Kashyap Disha, Cadeddu Martina, Oliver Peter L, Milne Thomas A, Booth Michael J
Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, U.K.
J Am Chem Soc. 2025 Aug 13;147(32):29478-29488. doi: 10.1021/jacs.5c09544. Epub 2025 Aug 4.
Antisense oligonucleotides (ASOs) are a promising class of therapeutics designed to modulate gene expression. Both key mechanisms of action for ASOs operate in the nucleus: splice-switching ASOs modify pre-mRNA, processed in the nucleus, and mRNA-degrading ASOs require RNase H, an enzyme predominantly active in the nucleus. Therefore, to achieve maximal therapeutic efficacy, ASOs require efficient nuclear delivery. In this work, we have synthesized ASO conjugates for active nuclear import, by covalent conjugation with a potent and proven small-molecule nuclear importer, (+)-JQ1. (+)-JQ1 is a well-characterized high-affinity binder for members of the BET bromodomain family of proteins and was recently shown to transport cytoplasmic proteins into the nucleus. Our (+)-JQ1-ASO conjugates outperformed their unmodified counterparts for both splice-switching and mRNA knockdown in the nucleus, across different molecular targets, backbone chemistries, and cell lines. In addition, we show that the improvement in on-target efficacy correlates with increased nuclear localization of the (+)-JQ1-modified ASOs by subcellular fractionation and immunocytochemistry. Notably, we improved the performance of Oblimersen, a BCL-2 ASO drug that failed in phase-III clinical trials. (+)-JQ1-Oblimersen showed increased effectiveness in an acute myeloid leukemia cell model, showing that this therapeutic may merit re-evaluation. This work demonstrates that the covalent modification of ASOs with a small-molecule nuclear importer can significantly improve target engagement and pave the way for more effective therapeutics.
反义寡核苷酸（ASOs）是一类很有前景的旨在调节基因表达的治疗药物。ASOs的两个关键作用机制都在细胞核中发挥作用：剪接转换ASOs修饰在细胞核中加工的前体mRNA，而mRNA降解ASOs需要核糖核酸酶H，这是一种主要在细胞核中具有活性的酶。因此，为了实现最大的治疗效果，ASOs需要高效的细胞核递送。在这项工作中，我们通过与一种强效且经过验证的小分子核输入剂（+）-JQ1共价结合，合成了用于主动核输入的ASO偶联物。（+）-JQ1是一种对BET溴结构域蛋白家族成员具有良好特征的高亲和力结合剂，最近被证明能将细胞质蛋白转运到细胞核中。我们的（+）-JQ1-ASO偶联物在细胞核中的剪接转换和mRNA敲低方面，在不同的分子靶点、骨架化学结构和细胞系中都优于未修饰的对应物。此外，我们通过亚细胞分级分离和免疫细胞化学表明，靶向疗效的提高与（+）-JQ1修饰的ASOs核定位增加相关。值得注意的是，我们提高了奥布利默森（Oblimersen）的性能，这是一种在III期临床试验中失败的BCL-2 ASO药物。（+）-JQ1-奥布利默森在急性髓性白血病细胞模型中显示出增强的有效性，表明这种治疗方法可能值得重新评估。这项工作表明，用小分子核输入剂对ASOs进行共价修饰可以显著改善靶点结合，并为更有效的治疗方法铺平道路。
