Allele-Specific RNAi to treat Genetic Prion Disease - Resubmission 01

描述（由申请人提供）：朊病毒疾病是致命的神经退行性疾病，由朊病毒蛋白（PrPSc）的错误折叠异构体积累引起。高达15%的病例是由朊蛋白基因（PRNP）的常染色体显性等位基因突变引起的。PRNP的Ala117Val突变与Gerstmann-Strussler-Scheinker病（GSS）有关，GSS是一种遗传性朊病毒疾病，其特征是进行性共济失调、痴呆和大脑内突出的PrP淀粉样斑块沉积。我们构建了表达人类PrP-A117V的小鼠同源基因的转基因小鼠系，命名为Tg（PrP-A116V）。这些小鼠再现了GSS的所有主要特征，包括进行性共济失调和小脑和海马内的PrP淀粉样蛋白沉积。最近的研究表明，RNA抑制（RNAi）是一种很有前途的治疗神经退行性疾病的方法，尽管重点主要局限于敲低野生型（wt）基因。然而，与受散发性朊病毒疾病影响的个体相比，PRNP突变的携带者可以在预测疾病发作之前被识别出来，这使得它们成为预防性治疗的理想候选者，可以在显著的神经元死亡发生之前很久就进行治疗。由于长期降低wt PrP对健康成人的影响尚不清楚，因此遗传性朊病毒疾病的理想治疗方法应该是在不影响正常等位基因的情况下选择性地敲除突变基因。作为开发等位基因特异性RNAi作为遗传朊病毒疾病治疗方法的概念证明，

DESCRIPTION (provided by applicant): Prion diseases are fatal neurodegenerative diseases that result from the accumulation of a misfolded isoform of the prion protein (PrPSc). Up to 15% of cases result from an autosomal dominant allelic mutation of the prion protein gene (PRNP). The Ala117Val mutation of PRNP is linked to Gerstmann-Strussler-Scheinker disease (GSS), a genetic prion disease characterized by progressive ataxia, dementia, and prominent PrP amyloid plaque deposits within the brain. We constructed a transgenic mouse line that expresses the mouse homolog of human PrP-A117V, designated Tg(PrP-A116V). These mice reproduce all the major features of GSS, including progressive ataxia and PrP amyloid deposits within the cerebellum and hippocampus. Recent work suggests RNA inhibition (RNAi) is a promising therapeutic approach for neurodegenerative disease, although the focus has been largely confined to knock down of wild type (wt) genes. However, in contrast to individuals affected by sporadic prion disease, carriers of PRNP mutations can be identified far in advance of the predicted onset of disease, making them ideal candidates for preventative therapies that can be administered long before significant neuronal death has occurred. Since the effect of long-term reduction of wt PrP in a healthy adult is not known, the ideal therapy for genetic prion disease should act to selectively knock down the mutated gene without affecting the normal allele. As proof of concept to develop allele-specific RNAi as a therapy for genetic prion disease, we will design and test siRNAs that selectively knock down PrP-A116V expression in vitro and in vivo, using our Tg(PrP-A116V) mice. Initial studies will design and test several siRNAs active against selected PrP mutations, with a special focus on A116V, using cell lines stably expressing mutant or wt PrP, to optimize allele selectivity. The siRNA sequence with the greatest selectivity for PrP-A116V will be packaged in a lentiviral vector as shRNA and delivered to the cerebellum in Tg(PrP- A116V/wt-PrP) heterozygous mice, to confirm efficient neuronal uptake and selective reduction of PrP-A116V expression. Once efficacy is confirmed, the same viral vector containing shRNA or control shRNA, will be injected into cerebellum of heterozygous mice and the resultant effect on PrP expression, disease onset, clinical symptoms, and histopathologic features of disease, will be assessed using quantitative and semi- quantitative measures. A significant delay in disease onset and a reduction of histopathologic features at specific disease intervals will provide strong support for allele-specific knockdown as a potential therapy in human genetic prion disease. Moreover, it will lay the foundation for the development of several allele-specific shRNAs for other PRNP mutations and, potentially, other genetic diseases.