RNAi as a Potential Therapy for ALS

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种中老年发病的致死性神经退行性疾病。肌萎缩侧索硬化症是一种进行性疾病——发病后，患者的肌肉逐渐减弱，最终瘫痪。瘫痪是由脊髓和运动皮层的运动神经元不断死亡引起的。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，其中，Cu， Zn超氧化物歧化酶（SODl）基因的功能突变占25%。目前已发现超过90种不同的SOD 1突变可导致ALS，其中绝大多数为点突变。大量证据表明，突变的SOD1通过获得毒性导致运动神经元变性（Xu, 2000）。因此，携带一个突变体和一个SOD 1野生型拷贝的杂合子仍然会发展为ALS。对于功能获得性SOD 1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SOD 1野生型拷贝的表达。

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.