CRISPR-mediated engineering and pilot study of mouse mutants of the bitter taste receptor genes

摘要：本R03建议书是根据RFA-RM-21-012《试点项目》编写的 研究未被充分研究的G蛋白偶联受体、离子通道和蛋白激酶。我们的研究将 关注G蛋白偶联受体家族中的人类苦味受体(TAS2Rs)，其表达为 在呼吸系统中检测到。这些受体已被指定为开放研究的合格蛋白质 NIH阐明了与这种RFA相关的可药物基因组(IDG)计划，以及他们在BITH中的作用 基于Tastant/TAS2R激动剂的哮喘治疗需要阐明。哮喘是一个主要的公共卫生问题。 挑战和儿科人群中最常见的慢性病。在美国，超过2500万人 包括8.4%的儿童在内的人们目前患有哮喘，估计每年的成本约为82美元 十亿美元。目前主要的治疗策略是基于β2-肾上腺素能受体激动剂、皮质类固醇、 和单抗。然而，这些药物的长期有效性和安全性仍然存在疑问。 接近了。此外，目前仍没有有效的治疗方法来治疗进行性呼吸道重塑，这对 在哮喘相关死亡中的关键作用。所有这些因素都为苦涩的调查提供了强大的推动力 Tatants/TAS2R激动剂用于治疗哮喘，因为最近研究表明，这些药物具有 在动物模型中治疗哮喘的卓越疗效，在动物模型中，数据表明这些激动剂可能 克服与当前治疗方法相关的严重缺陷，例如与渐进性呼吸道治疗有关的缺陷 改建。我们努力的长期目标是从机械的角度进一步理解TAS2Rs 与哮喘治疗相关的生理、病理和治疗过程。在这个试点项目中，我们 建议检验苦味受体在苦味/TAS2R激动剂中起重要作用的假设- 基于哮喘的治疗策略。因此，我们将设计出缺乏Tas2r基因的小鼠突变体 或通过使用基于CRISPR的高效技术携带单个Tas2r基因。我们还将比较 苦味剂/TAS2R激动剂对纯合子哮喘患者的疗效及相关信号转导 突变体和野生型窝种。这些实验将帮助我们更好地理解分子 基于苦味/TAS2R激动剂的哮喘治疗的潜在机制。我们的研究还将揭示如果 TAS2Rs对于小鼠的生存是必不可少的。最后，在本研究期间开发的遗传资源将是 强大的工具解开TAS2Rs与其他相关基因的生理、病理和治疗作用 健康状况，如肥胖、糖尿病和早产。

ABSTRACT: This R03 proposal has been prepared in response to RFA-RM-21-012 entitled “Pilot Projects Investigating Understudied G Protein-Coupled Receptors, Ion Channels, and Protein Kinases.” Our research will focus on human bitter taste receptors (TAS2Rs) in the G protein-coupled receptor family, whose expression is detected in the respiratory system. These receptors have been designated as eligible proteins open for study by NIH’s Illuminating the Druggable Genome (IDG) Program associated with this RFA, and their roles in bitter tastant/TAS2R-agonist-based therapies for asthma need to be elucidated. Asthma is a major public health challenge and the most common chronic disease in the pediatric population. In the United States, over 25 million people, including 8.4% of all children, are currently suffering from asthma with an estimated annual cost of ~$82 billion. At present, the main treatment strategies are based on β2-adrenergic receptor agonists, corticosteroids, and monoclonal antibodies. However, questions remain about the long-term efficacy and safety of these approaches. In addition, there is still no effective treatment for progressive airway remodeling, which plays a critical role in asthma-related deaths. All these factors provide a strong impetus for investigations of bitter tastants/TAS2R agonists for treating asthma because it has been recently shown that these agents have a superior efficacy for asthma treatment in animal models, in which data suggest that these agonists can potentially overcome critical deficiencies associated with current therapeutics such as those related to progressive airway remodeling. The long-term goal of our efforts is to further understand TAS2Rs mechanistically in terms of the physiological, pathological, and therapeutic processes associated with asthma treatment. In this pilot project, we propose to test the hypothesis that bitter taste receptors play an essential role in bitter tastant/TAS2R-agonist- based therapeutic strategies for asthma. Accordingly, we will engineer mouse mutants deficient for Tas2r genes or carrying a single Tas2r gene by using highly efficient CRISPR-based technology. We will also compare the outcomes of bitter tastants/TAS2R-agonist-based treatment of asthma and associated signaling in homozygous mutants and wild-type littermates. These experiments will help us to better understand the molecular mechanisms underlying bitter tastant/TAS2R-agonist-based therapies for asthma. Our study will also reveal if TAS2Rs are essential for the survival of mice. Lastly, the genetic resources developed during this study will be powerful tools for unraveling the physiological, pathological, and therapeutic roles of TAS2Rs related to other medical conditions, such as obesity, diabetes, and preterm labor.