Design of molecules that promote SMN2 exon 7 inclusion

促进 SMN2 外显子 7 包含的分子设计

• 批准号: 6540449
• 负责人: Adrian R Krainer
• 金额: $ 40.84万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6540449
• 关键词: HeLa cells; Mus musculus; RNA splicing; antisense nucleic acid; autosomal recessive trait; binding proteins; chemical synthesis; degenerative motor system disease; disease /disorder model; gene induction /repression; gene mutation; gene targeting; genetic models; genetically modified animals; inclusion body; laboratory mouse; model design /development; motor neurons; nucleic acid sequence; oligonucleotides; peptide nucleic acids; precursor mRNA; progressive spinal muscular atrophy

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.

描述（由申请人提供）： 该项目的目的是开发一种可以应用的新方法， 从长远来看，脊柱肌肉萎缩（SMA）的治疗干预。 SMA是一种常染色体不必要的儿科神经肌肉疾病，其特征是 通过脊柱运动神经元的变性。由 SMA确定的基因SMN1对于运动神经元的存活是必要的。这 SMA患者的基因有缺陷或不存在，其小鼠同源物是必不可少的 用于胚胎开发。在人类中，第二个，几乎相同的基因Smn2， 允许受影响的个体生存，但是在大多数患者中，它无法表达 足够量的活性蛋白质充分补偿 SMN1。 SMN2前MRNA的剪接主要是通过外显子7的跳过 而该外显子组成性地包括在剪接的SMN1 mRNA中。只有 正确剪接的SMN2 mRNA的小部分编码功能性SMN蛋白。一个 外显子7- in的位置6的两个基因之间的单核苷酸差异 同义密码子 - 负责剪接模式的差异。 增加外显子包含在SMN2转录本中的程度应产生 较高水平的功能蛋白，因此有望具有 治疗价值。外显子7中的SF2/ASF依赖外显剪接增强剂 在SMN2中似乎有缺陷，并且对外显子的识别非常敏感 微妙的扰动。新颖的小反义分子将开发为 在SMN2前MRNA剪接过程中促进外显子7包含。理性的设计 这些分子基于MRNA剪接研究的最新进展 外显子定义涉及的因素和信号。新化合物将是 使用SMN2 Pre-MRNA的体外剪接进行了测试和优化，并交付 进入培养的细胞。最好的化合物将用于治疗作用， 并将通过系统地和本地交付将它们进一步优化 老鼠。这些实验将利用最近开发的鼠标SMN 敲除菌株，由人类SMN2转基因营救，这是一种有用的动物 SMA的模型。