Mouse modeling of a human STING gene variant for infectious disease

描述（由申请人提供）：传染病导致的死亡人数超过任何其他单一原因。全基因组关联——对人类的研究表明，参与宿主防御的基因的遗传变异会影响个体对感染的易感性。在大多数情况下，这种理解遗传对疾病的贡献的反向遗传方法依赖于动物模型，这种模型允许在没有其他变量的情况下分析遗传变异的影响。缺乏这样的模型和它们所产生的知识，对携带这些遗传变异的个体的个性化治疗和保健的未来发展构成了重大障碍。在这里，我们提出构建小鼠基因敲入模型来研究编码STING的人类基因的两个变体的功能，STING是一种I型IFN刺激因子，在小鼠中对防御病毒感染至关重要。我们最近报道了人类中存在两个STING变异，每个变异编码三个氨基酸变化（R71H-G230A-R293Q）。我们将其称为WT和HAQ。我们的体外研究表明，相对于WT， HAQ-STING变异体在病原体感染后已经失去了90%刺激I型IFN产生的能力。此外，HAQ-STING变体在体外具有显性的负功能效应。令人惊讶的是，大约20%的美国人携带至少一个HAQ-sting变种。我们的长期目标是了解这种常见的、可能无功能的人类STING变异的分子机制和体内功能，它可能影响数百万美国人的疾病易感性。为了实现这一目标，我们建议建立两种人类STING变异的小鼠模型。这两种模型将包括：1)极简敲入小鼠基因的snp； 2)敲入具有潜在重要功能的独特人类背景序列的snp。值得注意的是，小鼠和人的STING蛋白具有83%的氨基酸序列同源性。因此，在第一个模型（mHAQ小鼠）中，我们将进行STING敲入，其中小鼠的等效氨基酸将改变为人类HAQ-STING中的氨基酸（目的1）。在第二种模型中，我们将考虑有限的非同源区域，但利用小鼠和人类STING基因具有相同的外显子-内含子结构这一事实。我们将制作一个表达嵌合形式的人类小鼠STING的STING敲入，其中整个n末端172aa被人类STING （cHAQ小鼠）的173aa的n末端取代（Aim 2）。该鼠标还将包含G230A和R293Q。同时构建合适的WT小鼠。如果时间允许，我们将使用这些模型来研究HAQ对感染抗性和STING信号功能的影响。

DESCRIPTION (provided by applicant): Infectious diseases kill more people than any other single cause. Genome-Wide-Association-Studies in humans have demonstrated that genetic variations in genes involved in host defense can affect individuals' susceptibility to infection. I most cases, such reverse genetic approaches to understanding genetic contributions to disease are dependent on animal models that allow analysis of the effects of genetic variance in the absence of other variables. The lack of such models and the knowledge they yield poses a significant barrier for the future development of personalized treatment and health care for individuals carrying these genetic variations. Here we propose the construction of mouse gene knockin models to study the function of two variants of the human gene that encodes STING, a type I IFN stimulator that in mice is essential for defense against viral infections. We recently reported that there are two STING variants in man, each encoding three amino-acid changes (R71H-G230A-R293Q). We refer to these as WT and HAQ. Our in vitro studies have demonstrated that relative to WT the HAQ-STING variant has lost >90% of the ability to stimulate type I IFN production upon pathogen infection. Furthermore, the HAQ-STING variant has a dominant negative functional effect in vitro. Surprisingly, ~20% of Americans carry at least one copy of the HAQ-sting variant. It is our long-term goal to understand the molecular mechanisms and in vivo function of this frequent, potentially nonfunctional, human STING variant that may affect disease susceptibility of millions of Americans. To achieve this goal, we propose to develop two mouse models for the human STING variants. The two models will include 1) a minimalist knockin of SNPs into the mouse gene and 2) a knockin of SNPs plus unique human contextual sequence of potential importance in function. It is noteworthy that mouse and human STING proteins share 83% amino acid sequence homology. Thus in the first model (mHAQ mouse), we will make a STING knock-in in which the mouse equivalent amino acids will be changed to those in human HAQ-STING (Aim 1). In the second model we will accommodate the limited regions of non-homology, yet take advantage of the fact that mouse and human STING genes have the same exon - intron structure. We will make a STING knock-in that expresses a chimeric form of human-mouse STING, in which the entire N-terminal 172aa is replaced by the N-terminal of 173aa of human STING (cHAQ mouse) (Aim 2). This mouse will also contain G230A and R293Q. Appropriate WT mice will also be constructed. As time permits we will use these models to study the effects of HAQ on resistance to infection and on STING signaling function.