Development of a Biochemical Diagnosis for Creutzfeldt-Jakob disease

描述（由申请人提供）：人朊病毒病是一种传染性的、总是致命的神经退行性疾病，包括最常见的散发性克雅氏病（sCJD）和变异型克雅氏病（vCJD），后者与食用受牛海绵状脑病感染的牛肉有关。目前，对这些疾病还没有敏感、客观和非侵入性的生化诊断，更没有在症状前阶段检测潜伏期的程序。这是公共卫生的一个主要问题，因为已知朊病毒疾病在人与人之间通过医源性传播，而且由于这种疾病的症状前阶段很长可能长达50年无症状携带者的数量远远超过临床受影响的个体。使情况更加复杂的是，引起这些疾病的感染因子，称为朊病毒，完全由一种天然存在的蛋白质的构象改变形式组成，称为PrPSc，它具有感染个体并在体内繁殖的独特能力，而不需要遗传物质。PrPSc不仅是神经变性的感染因子和可能的罪魁祸首，而且是疾病的最佳替代标记物。挑战在于其数量仅在疾病晚期的大脑中较高，然而，令人信服的证据表明PrPSc以微量存在于各种外周组织和生物液体中。这项提案的主要目标是开发一种血液- 脑脊液（CSF）为基础的检测试验与sCJD和vCJD相关的PrPSc。我们的策略利用了我们实验室开发的两种开创性的专有技术：首先是蛋白质错误折叠循环扩增（PMCA）技术，该技术能够扩增样品中存在的PrPSc的量，以检测到少至单个分子的PrPSc。PMCA具有与PCR相似的扩增能力，使我们能够首次检测血液中的朊病毒 甚至在疾病的症状前阶段。第二，在PrP基因敲除小鼠中，针对朊病毒感染的脑匀浆产生PrPSc特异性单克隆抗体（称为PrioC）。在这个项目中，我们将优化的技术相结合的PMCA扩增PrPSc的检测通过夹心ELISA使用PrioC构象抗体。将使用动物和人类样本对该检测方法进行优化，以高通量检测生物体液中的朊病毒，并将根据监管机构的要求对灵敏度和特异性进行验证，以获得商业化批准。在这个项目中产生的结果可能导致第一个生物化学测试验证CJD的诊断。Amprion公司将利用这一经过验证的技术建立一个检测和根除人类朊病毒疾病的国际参考实验室。

DESCRIPTION (provided by applicant): Human prion diseases are infectious and invariably fatal forms of neurodegenerative diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), the most common form, and variant CJD (vCJD) which is associated to consumption of cattle meat infected by bovine spongiform encephalopathy. Currently there is not sensitive, objective and non-invasive biochemical diagnosis for these diseases, and even less a procedure to detect people incubating the disease in the pre-symptomatic period. This is a major problem for public health, because prion diseases are known to transmit iatrogenically between human-to-human and because due to the long pre-symptomatic stage of the disease-which may span five decades-the asymptomatic carriers far outnumber the clinically affected individuals. To make the situation even more complicated, the infectious agent responsible for these diseases, termed prion, is composed exclusively of a conformationally altered form of a naturally occurring protein, named PrPSc, which has the unique ability to infect individuals and propagate in the body without the need for genetic material. PrPSc is not only the infectious agent and the likely culprit of neurodegeneration, but also the best surrogate marker for the disease. The challenge is that its quantity is high only in the brain at late stages of the disease However, compelling evidences indicate that PrPSc is present in minute amounts in various peripheral tissues and biological fluids. The main goal of this proposal is to develop a blood- and cerebrospinal fluid (CSF)-based detection assay for PrPSc associated with sCJD and vCJD. Our strategy utilizes two pioneering proprietary technologies developed in our lab: First the protein misfolding cyclic amplification (PMCA) technique which enables to amplify the amount of PrPSc present in the sample to detect as little as a single molecule of PrPSc. PMCA, has a similar power of amplification as PCR and allowed us to detect, for the first time, prions in blood and urine, even at the pre-symptomatic stages of the disease. Second, PrPSc-specific monoclonal antibodies (called PrioC) raised against prion-infected brain homogenates in PrP knock out mice. In this project we will optimize a technology combining PMCA amplification of PrPSc with detection by sandwich ELISA using the PrioC conformational antibodies. The assay will be optimized using animal and human samples for high throughput detection of prions in biological fluids, and will be validated for sensitivity and specificity according to the requiremets of the regulatory authorities with the aim to obtain approval for commercialization. The results generated in this project may lead to the first biochemical test validated for the diagnosis of CJD. With this validated technology, Amprion will establish an International Reference Laboratory for the Detection and Eradication of human prion diseases.