Department of Biochemistry, University of Leicester, Leicester LE1 9HN, Dubowitz Neuromuscular Centre, Institute of Child Health, UCL, London WC1N 1EH, UK.
Nucleic Acids Res. 2011 Sep 1;39(16):7194-208. doi: 10.1093/nar/gkr152. Epub 2011 May 20.
Controlling the patterns of splicing of specific genes is an important goal in the development of new therapies. We have shown that the splicing of a refractory exon, SMN2 exon 7, could be increased in fibroblasts derived from patients with spinal muscular atrophy by using bifunctional targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides that anneal to the exon and contain a 'tail' of enhancer sequences that recruit activating proteins. We show here that there are striking agreements between the effects of oligonucleotides on splicing in vitro and on both splicing and SMN2 protein expression in patient-derived fibroblasts, indicating that the effects on splicing are the major determinant of success. Increased exon inclusion depends on the number, sequence and chemistry of the motifs that bind the activator protein SRSF1, but it is not improved by increasing the strength of annealing to the target site. The optimal oligonucleotide increases protein levels in transfected fibroblasts by a mean value of 2.6-fold (maximum 4.6-fold), and after two rounds of transfection the effect lasted for a month. Oligonucleotides targeted to the upstream exon (exon 6 in SMN) are also effective. We conclude that TOES oligonucleotides are highly effective reagents for restoring the splicing of refractory exons and can act across long introns.
控制特定基因剪接模式是开发新疗法的重要目标。我们已经证明，通过使用双功能靶向剪接增强子寡核苷酸（TOES）寡核苷酸，能够增加来自脊髓性肌萎缩症患者的成纤维细胞中难治性外显子 SMN2 外显子 7 的剪接。这些寡核苷酸与外显子退火，并包含募集激活蛋白的增强子序列“尾巴”。我们在这里表明，寡核苷酸在体外对剪接的影响以及对患者来源的成纤维细胞中剪接和 SMN2 蛋白表达的影响之间存在惊人的一致性，这表明对剪接的影响是成功的主要决定因素。外显子包含的增加取决于结合激活蛋白 SRSF1 的基序的数量、序列和化学性质，但通过增加与靶位点的退火强度并不能提高。最佳的寡核苷酸通过平均增加 2.6 倍（最大 4.6 倍）来增加转染成纤维细胞中的蛋白水平，并且在两轮转染后，该效果持续一个月。靶向上游外显子（SMN 中的外显子 6）的寡核苷酸也有效。我们得出结论，TOES 寡核苷酸是恢复难治性外显子剪接的高效试剂，并且可以在长内含子中发挥作用。
