Determinants that regulate splicing of SMN

调节 SMN 剪接的决定因素

• 批准号: 7142158
• 负责人: Christian L. Lorson
• 金额: $ 33.64万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7142158
• 关键词: RNA splicing; animal genetic material tag; binding sites; disease /disorder model; gene expression; gene induction /repression; gene mutation; genetic enhancer element; genetic regulatory element; introns; mutant; nucleic acid sequence; precursor mRNA; progressive spinal muscular atrophy; transcription factor

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is the leading genetic cause of infantile death, yet there currently is no cure. SMA is a neuromuscular disorder resulting from the loss of survival motor neuron 1 (SMN1). A nearly identical copy gene exists, SMN2, however, it cannot provide protection from disease development in the absence of SMN1. Remarkably, both SMN1 and 2 encode identical proteins, however, due to a single silent mutation, the vast majority of SMN2 transcripts are alternatively spliced and the final coding exon (exon 7: 54 nts) is removed. Therefore, the molecular basis for this devastating disease is an alternative splicing event that results in the production of a truncated and unstable SMN protein (called SMN-delta7). The restoration of full-length SMN expression by modulating SMN2 splicing patterns represents an exciting prospect for therapeutic intervention. The molecular genetics of SMA make this disease especially amenable to therapeutic strategies that promote full-length expression from SMN2. 1) nearly 99% of all SMA cases are caused by a single gene; 2) SMN2 encodes an identical protein; 3) the SMA population is remarkably homogenous with regards to SMN2. Individuals have not been identified that are homozygous null for SMN1 and SMN2 - presumably because this condition would be lethal (consistent with the knock- out mouse model). Therefore, essentially all SMA patients carry at least one SMN2 gene; and 4) transcripts generated from SMN2 are stable. The primary goal of this proposal is to develop recombinant adeno-associated virus (rAAV) vectors that express short RNAs that promote stimulate full-length SMN expression by promoting the inclusion of SMN2 exon 7. A step-wise evaluation process will be used to identify the most effective rAAV-derived RNAs in cell- based models. Finally, the most effective rAAV vectors will be evaluated in a mild SMA mouse model to determine whether SMN2 splicing can be altered in vivo and whether this increase ameliorates the well- characterized SMA phenotype. While the experiments described in this proposal have immediate implications for the development of a SMA therapy, the results could be used as a model for a broad range of genetic disorders in which correcting a splicing defect would restore functionality to a disease-causing gene.

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是婴儿死亡的主要原因，但目前尚无治愈方法。 SMA 是一种因存活运动神经元 1 (SMN1) 丧失而导致的神经肌肉疾病。存在几乎相同的拷贝基因 SMN2，但是，如果没有 SMN1，它就无法提供针对疾病发展的保护。值得注意的是，SMN1 和 2 都编码相同的蛋白质，然而，由于单个沉默突变，绝大多数 SMN2 转录物被选择性剪接，并且最终的编码外显子（外显子 7：54 nts）被删除。因此，这种毁灭性疾病的分子基础是选择性剪接事件，导致产生截短且不稳定的 SMN 蛋白（称为 SMN-delta7）。通过调节 SMN2 剪接模式恢复全长 SMN 表达代表了治疗干预的令人兴奋的前景。 SMA 的分子遗传学使得这种疾病特别适合促进 SMN2 全长表达的治疗策略。 1) 近99%的SMA病例是由单一基因引起的； 2) SMN2编码相同的蛋白质； 3) SMA 群体在 SMN2 方面具有显着的同质性。尚未鉴定出 SMN1 和 SMN2 纯合无效的个体 - 可能是因为这种情况将是致命的（与敲除小鼠模型一致）。因此，基本上所有 SMA 患者都至少携带一种 SMN2 基因； 4) SMN2 生成的转录本是稳定的。该提案的主要目标是开发重组腺相关病毒 (rAAV) 载体，该载体表达短 RNA，通过促进包含 SMN2 外显子 7 来刺激全长 SMN 表达。将使用逐步评估过程来识别细胞模型中最有效的 rAAV 衍生 RNA。最后，将在轻度 SMA 小鼠模型中评估最有效的 rAAV 载体，以确定 SMN2 剪接是否可以在体内改变，以及这种增加是否可以改善已明确表征的 SMA 表型。虽然该提案中描述的实验对 SMA 疗法的开发具有直接影响，但其结果可用作广泛遗传性疾病的模型，在这些疾病中，纠正剪接缺陷将恢复致病基因的功能。