Characterization of de novo generation of infectious prions

描述（由申请人提供）：传染性海绵状脑病（tse）是一组影响人类和动物的神经退行性疾病。目前还没有针对这些致命疾病的有效治疗方法。tse可以有多种起源，包括遗传性，散发性（假定自发）和传染性。人类（散发性CJD）和其他物种（即美国牛的牛海绵状脑病（BSE）和新西兰羊的Nor98（非典型痒病））中自发发生的朊病毒疾病病例的发生，提供了这种罕见的自发现象可能自然发生的证据。在实验野生型啮齿动物模型中，没有报道自发性（从头开始）朊病毒疾病的病例。与tse相关的感染因子，朊病毒，似乎是由一种天然朊病毒蛋白（PrPC）的构象修饰版本（PrPSc）的蛋白质组成的。该疾病通过宿主PrPC转化为PrPSc而传播，PrPSc是由感染因子中含有的少量PrPSc诱导的。我们最近描述了一种新技术，蛋白质错误折叠循环扩增（PMCA），它在体外模拟了这一过程，但动力学大大加快，使PrPSc在试管中扩增。该技术已被用于有效地扩增来自小鼠、仓鼠、田鼠、鹿、牛、羊和人类的多种朊病毒株。此外，体外生成的朊病毒具有关键的朊病毒特征，即在体内具有传染性并保持其朊病毒株特异性。然而，以前不可能从健康材料开始产生新生感染性。换句话说，据推测，朊病毒的自发产生仅限于少数物种体内罕见的现象。在体外模拟感染性的自发产生是朊病毒领域目前面临的最重要挑战之一。在初步研究中，我们从非传染性的大脑开始，从十种不同的啮齿动物（包括野生动物）中产生了传染性朊病毒。该项目的主要目标是利用几种体外和体内方法重新生成和表征感染性朊病毒菌株的多样性，并通过区分最小的预先存在的种子PrPSc扩增与“体外自发”生成来揭示所涉及的内在机制。最后，我们将把这项研究扩展到其他物种，以模仿自然界中可能已经发生的事情。公共卫生相关性：朊病毒疾病是一组影响人类和动物的致命和传染性神经退行性疾病，目前尚无治疗方法。传染因子的性质是激烈争论的焦点。朊病毒在体内传播的机制正受到密切的研究。本病最常见的起源是一种不熟悉的自发现象。不幸的是，在实验模型中没有报道自发发生（新生）朊病毒疾病的病例，这使得对这一有趣事件的研究更加困难。在这个提议中，我们展示了强有力的证据，使用啮齿动物模型，这种现象可以在体外模拟。这一发现为理解朊病毒疾病和设计迄今为止难以想象的新疗法开辟了巨大的可能性。

DESCRIPTION (provided by applicant): Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative disorders affecting both humans and animals. There is no available treatment or therapy for these fatal diseases. TSEs can have diverse origins, including hereditary, sporadic (putatively spontaneous), and infectious. The occurrence of spontaneous cases of prion diseases in humans (sporadic CJD) and in other species, i.e. Bovine spongiform encephalopathy (BSE) in US cattle and Nor98 (atypical scrapie) in New Zealand sheep, provide evidence that this infrequent spontaneous phenomenon may occur naturally. There are no reported cases of spontaneously- occurring (de novo) prion disease in experimental wild-type rodent models. The infectious agent associated with TSEs, the prion, appears to be composed uniquely of a protein which is a conformationally-modified version (PrPSc) of a natural prion protein (PrPC). The disease is transmitted by the conversion of host PrPC into PrPSc induced by small quantities of PrPSc contained in the infectious agent. We have recently described a novel technique, Protein Misfolding Cyclic Amplification (PMCA), which mimics this process in vitro, but with greatly accelerated kinetics, enabling PrPSc amplification in the test tube. This technique has been used to efficiently amplify a variety of prion strains from mice, hamsters, bank voles, deer, cattle, sheep, and humans. In addition, the in vitro generated prions possess key prion features, i.e., they are infectious in vivo and maintain their prion strain specificity. However, it has not been previously possible to generate infectivity de novo, starting with healthy material. In other words, the spontaneous generation of prions has been hypothesized to be restricted to an infrequent in vivo phenomenon in only a few species. To mimic spontaneous generation of infectivity in vitro is one of the most important challenges that the prion field currently faces. In preliminary studies, we have generated infectious prions from ten different rodent species (including wild animals) starting with non-infectious brains. The major goal of this project is the de novo generation and characterization of a diversity of infectious prion strains using several in vitro and in vivo approaches as well as revealing the intrinsic mechanisms involved by distinguishing amplification of minimal preexisting seed PrPSc from "in vitro spontaneous" generation. Finally, we will extend this study to other species to mimic what may already be happening in nature. PUBLIC HEALTH RELEVANCE: Prion diseases are a group of fatal and infectious neurodegenerative disorders affecting humans and animals for which no therapy is available. The nature of the infectious agent is at the center of passionate controversy. The mechanism by prion agent propagates the disease in vivo is being subject to intense investigation. The most common origin of the disease is an unfamiliar spontaneous phenomenon. Unfortunately, there are no reported cases of spontaneously-occurring (de novo) prion diseases in experimental models making the study of this intriguing occurrence more difficult. In this proposal, we show strong evidence using rodent models where this phenomenon can be mimicked in vitro. This find opens enormous possibilities for understanding the prion diseases and for designing new therapies inconceivable until now.