RNAi as a Potential Therapy for ALS

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种中老年起病的致命性神经退行性疾病。肌萎缩侧索硬化症是一种进行性疾病--发病后，患者的肌肉会逐渐变弱，最终变得瘫痪。瘫痪是由脊髓和运动皮质中运动神经元死亡的持续进展引起的。目前，还没有办法阻止这种运动神经元的渐进性丧失。10%的ALS患者是家族性ALS患者，其中，铜锌超氧化物歧化酶(SODl)基因的功能获得突变占25%。目前已发现90多种不同的SOD1突变可导致肌萎缩侧索硬化症，其中绝大多数是点突变。大量证据表明，突变的SOD1通过获得一种有毒的性质而导致运动神经元退化(Xu，2000)。因此，携带一个突变型和一个野生型SOD1拷贝的杂合子仍然会发生ALS。对于由功能获得的SOD1突变引起的ALS，理想的治疗方法是选择性地消除突变蛋白，同时保留SOD1的野生型拷贝的表达。 在多种真核生物中，通过引入与靶基因相对应的双链RNA，可以实现序列选择性的、转录后的基因表达失活，这种现象被称为RNA干扰(RNAi)。RNAi方法最近已将RNAi方法扩展到培养哺乳动物细胞。RNAi途径中的中间产物小干扰RNA(SiRNA)双链导入培养细胞后，会引发与siRNA序列对应的mRNA的降解。这增加了siRNA用于选择性地阻断突变的SO1表达的可能性。为了测试这种方法的可行性，我们在R21阶段提出了(1)体外确定策略，通过该策略，siRNA可以选择性地抑制带有单个碱基突变的突变的SOD1mRNA的表达，同时允许野生型SOD1等位基因的表达；(2)确定在体外是否可以保持这种选择性在体外被针对突变的SOD1的siRNA转染的人细胞中。这些实验有望为使用基于RNAi的疗法治疗人类疾病(如ALS和其他神经退行性疾病)开辟一个全新的研究方向--这些疾病是由功能点突变引起的。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a middle to old age onset. ALS is progressive disease-after onset, patients' muscles progressively weaken and eventually become paralyzed. Paralysis is caused by the relentless progression of motoneuron death in the spinal cord and, motor cortex. At present, there is no means to stop this progressive loss of motoneurons. Ten percent of ALS cases are familial, and of those, gain-of function mutations in the Cu, Zn superoxide dismutase (SODl) gene account for 25%. More than 90 different SOD 1 mutations have been identified that cause ALS, the vast majority of which are point mutations. Overwhelming evidence has demonstrated that mutant SOD1 causes motorneuron degeneration by a gain of a toxic property (Xu, 2000). Thus, heterozygotes bearing one mutant and one wild-type copy of SOD 1 nevertheless develop ALS. The ideal therapy for ALS caused by a gain-of function SOD 1 mutation would be to selectively eliminate the mutant protein while retaining expression of the wild-type copy of SOD 1. Sequence-selective, post-transcriptional inactivation of gene expression can be achieved in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the targeted gene, a phenomenon termed RNA interference (RNAi). The RNAi method has recently been extended the RNAi methodology to cultured mammalian cells. The introduction into cultured cells of an intermediate in the RNAi pathway, small interfering RNA (siRNA) duplexes, triggers the degradation of mRNA corresponding to the siRNA sequence. This raises the possibility that siRNA may be used to selectively block the expression of mutant SOD 1. To test the feasibility of this approach, we propose in the R21 phase (1) to determine in vitro the strategy whereby siRNA can be used to selectively inhibit the expression of a mutant SOD1 mRNA bearing a single base mutation while permitting expression of the wild-type SOD 1 allele; (2) to determine whether this in vitro selectivity is maintained in cultured human cells transfected with siRNA targeting mutant SOD1. These experiments promise to open up an entirely new direction of study for using RNAibased therapeutics to treat human diseases such as ALS and other neurodegenerative disorders-caused by gain-of-function point mutations.