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，对脊髓性肌萎缩症（SMA）进行治疗干预。 SMA 是一种常染色体隐性遗传的小儿神经肌肉疾病，其特征是 通过脊髓运动神经元的退化。所编码的蛋白质 SMA 决定基因 SMN1 对于运动神经元的生存是必需的。这个 SMA 患者基因有缺陷或缺失，其小鼠同源物至关重要 用于胚胎发育。在人类中，有第二个几乎相同的基因 SMN2， 允许受影响的个体生存，但在大多数患者中它不能表达 足够量的活性蛋白以完全补偿缺乏 SMN1。 SMN2 前体 mRNA 的剪接主要是通过外显子 7 的跳跃， 而该外显子组成型地包含在剪接的 SMN1 mRNA 中。只有 正确剪接的 SMN2 mRNA 的一小部分编码功能性 SMN 蛋白。一个 两个基因之间的单核苷酸差异位于外显子 7 的位置 6 - in 同义密码子 - 造成剪接模式的差异。 增加 SMN2 转录本中外显子包含的程度应该会产生 更高水平的功能性蛋白质，因此预计具有 治疗价值。外显子 7 中依赖于 SF2/ASF 的外显子剪接增强子 SMN2 似乎有缺陷，该外显子的识别非常敏感 到微妙的扰动。将开发新颖的小反义分子 在 SMN2 pre-mRNA 剪接过程中促进外显子 7 的包含。合理的设计 这些分子基于前 mRNA 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 pre-Mrna 的体外剪接进行测试和优化，并交付 进入培养细胞。最好的化合物将被测试其治疗效果， 并将进一步优化，通过将它们系统地和局部地交付到 老鼠。这些实验将利用最近开发的小鼠 Smn 敲除菌株，由人类 SMN2 转基因拯救，是一种有用的动物 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.