RNAi as a Potential Therapy for ALS

描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种致命的神经退行性疾病，在中老年发病。ALS是一种进行性疾病，发病后，患者的肌肉逐渐减弱，最终瘫痪。瘫痪是由脊髓和运动皮层中运动神经元死亡的无情进展引起的。目前，还没有办法阻止这种运动神经元的逐渐丧失。10%的ALS病例是家族性的，并且其中，Cu，Zn超氧化物歧化酶（SODl）基因中的功能获得性突变占25%。目前已鉴定出90多种不同的SOD 1突变导致ALS，其中绝大多数是点突变。压倒性的证据表明，突变的SOD 1通过获得毒性特性导致运动神经元变性（Xu，2000）。因此，携带一个突变体和一个野生型拷贝的SOD 1的杂合子仍然发展ALS。对于由功能获得性SOD 1突变引起的ALS的理想疗法是选择性地消除突变蛋白，同时保留野生型SOD 1拷贝的表达。 序列选择性的、转录后的基因表达失活可以在多种真核生物中通过引入对应于靶基因的双链RNA来实现，这种现象称为RNA干扰（RNAi）。RNAi方法最近已经将RNAi方法扩展到培养的哺乳动物细胞。将RNAi途径中的中间体小干扰RNA（siRNA）双链体引入培养的细胞中，触发对应于siRNA序列的mRNA的降解。这增加了siRNA可用于选择性阻断突变SOD 1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：（1）在体外确定siRNA可用于选择性抑制携带单碱基突变的突变型SOD 1 mRNA表达同时允许野生型SOD 1等位基因表达的策略;（2）确定这种体外选择性是否在用靶向突变型SOD 1的siRNA转染的培养的人细胞中保持。这些实验有望开辟一个全新的研究方向，使用基于RNAi的治疗方法治疗人类疾病，如ALS和其他由功能获得性点突变引起的神经退行性疾病。

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.