A platform for genome mining of multidrug-resistant pathogens to develop therapeutic phages using synthetic biology

利用合成生物学开发治疗性噬菌体的多重耐药病原体基因组挖掘平台

基本信息

批准号：
10356122
负责人：
Yoann Stephane Le Breton
金额：
$ 16.91万
依托单位：
GENEVA FOUNDATION
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：
2021-02-18 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10356122
关键词：
Address Antibiotic Resistance Antibiotics Antimicrobial Resistance Bacteria Bacteriophages Biology Biomedical Engineering Cell Surface Receptors Cells Centers for Disease Control and Prevention (U.S.)Chromosomes Cytolysis DNA sequencing Development Disease Endotoxins Engineering Environment Essential Genes Experimental Designs Genes Genetic Genetic Engineering Genome Genome engineering Goals Gold Horizontal Gene Transfer Human Infection Knowledge Learning Life Life Cycle Stages Lytic Lytic Phase Medical Methods Microbial Biofilms Mining Modality Modeling Modern Medicine Modification Monoclonal Antibodies Multi-Drug Resistance Mutation Patients Penicillin Resistance Pharmaceutical Preparations Preparation Production Property Prophages Reporting Resistance Rogaine Safety Sepsis Series Source Specificity Streptococcal Infections Streptococcus pyogenes Structure Testing Therapeutic Vaccines Virulence Virulence Factors Wound Infection antimicrobial arm bacterial resistance base design design-build-test efficacy testing experience functional genomics gene interaction global health high throughput analysis human pathogen in vivo in vivo Model insight interest multi-drug resistant pathogen pathogen pathogenic bacteria receptor research and development small molecule synthetic biology therapeutic target tool

项目摘要

PROJECT SUMMARY/ABSTRACT Multidrug resistant [MDR] pathogens represent a global health threat and a challenge for modern medicine; and, as bacterial resistance to new antibiotics is now outpacing the antibiotic development effort, it is critical to develop new effective antimicrobial alternatives. The antimicrobial resistance crisis is bringing new interests worldwide to develop phage-based therapies. Over the last decade, efforts in the U.S. to produce phage therapeutics targeting different bacterial pathogens have shown promising results, including successful treatments of life-threatening infections in human patients. Safety of phage therapy is still a concern in the U.S.; however FDA has highlighted requirements for phage preparation: they need to be safe, pure, potent, exclusively lytic, non-transducing, and lacking undesirable genes (antibiotic resistance, virulence factors) and bacterial endotoxins. If bacteriophages for therapy have historically been isolated from natural environments, recent progresses in phage genetics and genome engineering have proven successful to generate synthetic, strictly lytic derivatives targeting pathogens. The development of synthetic phages against MDR pathogens would require pipelines to accelerate our knowledge on newly discovered phages and their potential for synthetic biology. Critical insights into their biology, i.e. genome structure, phage replication cycle, genetic content (essential genes versus dispensable [antibiotic resistance and virulence genes]), interaction with the target host, are a prerequisite. Here, we propose a platform to (i) mine the genomes of MDR pathogens, a gold mine to identify dormant lysogenic phages directly from within their natural host; and (ii) develop high-throughput pipelines to quickly gain knowledge on the phage biology to guide our efforts to engineer synthetic phages as therapeutics. We will use the Group A Streptococcus (GAS), a “Concerning Threat” on the 2019 CDC “18 MDR pathogens” Watch List, as our model. We showed that Tn-seq could identify functional lysogenic phages from cryptic ones in GAS genomes, and mutations to reboot dormant prophages into their lytic cycle. In Aim 1, we will produce the critical knowledge to guide decision on what phages to select for therapeutic potential using synthetic biology: we will experimentally assess phage genome organization, phage replication/transduction mechanisms, host range and cell surface receptor(s). In Aim 2, we will implement a design-build-test-learn cycle" pipeline to optimize the synthetic biology effort, i.e. deletion of undesirable genes and addition of “payload” genes, to enhance their potential as therapy phages. Finally, we will use in vivo model of wound infection to test the efficacy of the synthetic phages we generated. Our overarching goal is to develop the tools and experience to apply our synthetic biology phage-engineering platform to other MDR pathogens.

项目概要/摘要多重耐药[MDR]病原体对全球健康构成威胁，也是对现代医学的挑战；而且，由于细菌对新抗生素的耐药性现在已经超过了抗生素的开发速度，因此至关重要开发新的有效抗菌药物替代品抗菌药物耐药性危机带来了新的兴趣。在过去的十年中，美国在全球范围内开发基于噬菌体的疗法。针对不同细菌病原体的疗法已显示出有希望的结果，包括成功的噬菌体疗法的安全性仍然是人类患者危及生命的感染的治疗方法。美国；然而 FDA 强调了噬菌体制备的要求：它们需要安全、纯净、有效、完全裂解、非转导且缺乏不良基因（抗生素耐药性、毒力因子）和细菌内毒素。如果用于治疗的噬菌体历史上是从自然环境中分离出来的，那么最近在噬菌体遗传学和基因组工程已被证明可以成功地产生合成的、严格裂解的衍生物针对 MDR 病原体的合成噬菌体的开发需要管道加速我们对新发现的噬菌体及其合成生物学潜力的了解。进入它们的生物学，即基因组结构、噬菌体复制周期、遗传内容（必需基因与可有可无的[抗生素耐药性和毒力基因]），与目标宿主的相互作用是先决条件。在这里，我们提出了一个平台来（i）挖掘 MDR 病原体的基因组，这是识别休眠病原体的金矿直接从其天然宿主内溶源噬菌体；和（ii）开发高通量管道以快速获得有关噬菌体生物学的知识，以指导我们设计合成噬菌体作为治疗药物。我们将使用A组链球菌（GAS），这是2019年CDC“18种MDR病原体”上的“令人担忧的威胁” 观察列表，作为我们的模型，我们证明 Tn-seq 可以从隐性噬菌体中识别出功能性溶源噬菌体。在 GAS 基因组中，以及将休眠的原噬菌体重新启动到其裂解周期中的突变。在目标 1 中，我们将产生。决定使用合成噬菌体选择哪些噬菌体以实现治疗潜力的关键知识生物学：我们将通过实验评估噬菌体基因组组织、噬菌体复制/转导在目标 2 中，我们将实施设计-构建-测试-学习。循环”管道来优化合成生物学工作，即删除不需要的基因并添加 “有效负载”基因，以增强其作为治疗噬菌体的潜力。最后，我们将使用伤口的体内模型。我们的首要目标是开发工具。以及将我们的合成生物学噬菌体工程平台应用于其他耐多药病原体的经验。