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

摘要 耐药感染是美国和全球的主要公共卫生威胁，假单胞菌 铜绿假单胞菌(铜绿假单胞菌)是最受关注的病原体之一。噬菌体疗法是一种很有前途的治疗方法 这些感染，加上物种靶向活动的好处，使宿主微生物群得以幸免，具有穿透 生物被膜和杀死代谢不活跃的周围细胞，以及与抗生素不同的作用机制。 然而，FDA批准的噬菌体疗法的关键障碍是无法精确地从基因上操纵 并改造裂解噬菌体以解决它们的局限性。不能对裂解噬菌体进行基因工程类似于 试图开发小分子抗生素，但没有能力精确地修饰功能性 组。噬菌体用于临床试验和商业治疗的关键限制是：1)不能 在生产和研究中区分治疗噬菌体和潜在的天然污染物，2)有限 寄主范围，需要配制包含许多噬菌体的复杂鸡尾酒，以及3)能力有限 询问噬菌体生物学以改善诸如热稳定性、保质期和在感染部位的持久性等特征。 如果存在可泛化的工具，这些属性中的每一个都可以解决。 Bondy-Denomy实验室开发了使用CRISPR-CAS系统在细胞中选择工程噬菌体的工具 和同源的抗CRISPR基因作为选择标记。此外，费利克斯生物技术公司还开发了一些工具 使用体外基因组组装创造噬菌体变异体，并基于 关于寄主范围、基因组大小和组成，以及初步的安全性和有效性数据。这项建议结合了 这些工具用于创建基于反CRISPR的工程(ACE)，从而实现对各种裂解的精确工程 噬菌体，最初的重点是针对Pa.的噬菌体。使用ACE，Bondy-Denomy和Felix团队将设计 Felix的治疗噬菌体候选，可在更广泛的宿主范围内改善可追溯性和有效性。这部作品 将产生工程化噬菌体疗法候选者，其修饰可提高可追溯性并克服宿主- 防御系统。它还将使Felix的噬菌体候选通过以下方式进一步治疗成熟 在体内第二阶段PK/PD研究中测定噬菌体丰度的方法的发展。最后，我们的工程 这项工作将确定噬菌体基因组中允许的整合位点，以用于未来的增强。归根结底，这 该提案将产生一种FDA批准的商业噬菌体疗法来治疗铜绿假单胞菌感染和 在其他病原体物种中设计裂解噬菌体的工程工具。

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.