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，但目前尚无治愈方法。SMA是一种由存活的运动神经元1 （SMN1）缺失引起的神经肌肉疾病。一个几乎相同的复制基因存在，SMN2，然而，它不能在缺乏SMN1的情况下提供防止疾病发展的保护。值得注意的是，SMN1和2编码相同的蛋白质，然而，由于单个沉默突变，绝大多数SMN2转录本被选择性剪接，最终编码外显子（外显子7,54 nts）被移除。因此，这种毁灭性疾病的分子基础是一种选择性剪接事件，该事件导致产生截断且不稳定的SMN蛋白（称为SMN-delta7）。通过调节SMN2剪接模式来恢复全长SMN的表达是治疗干预的一个令人兴奋的前景。SMA的分子遗传学使这种疾病特别适合于促进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；3) SMA种群在SMN2方面具有显著的同质性。尚未发现SMN1和SMN2纯合子为零的个体——可能是因为这种情况是致命的（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SMN2基因；4) SMN2生成的转录本是稳定的。本研究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SMN2外显子7的包含来促进全长SMN的表达。逐步评估过程将用于在基于细胞的模型中识别最有效的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表征的SMA表型。虽然本提案中描述的实验对SMA治疗的发展具有直接意义，但其结果可以用作广泛的遗传疾病的模型，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.