Enhancement of SMN2 exon 7 inclusion by antisense oligonucleotides targeting the exon.
Enhancement of SMN2 exon 7 inclusion by antisense oligonucleotides targeting the exon.
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DOI：
10.1371/journal.pbio.0050073
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发表时间：
2007-04
期刊：
影响因子：
9.8
通讯作者：
Krainer, Adrian R
中科院分区：
文献类型：
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作者：
Hua, Yimin;Vickers, Timothy A;Baker, Brenda F;Bennett, C Frank;Krainer, Adrian R
已经采取了几种策略来增加SMN 2（运动神经元存活2）转录物剪接期间外显子7包含的程度，以最终用于脊髓性肌萎缩症（SMA）（一种遗传性神经肌肉疾病）的治疗。靶向外显子或其侧翼剪接位点的反义寡核苷酸（ASO）通常促进外显子跳跃。在此，我们系统检测了大量具有2′-O-甲氧基乙基核糖（莫伊）骨架的ASO，这些ASO与SMN 2外显子7的不同位置杂交，并鉴定了几种促进更多外显子包含的ASO，其他促进外显子跳跃的ASO，以及对两种选择性剪接产物蓄积具有复杂影响的ASO。这种方法提供了位置信息的假定外显子元件或二级结构的积极或消极的影响外显子包含。ASO不仅在无细胞剪接测定中有效，而且当转染到培养的细胞中时也有效，在培养的细胞中它们影响内源性SMN转录物的剪接。促进外显子7包含的ASO增加全长SMN蛋白水平，表明它们不干扰mRNA输出或翻译，尽管与外显子杂交。我们鉴定的一些ASO具有足够的活性以在SMA小鼠模型中进行实验。脊髓性肌萎缩症（SMA）是一种严重的遗传性疾病，导致运动神经元变性。SMA患者缺乏功能性SMN 1（运动神经元存活1）基因，但他们拥有完整的SMN 2基因，尽管与SMN 1几乎相同，但只有部分功能。SMN 2基因表达的缺陷在前体mRNA剪接水平（外显子7跳跃），所有SMA患者中存在该基因使其成为潜在治疗的有吸引力的靶点。在这里，我们已经调查了大量的反义寡核苷酸（ASO）的SMN 2 mRNA中的外显子7的不同区域互补。这些ASO中的一些能够纠正前体mRNA剪接缺陷，可能是因为它们结合到形成RNA结构的外显子7的区域，或提供蛋白质结合位点，这通常会削弱细胞核中剪接机制对该外显子的识别。我们描述了促进SMA患者培养细胞中SMN 2 mRNA正确表达并因此促进正常SMN蛋白表达的最佳ASO。这些ASO现在可以在SMA小鼠模型中进行测试，并且可能对SMA治疗有用。SMN 1突变导致脊髓性肌萎缩;一个几乎相同的基因没有功能，但在加入反义寡核苷酸后在体外和体内变得有功能。
Several strategies have been pursued to increase the extent of exon 7 inclusion during splicing of SMN2 (survival of motor neuron 2) transcripts, for eventual therapeutic use in spinal muscular atrophy (SMA), a genetic neuromuscular disease. Antisense oligonucleotides (ASOs) that target an exon or its flanking splice sites usually promote exon skipping. Here we systematically tested a large number of ASOs with a 2′-O-methoxy-ethyl ribose (MOE) backbone that hybridize to different positions of SMN2 exon 7, and identified several that promote greater exon inclusion, others that promote exon skipping, and still others with complex effects on the accumulation of the two alternatively spliced products. This approach provides positional information about presumptive exonic elements or secondary structures with positive or negative effects on exon inclusion. The ASOs are effective not only in cell-free splicing assays, but also when transfected into cultured cells, where they affect splicing of endogenous SMN transcripts. The ASOs that promote exon 7 inclusion increase full-length SMN protein levels, demonstrating that they do not interfere with mRNA export or translation, despite hybridizing to an exon. Some of the ASOs we identified are sufficiently active to proceed with experiments in SMA mouse models. Spinal muscular atrophy (SMA) is a severe genetic disease that causes motor-neuron degeneration. SMA patients lack a functional SMN1 (survival of motor neuron 1) gene, but they possess an intact SMN2 gene, which though nearly identical to SMN1, is only partially functional. The defect in SMN2 gene expression is at the level of pre-mRNA splicing (skipping of exon 7), and the presence of this gene in all SMA patients makes it an attractive target for potential therapy. Here we have surveyed a large number of antisense oligonucleotides (ASOs) that are complementary to different regions of exon 7 in the SMN2 mRNA. A few of these ASOs are able to correct the pre-mRNA splicing defect, presumably because they bind to regions of exon 7 that form RNA structures, or provide protein-binding sites, that normally weaken the recognition of this exon by the splicing machinery in the cell nucleus. We describe optimal ASOs that promote correct expression of SMN2 mRNA and, therefore, normal SMN protein, in cultured cells from SMA patients. These ASOs can now be tested in mouse models of SMA, and may be useful for SMA therapy. Mutations inSMN1 cause spinal muscular atrophy; a nearly identical gene is not functional, but becomes functional in vitro and in vivo after addition of antisense oligos.