct. As a model system for our technology we have created 3ASO constructs designed to specifically inhibit the expression of HER2, Akt and Hsp27 in HER2+ breast cancer cells. These trifunctional ON tools displayed very low toxicity and good levels of antiproliferative activity in HER2+ breast cancer cells. The present study will be of great potential in the fight against complex pathologies involving multiple mRNA targets, as the proposed cleavable designs will allow the efficient single-dose administration of different ON drugs simultaneously.Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.