Cyclic Amplification of Prion Protein Misfolding

描述（由申请人提供）：朊病毒疾病是一组影响人类和动物的感染性神经退行性疾病。虽然是罕见的疾病，但最近爆发的牛海绵状脑病和慢性消耗性疾病以及该疾病从牛向人类的传播引起了人们对克雅氏病可能流行的极大关注。传染因子的空前性质、其传播机制和似乎控制朊病毒传播的物种屏障等诸多不确定性加剧了这一问题。最被接受的假设提出，传染因子（称为朊病毒）完全由正常蛋白质的错误折叠版本组成，不含任何核酸。根据这一假设，疾病是通过将与疾病相关的异构体（称为PrPres）的错误折叠繁殖到正常宿主蛋白（称为PrPc）而传播的，后者转化为病理形式。我们最近描述了一种在体外诱导PrPc转化为prPres的程序，从微量的脑prPres开始。这一过程被称为蛋白质错误折叠循环扩增（PMCA），它模拟了朊病毒在体内的复制过程，但速度加快，导致PrPres初始量呈指数级扩增。该项目的主要目标是利用PMCA技术，尝试开发一种高度敏感和无创的朊病毒疾病诊断方法，并研究与感染因子性质、朊病毒转化和物种间传播相关的其他因素有关的各个方面。在具体目标1中，我们将研究体外生成的prPres的感染性和结构特性，目的是尝试在试管中繁殖和产生感染性。这个实验被广泛认为是朊病毒假说的最终证据。在特定目标2中，我们将尝试确定在体内朊病毒复制中似乎起主要作用的细胞蛋白因子。具体目的3提出研究物种屏障现象及PrP多态性对朊病毒体外复制效率的影响。在具体目标4中，我们将开发一种高灵敏度的诊断测试，通过将血液中存在的微量病理蛋白扩增到可重复检测的水平，来检测实验动物、牛和人类血液中的PrPres。因此，本项目提供了一个平衡的结合，基础科学研究旨在了解朊病毒领域最相关的科学问题，应用研究解决与这些疾病相关的主要实际问题，即缺乏高灵敏度的症状前血液诊断来限制这些不治之症的传播。

DESCRIPTION (provided by applicant): Prion diseases are a group of infectious neurodegenerative disorders affecting humans and animals. Although rare diseases, the recent outbreak of Bovine Spongiform Encephalopathy and Chronic Wasting disease and the transmission of the disease from cattle to humans have risen a great concern about a possible epidemic of Creutzfeldt-Jakob disease. This problem is aggravated by many uncertainties surrounding the unprecedented nature of the infectious agent, its mechanism of propagation and the species barrier that seems to control prion transmission. The most accepted hypothesis proposes that the infectious agent (termed prion) is composed exclusively by a misfolded version of a normal protein and does not contain any nucleic acid. According to this hypothesis, the disease is transmitted by propagation of the misfolding from the disease associated isoform (termed PrPres) to the normal host protein (termed PrPc), which become converted into the pathological form. We have recently described a procedure to induce the conversion of PrPc into prPres in vitro starting with minute quantities of brain PrPres. This procedure, named Protein Misfolding Cyclic Amplification (PMCA) mimics the process of prion replication in vivo, but at an accelerated speed resulting in an exponential amplification of the initial amount of PrPres. The major goal of this project is to take advantage of the PMCA technology to attempt developing a highly sensitive and noninvasive diagnosis of prion diseases as well as to study diverse aspects related to the nature of the infection agent, other factors involved in prion conversion and the transmission between species. In specific aim 1 we will study the infectious and structural properties of prPres generated in vitro with the objective to attempt multiplying and producing infectivity in the test tube. This experiment is widely considered as the final pending proof for the prion hypothesis. In specific aim 2 we will attempt to identify cellular protein factors that seem to play a major role in prion replication in vivo. Specific aim 3 proposes to study the species barrier phenomenon and the influence of PrP polymorphisms in the efficiency of prion replication in vitro. In specific aim 4 we will develop a highly-sensitive diagnostic test to detect PrPres in blood of experimental animals, cattle and humans, based on amplifying minute quantities of the pathological protein present in blood to a level that enable reproducible detection. Therefore, this project offers a balanced combination between basic science studies aimed to understand the most relevant scientific problems in the prion field and applied studies to resolve the main practical problem associated to these diseases, which is the lack of a highly-sensitive pre-symptomatic blood diagnosis to limit the spreading of these incurable illnesses.