Developing generalized engineering tools to create enhanced phage therapy for the clinic and commercialization

开发通用工程工具，为临床和商业化创建增强型噬菌体疗法

• 批准号: 10670407
• 负责人: Robert McBride
• 金额: $ 18.44万
• 依托单位: FELIX BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-22 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10670407
• 关键词: Address; Antibiotics; Bacteriophages; Bar Codes; Biological Assay; Biology; Biotechnology; Cells; Clinic; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Complex; DNA; Data; Development; Elements; Engineering; FDA approved; Formulation; Future; Genes; Genetic Engineering; Genome; Host Defense; In Vitro; Infection; Life; Lytic; Measures; Metabolic; Methods; Microbial Biofilms; Modification; Mutagenesis; Penetration; Phase; Positioning Attribute; Property; Proteins; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Public Health; Quality Control; RNA; Safety; Site; System; Technology; Therapeutic; Variant; Work; antibiotic resistant infections; assay development; clinical application; clinical development; commercialization; density; efficacious treatment; fitness; functional group; genetic manipulation; host microbiome; improved; in vivo; integration site; manufacture; pathogen; patient safety; permissiveness; pharmacokinetics and pharmacodynamics; research study; small molecule; therapeutic candidate; thermostability; tool; trait

ABSTRACT Antibiotic-resistant infections are a major public health threat in the U.S. and globally, with Pseudomonas aeruginosa (Pa) being one of the top pathogens of concern. Phage therapy is a promising approach to treat these infections, with benefits of species-targeted activity that spares the host microbiome, an ability to penetrate biofilms and kill metabolically-inactive persister cells, and a mechanism of action distinct from antibiotics. However, the key barrier to FDA-approved phage therapies is the inability to precisely genetically manipulate and engineer lytic phages to address their limitations. The inability to genetically engineer lytic phage is akin to attempting to developing small-molecule antibiotics but without the capability to precisely modify functional groups. Key among the limitations of phage for clinical trials and commercial therapy are 1) an inability to distinguish therapeutic phage from potential natural contaminants in manufacture and research studies, 2) limited host range that requires formulation of complex cocktails containing many phages, and 3) limited ability to interrogate phage biology to improve traits such as thermostability, shelf-life, and persistence at infection sites. Each of these properties could be tackled, if generalizable tools existed. The Bondy-Denomy lab has developed tools to select for engineered phages in cells using CRISPR-Cas systems and cognate anti-CRISPR genes as selectable markers. Additionally, Felix Biotechnology has developed tools to create phage variants using in vitro genome assembly and has identified therapeutic phage candidates based on host range, genome size and composition, and preliminary safety and efficacy data. This proposal combines these tools to create anti-CRISPR-based Engineering (ACE), which enables precise engineering of diverse lytic phages, with an initial focus on phage targeting Pa. Using ACE, the Bondy-Denomy and Felix team will engineer Felix’s therapeutic phage candidates for improved traceability and efficacy over a broader host range. This work will yield engineered phage therapy candidates with modifications that improve traceability and overcome host- defense systems. It will also position Felix’s phage candidates for further therapeutic maturation through development of assays to measure phage abundance in Phase II PK/PD studies in vivo. Lastly, our engineering work will identify permissive integration sites in phage genomes for future enhancements. Ultimately, this proposal will produce an FDA-approved, commercial phage therapy to treat P. aeruginosa infections and an engineering tool for engineering lytic phages in additional pathogen species.

抽象的 抗生素耐药性感染是美国和全球的主要公共卫生威胁 Aeruginosa（PA）是关注的最高病原体之一。噬菌体疗法是​​一种有前途的治疗方法 这些感染，具有以物种为目标的活性的好处，可以避免宿主微生物组，可以穿透的能力 生物膜并杀死了代谢不活跃的持久性细胞，以及一种与抗生素不同的作用机理。 但是，FDA批准的噬菌体疗法的关键障碍是无法精确操纵 和工程师裂解噬菌体以解决其局限性。一般无法设计的裂解噬菌体类似于 尝试开发小分子抗生素，但没有能力进行精确修改功能的能力 组。临床试验和商业疗法的噬菌体局限性的关键是1）无法 制造和研究中潜在自然污染物的独特治疗噬菌体噬菌体，2）限制 需要配制包含许多噬菌体的复杂鸡尾酒的宿主范围，3）有限的能力 询问噬菌体生物学以改善特征，例如在感染部位的热稳定性，保质期和持久性。 如果存在可推广的工具，则可以解决这些属性中的每一个。 债券 - 脱瘤实验室已经开发了使用CRISPR-CAS系统选择细胞中工程噬菌体的工具 和同源抗Crispr基因作为可选标记。此外，Felix Biotechnology开发了工具 使用体外基因组组装创建噬菌体变体，并确定了基于治疗​​的噬菌体候选者 在宿主范围，基因组大小和组成以及初步的安全性和效率数据上。该建议结合了 这些工具创建基于反危机的工程（ACE），从而可以精确的裂解工程 噬菌体，最初关注噬菌体针对PA的噬菌体。使用ACE，Bondy-densomy and Felix团队将设计 费利克斯（Felix）的治疗噬菌体候选者在更广泛的宿主范围内提高了可追溯性和效率。这项工作 将产生工程设计的噬菌体治疗候选者，并进行修改以提高可追溯性并克服宿主 - 国防系统。它还将定位Felix的噬菌体候选者，以通过 在体内II PK/PD研究中测量噬菌体丰度的测定的开发。最后，我们的工程 工作将确定噬菌体基因组中的允许整合位点，以实现未来的增强。最终，这个 提案将产生FDA批准的商业噬菌体疗法，以治疗铜绿假单胞菌感染和 工程工具用于其他病原体中的工程裂解噬菌体。