Development of a Biochemical Diagnosis for Creutzfeldt-Jakob disease

描述(由申请人提供)：人类普恩病毒疾病是一种传染性和致命性的神经退行性疾病，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。目前还没有对这些疾病进行敏感、客观和非侵入性的生化诊断，更没有一种程序来检测在症状前期潜伏着疾病的人。这是公共卫生的一个主要问题，因为已知普恩病毒疾病在人与人之间通过医学途径传播，而且由于该疾病的长期症状前阶段--可能跨越50年--无症状携带者的数量远远超过临床上受影响的个人。让情况变得更加复杂的是，导致这些疾病的感染性病原体被称为Prion，完全由一种自然产生的蛋白质的构象改变形式组成，称为PrPSc，它具有感染个人并在体内传播的独特能力，而不需要遗传物质。PrPSc不仅是神经退行性变的感染源和可能的罪魁祸首，而且是该病最好的替代标志物。然而，令人信服的证据表明，PrPSc在各种外周组织和生物液中以微量存在。这项提议的主要目标是发展一种血液和 脑脊液(CSF)检测与sCJD和vCJD相关的PrPSc我们的策略利用了我们实验室开发的两项开创性的专利技术：第一，蛋白质错折叠循环扩增(PMCA)技术，该技术能够放大样本中存在的PrPSc的数量，以检测到小至单个PrPSc分子。PMCA具有类似于聚合酶链式反应的扩增能力，使我们第一次能够检测血液中的普鲁恩 和尿液，即使在疾病的症状前期也是如此。其次，在PrP基因敲除小鼠中，针对PrP感染的脑组织匀浆产生PrPSc特异性单抗(称为PrioC)。在这个项目中，我们将优化一种结合PMCA扩增PrPSc和使用PrioC构象抗体的夹心ELISA法检测PrPSc的技术。该检测将使用动物和人体样本进行优化，以高通量检测生物液中的普恩病毒，并将根据监管机构的要求进行灵敏度和特异性验证，以期获得商业化批准。该项目产生的结果可能导致第一个被证实为CJD诊断的生化测试。有了这项经过验证的技术，Amprion将建立一个检测和根除人类Pron疾病的国际参考实验室。 与公共卫生相关：开发一种生化检测方法，用于敏感、早期和非侵入性地检测受人类普恩病毒疾病影响的患者的生物液中的普恩病毒，是减少这些传染病的进一步传播和帮助有效治疗的首要医学优先事项。该项目结合了两项开创性的技术，生产出一种具有独特可能性的检测方法，可以检测患者血液和脑脊液中的普恩病毒，甚至在感染普恩病毒的无症状携带者中也能检测到。在这个项目中，我们汇集了相关的技术和业务专业知识，确保了关键样品的可用性，并已开始与监管机构合作，以获得测试的成功验证和批准。

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. PUBLIC HEALTH RELEVANCE: Development of a biochemical assay for the sensitive, early and non-invasive detection of prions in biological fluids of patients affected by human prion diseases is a top medical priority to decrease further spreading of these infectious diseases and to help efficient treatment. This project combines two pioneer technologies to produce an assay that has the unique possibility to detect prions in blood and cerebrospinal fluid of patients and even in asymptomatic carriers of infectious prions. In this project we have put together the relevant technical and business expertise, secured the availability to key samples and already began working with regulatory authorities to get the successful validation and approval of the test.