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

摘要 抗生素耐药性感染是美国和全球的一个主要公共卫生威胁，假单胞菌属 铜绿假单胞菌（Pa）是最受关注的病原体之一。噬菌体治疗是一种很有前途的治疗方法， 这些感染，具有物种靶向活性的好处，可以使宿主微生物组免受感染， 生物膜和杀死代谢不活跃的持续细胞，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遗传操纵 并设计裂解酶来解决它们的局限性。不能对裂解性噬菌体进行基因工程改造类似于 试图开发小分子抗生素，但没有能力精确修饰功能性抗生素， 组噬菌体用于临床试验和商业治疗的局限性中的关键是：1）不能 在生产和研究中区分治疗性噬菌体与潜在的天然污染物，2）有限 宿主范围，需要配制含有多种维生素的复杂鸡尾酒，和3）有限的能力， 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性状。 如果存在可推广的工具，这些特性中的每一个都可以处理。 Bondy-Denomy实验室开发了使用CRISPR-Cas系统选择工程化细胞的工具 和同源抗CRISPR基因作为选择标记。此外，Felix Biotechnology还开发了 使用体外基因组组装来产生噬菌体变体，并且已经鉴定了基于 宿主范围、基因组大小和组成以及初步的安全性和有效性数据。该提案结合了 这些工具可以创建基于抗CRISPR的工程（ACE），从而实现对不同裂解酶的精确工程化。 的噬菌体靶向Pa。使用ACE，Bondy-Denomy和Felix团队将设计 Felix的治疗性噬菌体候选物可在更广泛的宿主范围内提高可追溯性和功效。这项工作 将产生具有改进可追溯性和克服宿主- 防御系统。它还将定位Felix的噬菌体候选物，以通过 开发用于在体内II期PK/PD研究中测量噬菌体丰度的测定法。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，以用于未来的增强。最终这 该提案将生产一种FDA批准的商业噬菌体疗法来治疗铜绿假单胞菌感染，并 用于在其它病原体物种中工程化裂解酶的工程化工具。

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.