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

描述(申请人提供)：Prion病是一种致命的神经退行性疾病，由PrPSc蛋白错误折叠的异构体积累引起。高达15%的病例是由PrNP基因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PrP疾病，其特征是进行性共济失调、痴呆和大脑内显著的PrP淀粉样斑块沉积。我们构建了一个表达人PrP-A117V小鼠同源基因的转基因小鼠系，命名为TG(PrP-A116V)。这些小鼠复制了GSS的所有主要特征，包括进行性共济失调和小脑和海马区的PrP淀粉样沉积。最近的工作表明，RNA抑制(RNAi)是治疗神经退行性疾病的一种有前途的方法，尽管重点主要限于敲除野生型(Wt)基因。然而，与散发性Prion病患者相比，PRNP突变携带者可以在预测的疾病发作之前很久就被识别出来，这使他们成为预防性治疗的理想候选者，这些预防性治疗可以在重大神经元死亡发生之前很久就实施。由于长期降低wt PrP在健康成人中的效果尚不清楚，遗传性PrP疾病的理想治疗方法应该是选择性地敲除突变的基因，而不影响正常的等位基因。作为开发等位基因特异性RNAi作为遗传性Pron疾病治疗方法的概念证明， 我们将利用我们的TG(PrP-A116V)小鼠，在体外和体内设计并测试选择性下调PrP-A116V表达的siRNAs。初步研究将设计和测试几个针对选定PrP突变的siRNA，特别关注A116V，使用稳定表达突变或wt PrP的细胞系，以优化等位基因选择性。将对PrP-A116V具有最高选择性的siRNA序列包装在慢病毒载体中作为shRNA，并将其输送到TG(PrP-A116V/wt-PrP)杂合子小鼠的小脑，以证实神经元有效地摄取和选择性地降低PrP-A116V的表达。一旦疗效得到证实，相同的含有shRNA或对照shRNA的病毒载体将被注射到杂合小鼠的小脑中，并将使用定量和半定量方法评估其对PrP表达、疾病发病、临床症状和组织病理学特征的影响。疾病发作的显著延迟和特定疾病间隔的组织病理学特征的减少将为潜在的等位基因特异性基因敲除提供强有力的支持 人类遗传性Pron病的治疗。此外，它还将为开发其他PRNP突变的几个等位基因特异性shRNAs奠定基础，并可能开发其他遗传性疾病。

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.