Mouse modeling of a human STING gene variant for infectious disease

描述(申请人提供)：传染病导致的死亡人数超过其他任何单一原因。对人类的全基因组关联研究表明，参与宿主防御的基因的遗传变异可以影响个体对感染的易感性。在大多数情况下，这种理解疾病遗传因素的反向遗传方法依赖于动物模型，动物模型允许在没有其他变量的情况下分析遗传变异的影响。缺乏这样的模型及其产生的知识对携带这些基因变异的个人的个性化治疗和保健的未来发展构成了重大障碍。在这里，我们建议建立小鼠基因敲打模型来研究人类基因的两个变体的功能，该基因编码的是一种I型干扰素刺激物，在小鼠中对于抵御病毒感染是必不可少的。我们最近报道在人类中有两个STINT变体，每个变体编码三个氨基酸变化(R71H-G230A-R293Q)。我们将这些称为WT和HAQ。我们的体外研究表明，与WT相比，HAQ-STING变异体在病原体感染时已经失去了90%的刺激I型干扰素产生的能力。此外，HAQ-STING变异体在体外具有显性的负功能效应。令人惊讶的是，约20%的美国人至少携带一种Haq-sting变种。我们的长期目标是了解这种常见的、潜在的无功能的人类刺变异体的分子机制和体内功能，它可能会影响数百万美国人的疾病易感性。为了实现这一目标，我们建议开发两种人类叮咬变体的小鼠模型。这两个模型将包括1)最低限度的SNPs敲击小鼠基因和2)SNPs敲击加上在功能上具有潜在重要性的独特的人类上下文序列。值得注意的是，小鼠和人的刺蛋白有83%的氨基酸序列同源性。因此，在第一个模型(mHAQ小鼠)中，我们将进行敲击，其中小鼠的等量氨基酸将改变为人类HAQ-STING中的氨基酸(目标1)。在第二个模型中，我们将适应非同源性的有限区域，但利用了老鼠和人类的刺痛基因具有相同的外显子-内含子结构的事实。我们将制作一种表达人-鼠刺的嵌合形式的刺敲入，其中整个N-末端172aa被人刺(CHAQ小鼠)的173aa的N-末端取代(目标2)。这款鼠标还将包含G230A和R293Q。还将构建适当的WT小鼠。在时间允许的情况下，我们将使用这些模型来研究HAQ在抵抗感染和刺痛信号功能方面的作用。 与公共卫生相关：刺痛是一种重要的基因，其蛋白产物在控制我们对病毒和细菌感染的易感性方面发挥作用。HAQ-STING是一种变异基因，约20%的美国人携带。HAQ-STING编码的蛋白质似乎存在功能缺陷，因此HAQ-STING的携带者感染的风险可能会增加。在这个方案中，我们将构建一个动物模型来最终测试HAQ-STING编码蛋白的功能。除了提高我们对这种基因变异对人类健康构成的风险的理解外，这些动物模型应该有助于开发和测试个性化的治疗方法。

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. PUBLIC HEALTH RELEVANCE: Sting is an important gene whose protein product functions in the control of our susceptibility to viral and bacterial infections. HAQ-STING is variant gene carried by ~20% of Americans. The protein encoded by HAQ- STING appears to be functionally defective, and therefore carriers of HAQ-STING may be at increased risk of infection. In this proposal, we will construct an animal model to definitively test the function of HAQ-STING encoded protein. In addition to advancing our understanding of the risk to human health posed by this gene variant,these animal models should be useful for developing and testing personalized treatments.