Design of molecules that promote SMN2 exon 7 inclusion

描述(由申请人提供)： 这个项目的目标是开发一种可以应用的新方法， 从长远来看，对脊髓性肌萎缩症(SMA)的治疗干预。 SMA是一种常染色体隐性遗传的儿科神经肌肉疾病，其特征是 由脊髓运动神经元的退化引起。该基因编码的蛋白质 SMA决定基因SMN1是运动神经元生存所必需的。这 SMA患者的基因存在缺陷或缺失，其小鼠同源基因是必不可少的 用于胚胎发育。在人类中，第二个几乎相同的基因SMN2， 允许受影响的个体存活，但在大多数患者中无法表达 足够数量的活性蛋白质来完全弥补缺乏 SMN1。SMN2前-mRNA的剪接主要通过跳过外显子7， 而该外显子则结构性地包含在剪接的SMN1 mRNA中。只有 一小部分正确拼接的SMN2mRNA编码具有功能的SMN蛋白。一个 这两个基因外显子7-in第6位的单核苷酸差异 同义密码子--是造成剪接模式差异的原因。 增加SMN2转录本中外显子的包含程度应该会产生 更高水平的功能蛋白，因此预计会有 治疗价值。依赖SF2/ASF的外显子第7外显子剪接增强子 Smn2似乎有缺陷，对该外显子的识别非常敏感 到微妙的微扰。新型反义小分子将被开发成 在SMN2Pre-mRNA剪接过程中促进外显子7的包涵体。建筑结构的合理设计 这些分子是基于前信使核糖核酸剪接研究的最新进展。 外显子定义中涉及的因素和信号。新化合物将是 使用SMN2前-mRNA的体外剪接进行测试和优化，并交付 转化成培养的细胞。最好的化合物将接受治疗效果测试， 并将进一步优化，将它们系统地和本地地交付到 老鼠。这些实验将利用最近开发的小鼠SMN 被人类SMN2转基因拯救的基因敲除菌株，这是一种有用的动物 SMA的模型。

DESCRIPTION (provided by applicant): The objective of this project is to develop a new approach that can be applied, in the long term, to therapeutic intervention in spinal muscular atrophy (SMA). SMA is an autosomal-recessive, pediatric neuromuscular disorder, characterized by the degeneration of spinal motor neurons. The protein encoded by the SMA-determining gene SMN1 is necessary for the survival of motor neurons. This gene is defective or absent in SMA patients, and its mouse homolog is essential for embryonic development. In humans, a second, nearly identical gene, SMN2, allows affected individuals to survive, but in most patients it cannot express sufficient amounts of active protein to fully compensate for the absence of SMN1. Splicing of the SMN2 pre-mRNA is predominantly via skipping of exon 7, whereas this exon is constitutively included in spliced SMN1 mRNA. Only the small fraction of correctly spliced SMN2 mRNA encodes functional SMN protein. A single-nucleotide difference between the two genes at position 6 of exon 7 - in synonymous codons - is responsible for the difference in splicing patterns. Increasing the extent of exon inclusion in the SMN2 transcripts should generate higher levels of functional protein, and is therefore expected to have therapeutic value. An SF2/ASF-dependent exonic splicing enhancer in exon 7 appears to be defective in SMN2, and recognition of this exon is very sensitive to subtle perturbations. Novel, small antisense molecules will be developed to promote exon 7 inclusion during SMN2 pre-mRNA splicing. The rational design of these molecules is based on recent advances in studies of pre-mRNA splicing factors and signals involved in exon definition. The new compounds will be tested and optimized using in vitro splicing of SMN2 pre-Mrna, and delivered into cultured cells. The best compounds will be tested for therapeutic effects, and will be further optimized, by delivering them systemically and locally into mice. These experiments will make use of the recently-developed mouse Smn knockout strain, rescued by a human SMN2 transgene, which is a useful animal model of SMA.