Department of Environmental and Prevention Sciences, University of Ferrara, 44121 Ferrara, Italy.
Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, 44121 Ferrara, Italy.
Int J Mol Sci. 2022 Apr 12;23(8):4270. doi: 10.3390/ijms23084270.
Our groups previously reported that conjugation at 3'-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature () of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5'-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5'-UDC- and 5',3'-bis-UDC-ASO 51.
我们的团队之前曾报道过，在 3'末端与熊去氧胆酸（UDCA）缀合可显著增强针对人类 DMD 外显子 51 的 ASO 51 寡核苷酸的体外外显子跳跃特性。在这项研究中，我们设计了一系列 ASO 51 的亲脂性缀合物，以探索亲脂部分对外显子跳跃效率的影响。为此，我们对三种胆酸和两种脂肪酸进行了衍生化和/或修饰，并通过自动化固相合成将其与 ASO 51 缀合。我们测量了亲脂性缀合物的熔点（），以评估其与靶 RNA 形成稳定双链的能力。首先在转染剂存在的情况下，在肌源性细胞系中评估了外显子跳跃效率，然后在选择的缀合 ASO 51 上在非电转条件下进行评估。在 5'-UDC-ASO 51 的情况下，我们还评估了 PS 含量对外显子跳跃效率的影响；我们发现，它具有完整 PS 键合的更好的外显子跳跃性能。在提高外显子跳跃效率方面，更有效的化合物是 5'-UDC-和 5'，3'-双 UDC-ASO 51。
