G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of FXN expression through indirect effects.
G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of FXN expression through indirect effects.
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DOI：
10.1093/nar/gkac1108
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发表时间：
2022-12-09
影响因子：
14.9
通讯作者：
Watts, Jonathan K.
中科院分区：
文献类型：
--
作者：
Wang, Feng;Calvo-Roitberg, Ezequiel;Rembetsy-Brown, Julia M.;Fang, Minggang;Sousa, Jacquelyn;Kartje, Zachary J.;Krishnamurthy, Pranathi Meda;Lee, Jonathan;Green, Michael R.;Pai, Athma A.;Watts, Jonathan K.
弗里德赖希共济失调是一种由共济失调蛋白（FXN）缺乏引起的不治之症，其主要由FXN基因内含子1中的GAA重复扩增引起。在这里，我们确定了反义寡核苷酸（ASO），互补的FXN前mRNA的第一个内含子内的两个区域，这可以增加患者成纤维细胞中的FXN mRNA的102倍。FXN mRNA的增加通过在每个区域鉴定多个重叠的FXN活化ASO、两个独立的RNA定量测定和通过多个管家基因的标准化来证实。对缺失ASO结合位点的细胞的实验表明，ASO诱导的FXN活化是由间接效应驱动的。RNA测序分析显示，两种ASO诱导了类似的转录组范围的变化，这与野生型细胞的转录组不相似。该RNA-seq分析没有直接鉴定出ASO之间共有的碱基配对的脱靶基因。错配研究确定了FXN激活所需的ASO内的两个富含鸟苷的基序（CCGG和G4）。我们的ASO的磷酰二胺吗啉代寡聚体类似物不激活FXN，这表明PS骨架介导的作用。我们的研究表明，在寡核苷酸研究中，采用新的机制，如基因激活，多个，详细的控制实验和目标验证的重要性。
Friedreich’s ataxia is an incurable disease caused by frataxin (FXN) protein deficiency, which is mostly induced by GAA repeat expansion in intron 1 of the FXN gene. Here, we identified antisense oligonucleotides (ASOs), complementary to two regions within the first intron of FXN pre-mRNA, which could increase FXN mRNA by ∼2-fold in patient fibroblasts. The increase in FXN mRNA was confirmed by the identification of multiple overlapping FXN-activating ASOs at each region, two independent RNA quantification assays, and normalization by multiple housekeeping genes. Experiments on cells with the ASO-binding sites deleted indicate that the ASO-induced FXN activation was driven by indirect effects. RNA sequencing analyses showed that the two ASOs induced similar transcriptome-wide changes, which did not resemble the transcriptome of wild-type cells. This RNA-seq analysis did not identify directly base-paired off-target genes shared across ASOs. Mismatch studies identified two guanosine-rich motifs (CCGG and G4) within the ASOs that were required for FXN activation. The phosphorodiamidate morpholino oligomer analogs of our ASOs did not activate FXN, pointing to a PS-backbone-mediated effect. Our study demonstrates the importance of multiple, detailed control experiments and target validation in oligonucleotide studies employing novel mechanisms such as gene activation.
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影响因子：
14.9
作者：
Hagedorn PH;Hansen BR;Koch T;Lindow M
通讯作者：
Lindow M
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通讯作者：
Flicek P
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4.5
作者：
Groh M;Lufino MM;Wade-Martins R;Gromak N
通讯作者：
Gromak N
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作者：
Gagnon, Keith T.;Corey, David R.
通讯作者：
Corey, David R.
影响因子：
64.8
作者：
Carrieri, Claudia;Cimatti, Laura;Gustincich, Stefano
通讯作者：
Gustincich, Stefano