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

等位基因特异性 RNAi 治疗遗传性朊病毒病 - 重新提交 01

• 批准号: 8445972
• 负责人: JAMES A MASTRIANNI
• 金额: $ 23.7万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445972
• 关键词: Adult; Affect; Alleles; Animals; Ataxia; Brain; Cell Line; Cerebellum; Cessation of life; Characteristics; Clinical; Clinical Pathology; Codon Nucleotides; Cultured Cells; Dementia; Deposition; Development; Digestion; Disease; Disease Progression; Foundations; Gene Mutation; Genes; Genetic; Gerstmann-Straussler-Scheinker Disease; Goals; Hereditary Disease; Hippocampus (Brain); Histopathology; Homologous Gene; Human; Human Genetics; In Vitro; Individual; Lentivirus Vector; Life; Link; Long-Term Effects; Longitudinal Studies; Measures; Molecular Conformation; Monitor; Mus; Mutate; Mutation; Neurodegenerative Disorders; Neurons; Onset of illness; Other Genetics; Pathology; Patients; Plasmids; Positioning Attribute; PrP; PrP amyloid; PrPSc Proteins; Prion Diseases; Prions; Property; Protein Isoforms; RNA; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Risk; Rodent; Senile Plaques; Site; Small Interfering RNA; Subfamily lentivirinae; Susceptibility Gene; Symptoms; Techniques; Technology; Testing; Therapeutic; Transgenic Mice; Viral; Viral Vector; Work; base; design; gene therapy; human PrP; in vivo; knock-down; mouse model; mutant; nervous system disorder; prevent; small hairpin RNA; transduction efficiency; uptake; wild-type PrP

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. PUBLIC HEALTH RELEVANCE: Fifteen percent of prion disease cases are linked to a mutation of the prion protein gene (PRNP) that predisposes the prion protein to acquire a misfolded conformation, aggregate, and ultimately result in neurological disease. We have generated a transgenic mouse that displays all the characteristic clinical and pathological features associated with one form of genetic prion disease, known as Gerstmann-Strussler-Scheinker disease (GSS). In an effort to develop a therapy for these untreatable diseases, we will determine whether a technology known as RNA inhibition can be developed to selectively inhibit expression of only the mutated PRNP gene, while sparing the normal PRNP gene, and whether this technique can prevent or significantly mitigate disease in our transgenic mice.

描述（由申请人提供）：prion病是致命的神经退行性疾病，这些疾病是由于prion蛋白（PRPSC）的错误折叠同工型的积累而导致的。多达15％的病例是由普染色体蛋白基因（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，这是一种以进行性共济失调，痴呆症和著名的PRP淀粉样蛋白斑块沉积物为特征的遗传病毒疾病。我们构建了一条转基因小鼠系，该系列表示人类PRP-A117V的小鼠同源物，指定为TG（PRP-A116V）。这些小鼠重现了GSS的所有主要特征，包括在小脑和海马体内进行性共济失调和PRP淀粉样蛋白沉积物。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，尽管重点很大程度上被限制在击倒野生型（WT）基因。然而，与受零星pr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就可以鉴定出来，这使其成为预防性疗法的理想候选者，可以在发生重大神经元死亡之前进行。由于尚不清楚WT PRP长期降低WT PRP的影响，因此遗传病毒疾病的理想疗法应起到有选择地击倒突变基因而不会影响正常等位基因的理想疗法。作为开发等位基因特异性RNAi作为遗传prion疾病的疗法的概念证明， 我们将使用我们的TG（PRP-A116V）小鼠在体外和体内选择性地设计和测试siRNA。初步研究将使用稳定表达突变体或WT PRP的细胞系设计和测试针对选定的PRP突变的几个siRNA活跃的siRNA，以特别关注A116V，以优化等位基因选择性。对PRP-A116V具有最大选择性的siRNA序列将在慢病毒载体中包装为shRNA，并将其递送到TG（PRP-A116V/WT-PRP）杂合小鼠中的小脑中，以确认有效的神经元摄取和选择性降低PRP-A116V表达。一旦确认疗效，将使用定量和半定量测量方法评估相同的病毒载体或对照shRNA的相同病毒载体，将注射到杂合小鼠的小脑中，并对PRP表达，疾病发作，临床症状和疾病的组织病理学特征产生影响。疾病发作的显着延迟和在特定疾病间隔下的组织病理学特征的减少将为等位基因特异性敲低提供强烈的支持 人类遗传病毒疾病的治疗。此外，它将为用于其他PRNP突变以及其他遗传疾病的几个等位基因特异性shRNA的开发奠定基础。 公共卫生相关性：15％的病毒疾病病例与prion蛋白基因（PRNP）的突变有关，这些突变使prion蛋白易于获得错误折叠的构象，骨料，并最终导致神经系统疾病。我们已经产生了一种转基因小鼠，该小鼠显示了与一种形式的遗传病毒疾病相关的所有特征性临床和病理特征，称为Gerstmann-Strussler-Scheinker病（GSS）。为了为这些不可治疗的疾病开发一种治疗方法，我们将确定一种称为RNA抑制的技术是否可以选择性地抑制突变的PRNP基因的表达，同时保留正常的PRNP基因，以及该技术是否可以预防我们的转基因小鼠中的疾病或显着减轻疾病。