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Large-scale CRISPR screening of essential calcium-related genes in the human parasite Tr. cruzi

大规模 CRISPR 筛选人类寄生虫 Tr 中必需的钙相关基因。

基本信息

批准号：
10480891
负责人：
Noelia Lander
金额：
$ 24.9万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-03 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10480891
关键词：
Address Adult Affect Antiparasitic Agents Basic Science Bioenergetics Biological Biological Assay CRISPR screen CRISPR/Cas technology Calcium Calcium Signaling Calcium ion Cardiomyopathies Cell Line Cell physiology Cells Centers for Disease Control and Prevention (U.S.)Chagas Disease Characteristics Chemicals Chronic Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Country Development Disease Drug Targeting Environment Essential Genes Europe Exhibits Face Gene Expression Gene Silencing Generations Genes Genome Goals Guide RNA Health Heart Diseases Homeostasis Human Immunoprecipitation In Situ Infection Infrastructure Inositol Intervention Knock-out Knowledge Laboratories Latin America Libraries Life Life Cycle Stages Mammalian Cell Mediating Mentors Messenger RNA Metabolic Metabolic Pathway Mitochondria Modeling Molecular Movement Multigene Family North America Organism Osmoregulation Parasites Pathogenicity Pathology Patients Persons Pharmaceutical Preparations Phase Phenotype Population Prokaryotic Cells Proteins Recombinant Proteins Regulation Research Personnel Role Scientist Second Messenger Systems Signal Transduction Signaling Protein System Time Tissues Toxic effect Training Trypanosoma Trypanosoma cruzi Tumor stage United States Universities Vaccines Validation alternative treatment base calcium uniporter cell growth chemotherapy drug efficacy effective therapy environmental change fitness genetic manipulation genetically modified cells genome editing genome-wide improved inhibitor interest migration mutant new therapeutic target overexpression protein function receptor screening side effect therapy development tool

项目摘要

Abstract Chagas disease (CD) is a parasitic tropical pathology caused by the protozoan parasite Trypanosoma cruzi. It affects 6-7 million people worldwide, for which no vaccine or satisfactory treatment are available. Historically, CD has been considered endemic in Latin America, but more recently it has spread to Europe, North America, and the Pacific, due to global migrations. An estimated 300,000 people are currently infected in the United States, 45,000 of which exhibit cardiomyopathy, while most cases remain undiagnosed and untreated. There are only two accepted drugs to treat Chagas disease, but they are only available through the Centers for Disease Control and Prevention (CDC). Besides their adverse side effects, the efficacy of these drugs decreases the longer a person has been infected. Therefore, the development of new non-toxic chemotherapies to treat chronic patients of Chagas disease is highly demanded. We are interested in investigating proteins that can be used as chemotherapeutic targets for developing effective treatments against Chagas disease. Our approach is to identify metabolic pathways that are absent in human cells but necessary for parasite survival. Calcium ion (Ca2+) is an important second messenger in trypanosomatids that regulates a vast repertoire of cellular processes. In T. cruzi, Ca2+ signaling is particularly important for host cell invasion, differentiation, osmoregulation and cell bioenergetics. Several proteins involved in calcium homeostasis have been identified in trypanosomes. However, the T. cruzi genome contains a wide variety of calcium-interacting uncharacterized proteins. Using the CRISPR/Cas9 system, we have been able to perform knockout, complementation and in situ tagging of T. cruzi genes. Furthermore, we have established important differences between calcium signaling proteins in T. cruzi and their homologs in mammalian cells. The development of a CRISPR-based inducible silencing system is feasible in T. cruzi and we will use it to perform a large-scale screening of genes encoding calcium-interacting proteins in this organism. Then we will identify essential proteins involved in calcium signaling and perform their biological validation as drug targets, together with other essential proteins already identified. We finally expect to disclose relevant information on protein function, interacting partners, cellular localization and even inhibitors of essential calcium-related proteins. Taken together these results will eventually culminate in the chemical validation of them as target candidates for treatment development against Chagas disease. The mentored phase of this project is going to be developed in the laboratory of Dr. Roberto Docampo at the University of Georgia, which belongs to the Center for Tropical and Emerging Global Diseases. The academic environment, infrastructure and laboratory dynamics integrate the perfect conditions to complete my training plan, and will be determinant to achieve my actual goal: to become an independent researcher and a leader scientist, contributing to the generation of knowledge in the biomedical field.

摘要恰加斯病是由原虫锥虫克氏锥虫引起的一种热带寄生虫病。它影响全球600-700万人，目前尚无疫苗或令人满意的治疗方法。从历史上看， CD一直被认为是拉丁美洲的地方病，但最近它已经蔓延到欧洲、北美、和太平洋，由于全球移民。据估计，美国目前有30万人感染其中45,000个州表现出心肌病，而大多数病例仍未得到诊断和治疗。那里只有两种被接受的治疗恰加斯病的药物，但它们只在疾病控制和预防(CDC)。除了它们的副作用外，这些药物的疗效感染的时间越长，感染的时间就越长。因此，开发新型无毒食品治疗慢性恰加斯病患者的化疗需求很高。我们对研究可以作为化疗靶点的蛋白质很感兴趣针对恰加斯病的有效治疗方法。我们的方法是找出不存在的代谢途径人类细胞，但对寄生虫的生存是必要的。钙离子(Ca~(2+))是体内重要的第二信使调节大量细胞过程的锥虫。在克氏锥虫中，Ca~(2+)信号传导尤其强烈。对宿主细胞入侵、分化、渗透调节和细胞生物能量学具有重要作用。几种蛋白质已经在锥虫体内发现了与钙稳态有关的分子。然而，克氏锥虫的基因组包含种类繁多的钙相互作用的未鉴定蛋白质。利用CRISPR/CAS9系统，我们一直在能够进行克氏毛滴虫基因的敲除、互补和原位标记。此外，我们还拥有建立了克鲁兹毛滴虫及其同源物钙信号蛋白之间的重要差异哺乳动物细胞。基于CRISPR的可诱导沉默系统的开发是可行的。我们将使用它对编码钙相互作用蛋白的基因进行大规模筛选有机体。然后，我们将确定与钙信号有关的基本蛋白质，并执行它们的生物学作为药物靶点的验证，以及已经确定的其他基本蛋白质。我们最终预计会披露蛋白质功能、相互作用伙伴、细胞定位甚至抑制物的相关信息必需的钙相关蛋白。总而言之，这些结果最终将在化学物质中达到顶峰。确认它们是治疗恰加斯病的目标候选药物。该项目的指导阶段将在Roberto Docampo博士的实验室中开发，佐治亚大学，隶属于热带和新出现的全球疾病中心。这个学术环境、基础设施和实验室动力相结合的完美条件，完成我的培训计划，这将是实现我的实际目标的决定性因素：成为一名独立的研究人员和领先的科学家，为生物医学领域知识的产生做出了贡献。