Transgenetic studies of prion disease in cervids

描述（由申请人提供）：朊病毒在动物和人类中引起致命的、不可治愈的传染性神经退行性疾病。通过致病构象PrPSc破坏细胞朊病毒蛋白（PrPC）进行传播。朊病毒与基于核酸的病原体共享传播菌株信息的能力。我们的首要目标是了解朊病毒株变异的生物学和物理化学基础，以及在缺乏核酸的情况下菌株如何突变和获得适应性，这些现象与其潜在的发病机制和人畜共患潜力有关。我们的重点仍然主要是慢性消耗病（CWD），一个新兴的，高度传染性朊病毒疾病的圈养和野生鹿科动物。不同的慢性消耗病菌株的发现，以及鹿骨骼肌和麋鹿鹿茸中朊病毒的发现，对人类健康有着明确的意义。目前难以区分的PrPSc构象和不稳定的转换的CWD菌株是一致的，它们的分离相对较低的能量障碍。鹿和麋鹿的品系稳定性也不同。我们的总体假设是，主机PrPC的一级结构的影响组合的药物首选PrPSc构象，动力学选择朊病毒传播过程中。本研究的目的和方法包括：1.用鹿或麋鹿的Prnp基因替换小鼠的Prnp基因，制备基因靶向（Gt）小鼠。这将使我们能够探索的假设，残留物226，鹿和麋鹿PrP一级结构的唯一差异，驻留在一个关键的域影响PrP转换和应变稳定性。第二章：GT小鼠提供了一个资源，以精确地表征CWD菌株的属性，并评估PrP的一级结构对菌株稳定性的贡献。在这一目标下，还将扩大对包含鹿科动物残基226的结构域的转基因研究。第三章：我们将使用Gt小鼠和现有的转基因模型来评估CWD菌株在疾病期间是否干扰或协同作用，并探索菌株的外周发病机制，这可能是CWD无与伦比的传染性传播的因素。4：我们将开发创新的细胞培养资源，用于定量CWD朊病毒和表征菌株特性。我们假设朊病毒菌株受到选择压力，并且我们将评估朊病毒复制修饰剂对鹿PrPSc生物学和生化特性改变的菌株变体形成的影响。第五章：最后，我们假设CWD菌株的不稳定性，以及它们目前不可辨别的生化特性，反映了构成这些菌株的PrPSc之间的热力学相似性。我们将检查纯化菌株是否与蛋白酶敏感的PrPSc不同，并评估构象依赖性免疫测定，傅里叶变换红外光谱，发光共轭聚合物和蛋白质错误折叠循环扩增方法的灵敏度菌株歧视。我们预计，我们的工作将继续对这一领域产生持续、强大的影响，并可能涉及蛋白质错误折叠的相关疾病，以及澄清慢性消耗病生物学的许多剩余谜团。

DESCRIPTION (provided by applicant): Prions cause fatal, incurable, infectious neurodegenerative diseases in animals and humans. Transmission occurs by corruption of cellular prion protein (PrPC) by the pathogenic conformation, PrPSc. Prions share with nucleic acid based pathogens, the ability to propagate strain information. Our overarching goal is to understand the biological and physico-chemical basis of prion strain variation, and how strains mutate and acquire fitness in the absence of nucleic acids, phenomena that are linked to their underlying pathogenesis and zoonotic potential. Our focus remains largely on chronic wasting disease (CWD), a burgeoning, and highly contagious prion disorder of captive and wild cervids. The discoveries of distinct CWD strains, and of prions in deer skeletal muscle and elk antler velvet, have unmistakable relevance to human health. The currently indistinguishable PrPSc conformations and unstable transitions of CWD strains are consistent with their separation by relatively low energy barriers. Strain stability also differs between deer and elk. Our overarching hypothesis is that host PrPC primary structure influences the portfolio of thermodynamically preferred PrPSc conformations that are kinetically selected during prion propagation. The following Aims comprise our research design and methods: 1: To produce gene targeted (Gt) mice, in which the mouse Prnp coding sequence is substituted by that of deer or elk. This will enable us to explore the hypothesis that residue 226, the sole difference in deer and elk PrP primary structures, resides within a critical domain influencing PrP conversion and strain stability. 2: Gt mice provide a resource to precisely characterize the properties of CWD strains, and to assess the contributions of PrP primary structure on strain stability. Transgenetic studies of the domain encompassing cervid residue 226 will also be extended in this Aim. 3: We will use Gt mice and existing transgenic models to assess whether CWD strains interfere or synergize during disease, and to explore peripheral pathogenesis of strains, which are likely factors in the unparalleled contagious transmission of CWD. 4: We will develop innovative cell culture resources for quantifying CWD prions and characterizing strain properties. We hypothesize that prion strains are subject to selective pressure, and we will assess the effects of modifiers of prion replication on the formation of strain variants with altered biological and biochemical properties of cervid PrPSc. 5: Finally, we hypothesize that the instabilities of CWD strains, and their currently indiscernible biochemical properties, reflect thermodynamic similarities between PrPSc constituting these strains. We will examine whether purified strains differ with respect to protease-sensitive PrPSc, and assess the sensitivity of Conformational Dependent Immunoassay, Fourier-transform infrared spectroscopy, luminescent conjugated polymers, and Protein Misfolding Cyclic Amplification approaches for strain discrimination. We anticipate that our work will continue to have a sustained, powerful impact on this field, and likely related diseases involving protein misfolding, as well as clarifying many remaining enigmatic aspects of CWD biology.