<br>miRNA 触发的条件反义寡核苷酸,ACS Chemical Biology

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miRNA 触发的条件反义寡核苷酸
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2021-10-19 , DOI: 10.1021/acschembio.1c00387
Jiahui Zhang ₁ , Radhika Sharma ₂ , Kitae Ryu ₁ , Patrick Shen ₂ , Khalid Salaita _{1,

2} , Hanjoong Jo _{1,

3}

Affiliation

反义寡核苷酸 (ASO) 是单链短核酸，可沉默靶 mRNA 的表达并显示出增强的治疗潜力。由于 ASO 被许多细胞类型（包括正常细胞和患病细胞）内化，因此不需要的细胞中的基因沉默对其治疗用途来说是一个重大挑战。为了应对这一挑战，我们创建了条件 ASO，仅在检测到目标细胞特有的转录本时才会激活。作为概念验证，我们修改了 HIF1α ASO (EZN2968) 以生成 miRNA 特异性条件 ASO，它可以在肝细胞特异性 miRNA miR-122 存在的情况下通过立足点交换反应抑制 HIF1α。我们对核酸库进行了表征，测试了条件 ASO 的构象、热稳定性和化学成分如何影响响应 miR-122 作为触发信号的特异性和功效。优化设计的条件 ASO 证明，在转染外源 miR-122 的细胞和表达内源 miR-122 的肝细胞中 HIF1α 被敲低。我们证实条件 ASO 活性是通过 miR-122 和条件 ASO 双链体之间的立足点交换介导的，并且敲低的幅度取决于立足点长度和 miR-122 水平。使用相同的概念，我们进一步生成了另一个可以由 miR-21 触发的条件 ASO。我们的结果表明，可以使用任何 miRNA 定制设计条件 ASO，以控制靶细胞中的 ASO 激活，同时减少非靶细胞中的不良影响。

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Conditional Antisense Oligonucleotides Triggered by miRNA

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.

更新日期：2021-11-19

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