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）的错误折叠同种型积累引起。高达15%的病例是由朊病毒蛋白基因（PRNP）的常染色体显性等位基因突变引起的。PRNP的Ala 117 Val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、痴呆和脑内明显的PrP淀粉样斑块沉积。我们构建了一个转基因小鼠系，表达人PrP-A117 V的小鼠同源物，命名为Tg（PrP-A116 V）。这些小鼠重现了GSS的所有主要特征，包括小脑和海马内的进行性共济失调和PrP淀粉样蛋白沉积。最近的工作表明RNA抑制（RNAi）是神经退行性疾病的一种有前途的治疗方法，尽管焦点主要局限于敲除野生型（wt）基因。然而，与受散发性朊病毒疾病影响的个体相反，PRNP突变的携带者可以在预测的疾病发作之前被识别，使其成为预防性治疗的理想候选者，可以在发生显著的神经元死亡之前很久就给予预防性治疗。由于长期减少野生型朊蛋白在健康成人中的作用尚不清楚，因此遗传性朊病毒病的理想疗法应该是选择性地敲除突变基因而不影响正常等位基因。作为开发等位基因特异性RNAi作为遗传性朊病毒疾病治疗的概念证明， 我们将设计和测试siRNA，选择性地敲低PrP-A116 V表达在体外和体内，使用我们的Tg（PrP-A116 V）小鼠。最初的研究将设计和测试几种siRNA对选定的PrP突变有活性，特别关注A116 V，使用稳定表达突变体或野生型PrP的细胞系，以优化等位基因选择性。将对PrP-A116 V具有最大选择性的siRNA序列作为shRNA包装在慢病毒载体中，并递送至Tg（PrP-A116 V/wt-PrP）杂合小鼠的小脑，以确认有效的神经元摄取和PrP-A116 V表达的选择性降低。一旦确认疗效，将含有shRNA或对照shRNA的相同病毒载体注射到杂合小鼠的小脑中，并使用定量和半定量测量评估对PrP表达、疾病发作、临床症状和疾病的组织病理学特征的最终影响。疾病发作的显著延迟和在特定疾病间隔的组织病理学特征的减少将为等位基因特异性敲除作为潜在的免疫抑制剂提供强有力的支持。 治疗人类遗传性朊病毒病此外，它将为开发用于其他PRNP突变和潜在的其他遗传疾病的几种等位基因特异性shRNA奠定基础。

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.