Determinants that regulate splicing of SMN

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是婴儿死亡的主要遗传原因，但目前还没有治愈方法。SMA是一种神经肌肉疾病，由运动神经元存活1（SMN 1）缺失引起。存在一个几乎相同的拷贝基因SMN 2，然而，在没有SMN 1的情况下，它不能提供疾病发展的保护。值得注意的是，SMN 1和2编码相同的蛋白质，然而，由于单个沉默突变，绝大多数SMN 2转录物被选择性剪接，最终编码外显子（外显子7：54 nts）被删除。因此，这种毁灭性疾病的分子基础是一种选择性剪接事件，导致产生截短和不稳定的SMN蛋白（称为SMN-delta 7）。通过调节SMN 2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的分子遗传学使这种疾病特别适合促进SMN 2全长表达的治疗策略。1)几乎99%的SMA病例是由单一基因引起的; 2）SMN 2编码相同的蛋白质; 3）SMA群体与SMN 2具有显著的同源性。尚未鉴定出SMN 1和SMN 2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患者都携带至少一个SMN 2基因; 4）SMN 2产生的转录物稳定。该提案的主要目标是开发重组腺相关病毒（rAAV）载体，其表达通过促进SMN 2外显子7的纳入来促进刺激全长SMN表达的短RNA。逐步评估过程将用于在基于细胞的模型中鉴定最有效的rAAV衍生RNA。最后，将在轻度SMA小鼠模型中评价最有效的rAAV载体，以确定SMN 2剪接是否可以在体内改变，以及这种增加是否改善了充分表征的SMA表型。虽然本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疾病的模型，其中纠正剪接缺陷将恢复致病基因的功能。

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.