Wallace H. Coulter Department of Biomedical Engineering, Emory University and Georgia Institute of Technology, Atlanta, Georgia 30332, United States.
Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States.
ACS Chem Biol. 2021 Nov 19;16(11):2255-2267. doi: 10.1021/acschembio.1c00387. Epub 2021 Oct 19.
Antisense oligonucleotides (ASOs) are single-stranded short nucleic acids that silence the expression of target mRNAs and show increasing therapeutic potential. Since ASOs are internalized by many cell types, both normal and diseased cells, gene silencing in unwanted cells is a significant challenge for their therapeutic use. To address this challenge, we created conditional ASOs that become active only upon detecting transcripts unique to the target cell. As a proof-of-concept, we modified an HIF1α ASO (EZN2968) to generate miRNA-specific conditional ASOs, which can inhibit HIF1α in the presence of a hepatocyte-specific miRNA, miR-122, via a toehold exchange reaction. We characterized a library of nucleic acids, testing how the conformation, thermostability, and chemical composition of the conditional ASO impact the specificity and efficacy in response to miR-122 as a trigger signal. Optimally designed conditional ASOs demonstrated knockdown of HIF1α in cells transfected with exogenous miR-122 and in hepatocytes expressing endogenous miR-122. We confirmed that conditional ASO activity was mediated by toehold exchange between miR-122 and the conditional ASO duplex, and the magnitude of the knockdown depended on the toehold length and miR-122 levels. Using the same concept, we further generated another conditional ASO that can be triggered by miR-21. Our results suggest that conditional ASOs can be custom-designed with any miRNA to control ASO activation in targeted cells while reducing unwanted effects in nontargeted cells.
反义寡核苷酸（ASO）是单链短核酸，可沉默靶 mRNA 的表达，并显示出越来越大的治疗潜力。由于 ASO 被许多细胞类型（包括正常细胞和病变细胞）内化，因此在不需要的细胞中实现基因沉默是其治疗用途的一个重大挑战。为了解决这一挑战，我们创建了条件性 ASO，这些 ASO 只有在检测到靶细胞特有的转录本时才会变得活跃。作为概念验证，我们对 HIF1α ASO（EZN2968）进行了修饰，以生成 miRNA 特异性条件性 ASO，这些 ASO 可以通过引发置换反应，在存在肝细胞特异性 miRNA miR-122 的情况下抑制 HIF1α。我们对一组核酸进行了表征，测试了条件性 ASO 的构象、热稳定性和化学组成如何影响其对 miR-122 作为触发信号的特异性和疗效。经过优化设计的条件性 ASO 在外源转染 miR-122 的细胞和表达内源性 miR-122 的肝细胞中均能有效抑制 HIF1α。我们证实，条件性 ASO 的活性是通过 miR-122 与条件性 ASO 双链体之间的引发置换反应介导的，敲低的幅度取决于引发物的长度和 miR-122 的水平。基于相同的概念，我们进一步生成了另一种可以被 miR-21 触发的条件性 ASO。我们的结果表明，可以根据任何 miRNA 定制设计条件性 ASO，以控制靶向细胞中 ASO 的激活，同时减少对非靶向细胞的不良影响。
