Development of Prion Protein-Lowering Divalent siRNA Therapy for Prion Disease

降低朊病毒蛋白的二价siRNA治疗朊病毒病的研究进展 项目总结/摘要 朊病毒病是一种致命的、不可治愈的、快速进展的神经退行性疾病，由单一蛋白质引起， 朊病毒蛋白或PrP。在疾病中，PrP转化为致病性的“朊病毒”构象， 破坏其他PrP分子的构象来破坏大脑这个过程严格依赖于PrP的表达， 而PrP的减少，无论是遗传性的还是非遗传性的， 动物关键的是，即使持续50%的PrP敲低，所有动物最终都死于致命的 疾病PrP基因敲除模型表明，PrP的完全消融对朊病毒疾病具有完全保护作用， 即使是>90%的瞬时钳位抑制也可以使大脑清除朊病毒，导致一次性 “治愈”到目前为止，还没有药物形式可用于a）能够降低单一病原体的水平， 蛋白质，B）在整个大脑中有活性，并且实际上可递送到整个大脑，以及c）足够有效以测试上述 假说然而，最近描述的二价siRNA（di-siRNA）现在提供了第一个 这种方式。Di-siRNA是化学修饰的连接的siRNA双链体，能够到达整个啮齿动物。 和非人灵长类动物的大脑，并降低特定的致病水平， 亨廷顿蛋白等蛋白质含量> 99%。针对小鼠和人两者初步预筛选的Di-siRNA PrP RNA将由我们的合作者Anastasia Khvorova博士在麻省大学合成和提供。在R61 在奖励阶段，我们将：1）PrP靶向di-siRNA分子在细胞中的生物学效力，通过表征 PrP RNA和蛋白质敲低，以及在小鼠和人细胞中的剂量反应性。（二） 通过评估体内效力，表征未感染小鼠中的主要di-siRNA体内PK/PD特性， 耐受性、RNA和蛋白质减少之间的相关性、剂量反应性、脑分布和时间 以在野生型和“人源化”（表达人PrP）转基因小鼠中洗脱。3)表征时间， 病理学和终末疾病的人源化小鼠，通过感染一种新的转基因人源化小鼠， 模型与人类朊病毒和跟踪时间的生物标志物的变化，症状和终点。R33奖 阶段，我们将在感染小鼠和人朊病毒的小鼠中进行存活实验，以评估 多种临床相关治疗模式。1)免疫启动的慢性 剂量。我们将通过启动慢性预防性治疗， 在朊病毒感染前给予di-siRNA，并评估对存活率的影响; 2）在感染前给予单次剂量后的存活率。 病理前的时间点。我们将评估在朊病毒感染早期单次注射di-siRNA的影响 对大脑朊病毒滴度和存活率的影响3)在症状时间点单次给药后的生存期。我们 将通过在晚期疾病中递送单次推注剂量的di-siRNA来模拟肿瘤阶段治疗， 时间点，并将评估对朊病毒滴度和存活率的影响。总之，这些目标将决定PrP- 降低di-siRNA应该被推进到临床用于预防和/或治疗人朊病毒疾病。

Development of prion protein-lowering divalent siRNA therapy for prion disease Project Summary / Abstract Prion disease is a fatal, untreatable, rapidly progressive neurodegenerative disease caused by a single protein, the prion protein or PrP. In disease, PrP converts into a pathogenic “prion” conformation that spreads across the brain by conformationally corrupting other PrP molecules. This process is strictly reliant on PrP expression, and reduction of PrP, either genetically or pharmacologically, dose-dependently prion delays disease in animals. Critically, even with sustained 50% knockdown of PrP, all animals eventually succumb to fatal disease. PrP knockout models indicate that total ablation of PrP is fully protective against prion disease, and that even transient clamp suppression of >90% could allow the brain to clear prions, resulting in a one-time “cure.” To date, no drug modality has been available that is a) capable of reducing levels of a single pathogenic protein, b) active in, and realistically deliverable to, the whole brain, and c) potent enough to test the above hypothesis pharmacologically. However, recently described divalent si-RNAs (di-siRNAs) now offer the first such modality. Di-siRNAs are chemically modified linked siRNA duplexes capable of reaching the whole rodent and non-human primate brain following delivery into CSF, and reducing levels of specific disease-causing proteins such as Huntingtin by >99%. Di-siRNAs preliminarily pre-screened against both mouse and human PrP RNA will be synthesized and provided by our collaborator Dr. Anastasia Khvorova at UMass. In the R61 award phase, we will: 1) Validate potency of PrP-targeting di-siRNA molecules in cells, by characterizing PrP RNA and protein knockdown, and dose-responsiveness of effect, in mouse and human cells. 2) Characterize leading di-siRNA in vivo PK/PD properties in uninfected mice, by assessing in vivo potency, tolerability, correlation between RNA and protein reduction, dose-responsiveness, brain distribution, and time to washout in wild-type and “humanized” (human-PrP expressing) transgenic mice. 3) Characterize time to pathology and terminal disease in humanized mice, by infecting a novel transgenic humanized mouse model with human prions and tracking time to biomarker changes, symptoms and endpoint. In the R33 award phase, we will perform survival experiments in mice infected with both mouse and human prions to assess multiple clinically relevant treatment paradigms. 1) Survival following prophylactically initiated chronic dosing. We will model preventive treatment of individuals at risk for genetic prion disease by initiating chronic di-siRNA dosing before prion infection, and assess impact on survival; 2) Survival following a single dose at a pre-pathological timepoint. We will assess the impact of a single bolus of di-siRNA early in prion infection on both brain prion titer and survival. 3) Survival following a single dose at a symptomatic timepoint. We will model symptomatic-stage treatment by delivering a single bolus dose of di-siRNA at an advanced disease timepoint, and will assess impact on prion titer and survival. Together, these aims will determine whether PrP- lowering di-siRNAs should be advanced to the clinic for prevention and/or treatment of human prion disease.