Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in Transgenic mice
Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in Transgenic mice
复制标题
DOI：
10.1016/j.ajhg.2008.01.014
复制
发表时间：
2008-04-01
影响因子：
9.8
通讯作者：
Krainer, Adrian R.
中科院分区：
文献类型：
--
作者：
Hua, Yimin;Vickers, Timothy A.;Krainer, Adrian R.
运动神经元着丝粒存活基因2（SMN 2）是一种改变脊髓性肌萎缩症（SMA）严重程度的基因，SMA是一种运动神经元疾病，是婴儿死亡的主要遗传原因。增加SMN 2外显子7（主要是跳过的）的纳入有望治疗或可能治愈SMA;一种实用策略是破坏损害外显子7识别的剪接沉默子。通过使用反义寡核苷酸（阿索）平铺方法，我们系统地筛选了近端内含子区域侧翼外显子7，并确定了两个内含子剪接沉默子（ISS）：一个在内含子6和最近描述的一个在内含子7。我们分析了内含子7 ISS的诱变，加上剪接分析，RNA亲和层析，和蛋白质过表达，并发现两个串联hnRNP A1/A2基序内的ISS是负责其抑制特性。这两个基序中的突变或阻断它们的ASO促进非常有效的外显子7包含。我们在该区域筛选了31个ASO，并选择了两个最佳ASO在人SMN 2转基因小鼠中进行测试。两种ASO均显著增加了转基因动物肝脏和肾脏中的hSMN 2外显子7包涵体。我们的研究结果表明，高分辨率的ASO平铺方法可以识别顺式元件，调节剪接积极或消极。最重要的是，我们的研究结果突出了其中一些ASO在SMA背景下的治疗潜力。
survival of motor neuron 2, centromeric (SMN2) is a gene that modifies the severity of spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of infant mortality. Increasing inclusion of SMN2 exon 7, which is predominantly skipped, holds promise to treat or possibly cure SMA; one practical strategy is the disruption of splicing silencers that impair exon 7 recognition. By using an antisense oligonucleotide (ASO)-tiling method, we systematically screened the proximal intronic regions flanking exon 7 and identified two intronic splicing silencers (ISSs): one in intron 6 and a recently described one in intron 7. We analyzed the intron 7 ISS by mutagenesis, coupled with splicing assays, RNA-affinity chromatography, and protein overexpression, and found two tandem hnRNP A1/A2 motifs within the ISS that are responsible for its inhibitory character. Mutations in these two motifs, or ASOs that block them, promote very efficient exon 7 inclusion. We screened 31 ASOs in this region and selected two optimal ones to test in human SMN2 transgenic mice. Both ASOs strongly increased hSMN2 exon 7 inclusion in the liver and kidney of the transgenic animals. Our results show that the high-resolution ASO-tiling approach can identify cis-elements that modulate splicing positively or negatively. Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA.