Design of molecules that promote SMN2 exon 7 inclusion

描述（由申请人提供）： 该项目的目标是开发一种可以应用的新方法， 从长远来看，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，其特征在于 脊髓运动神经元的退化编码的蛋白质 SMA决定基因SMN 1是运动神经元存活所必需的。这 SMA患者中基因缺陷或缺失，其小鼠同源物是必需的 进行胚胎发育。在人类中，第二个几乎相同的基因SMN 2， 使受影响的人生存，但在大多数患者中，它不能表达 足够量的活性蛋白质，以完全补偿 SMN 1。SMN 2前体mRNA的剪接主要通过外显子7的跳跃， 而该外显子组成性地包含在剪接的SMN 1 mRNA中。只有 小部分正确剪接的SMN 2 mRNA编码功能性SMN蛋白。一 两个基因在外显子7的第6位的单核苷酸差异-在 同义密码子-负责剪接模式的差异。 增加SMN 2转录物中外显子包含的程度， 更高水平的功能性蛋白质，因此预计 治疗价值一个SF 2/ASF依赖的外显子7剪接增强子 SMN 2似乎有缺陷，并且对该外显子的识别非常敏感 微妙的扰动。将开发新型小反义分子， 在SMN 2前体mRNA剪接期间促进外显子7包含。的合理设计 这些分子是基于前体mRNA剪接研究的最新进展 因子和信号参与外显子的定义。新化合物将 使用SMN 2 pre-mRNA的体外剪接进行测试和优化， 转化为培养的细胞。最好的化合物将被测试治疗效果， 并将进一步优化，通过系统地和本地地将它们交付给 小鼠这些实验将利用最近开发的小鼠Smn 敲除菌株，由人SMN 2转基因拯救，其是有用的动物 SMA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.