Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Hyogo, Japan.
Department of Biochemistry, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Jalan Farmako, Yogyakarta 55281, Indonesia.
Genes (Basel). 2022 Apr 13;13(4):685. doi: 10.3390/genes13040685.
Spinal muscular atrophy (SMA) is caused by deletion. The encodes the same protein as does, but it has a splicing defect of exon 7. Some antisense oligonucleotides (ASOs) have been proven to correct this defect. One of these, nusinersen, is effective in SMA-affected infants, but not as much so in advanced-stage patients. Furthermore, the current regimen may exhibit a ceiling effect. To overcome these problems, high-dose ASOs or combined ASOs have been explored. Here, using SMA fibroblasts, we examined the effects of high-concentration ASOs and of combining two ASOs. Three ASOs were examined: one targeting intronic splicing suppressor site N1 (ISS-N1) in intron 7, and two others targeting the 3' splice site and 5' region of exon 8. In our experiments on all ASO types, a low or intermediate concentration (50 or 100 nM) showed better splicing efficiency than a high concentration (200 nM). In addition, a high concentration of each ASO created a cryptic exon in exon 6. When a mixture of two different ASOs (100 nM each) was added to the cells, the cryptic exon was included in the mRNA. In conclusion, ASOs at a high concentration or used in combination may show less splicing correction and cryptic exon creation.
脊髓性肌萎缩症（SMA）是由缺失引起的。该基因编码与基因相同的蛋白质，但它在外显子 7 处存在剪接缺陷。一些反义寡核苷酸（ASO）已被证明可以纠正这种缺陷。其中一种名为 nusinersen 的药物在受影响的婴儿 SMA 中有效，但在晚期患者中效果不那么明显。此外，目前的治疗方案可能存在上限效应。为了克服这些问题，已经探索了高剂量 ASO 或联合 ASO。在这里，我们使用 SMA 成纤维细胞研究了高浓度 ASO 和联合两种 ASO 的效果。我们检查了三种 ASO：一种针对内含子 7 中的内含子剪接抑制物位点 N1（ISS-N1），另外两种针对 3'剪接位点和 5'外显子 8 区域。在我们对所有 ASO 类型的实验中，低浓度或中浓度（50 或 100 nM）比高浓度（200 nM）显示出更好的剪接效率。此外，每种 ASO 的高浓度都会在外显子 6 中产生一个隐蔽外显子。当向细胞中添加两种不同 ASO（每种 100 nM）的混合物时，隐蔽外显子会包含在 mRNA 中。总之，高浓度的 ASO 或联合使用可能会显示出较少的剪接纠正和隐蔽外显子的产生。
