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，无法治愈的，传染性神经退行性疾病。通过致病性构象PrPSc破坏细胞朊病毒蛋白（PrPC）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菌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，这些现象与它们的潜在发病机制和人畜共患潜力有关。我们的重点仍然主要放在慢性消耗性疾病（CWD）上，这是一种新兴的、高度传染性的朊病毒疾病，发生在圈养和野生美洲豹中。不同CWD菌株的发现，以及鹿骨骼肌和麋鹿鹿茸中朊病毒的发现，与人类健康具有明确的相关性。目前难以区分的PrPSc构象和CWD菌株的不稳定转变与它们通过相对较低的能垒分离相一致。鹿和麋鹿的应变稳定性也不同。我们的总体假设是，宿主PrPC初级结构影响了在朊病毒传播过程中动力学选择的热动力首选PrPSc构象组合。我们的研究设计和方法如下：1 .用鹿或麋鹿的Prnp编码序列代替小鼠的Prnp编码序列，制备基因靶向小鼠（Gt）。这将使我们能够探索残基226（鹿和麋鹿PrP初级结构的唯一差异）存在于影响PrP转化和应变稳定性的关键区域的假设。2: Gt小鼠为精确表征CWD菌株的特性和评估PrP一级结构对菌株稳定性的贡献提供了资源。3 .我们将使用Gt小鼠和现有的转基因模型来评估CWD菌株在疾病期间是干扰还是协同作用，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：开发创新的细胞培养资源，用于CWD朊病毒的定量和菌株特性的表征。我们假设朊病毒菌株受到选择压力的影响，我们将评估朊病毒复制修饰剂对形成具有改变子宫颈PrPSc生物学和生化特性的菌株变体的影响。最后，我们假设CWD菌株的不稳定性，以及它们目前无法辨别的生化特性，反映了构成这些菌株的PrPSc之间的热力学相似性。我们将研究纯化菌株在蛋白酶敏感性PrPSc方面是否存在差异，并评估构象依赖免疫分析、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对菌株识别的敏感性。我们预计，我们的工作将继续对这一领域以及可能涉及蛋白质错误折叠的相关疾病产生持续的、强大的影响，并澄清CWD生物学中许多仍未解之谜的方面。

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. PUBLIC HEALTH RELEVANCE: Prions are proteinacious infectious agents causing fatal, currently untreatable neurodegenerative diseases in animals and humans. The transmission of bovine prions to humans exemplifies their zoonotic potential, and that the factors controlling interspecies transmission are poorly understood. The unprecedented contagious spread of chronic wasting disease (CWD), a burgeoning epidemic of cervids, the discovery of distinct but unstable CWD strains, and the presence of CWD prions in muscle and other animal derivatives destined for human consumption, places the human species barrier to CWD prions at the forefront of public health concerns.