Gifu University succeeds in modifying and assembling bacteriophage genomes in vitro - success in treating septic mice

A research group consisting of Researcher Shoichi Mitsunaka and Associate Professor Hiroki Ando of the Phage Biologics Research Course, Graduate School of Medicine, Gifu University, and their colleagues has developed a technique for phage synthesis and engineering that can modify, assemble and reboot bacteriophage (phage) genomes in vitro. Using this, they succeeded in rebooting numerous phages in practice, chemically synthesizing DNA from sequence data from a database, assembling this in vitro, and rebooting the synthetic phage.

Many lives are saved through phage therapy using wild-type phages. However, there are always issues with this therapy - for example, narrowness of host range and the appearance of phage-resistant bacteria. The research group engaged in this research with the belief that modifying phages could solve these issues.

Phage genomes from PCR products were assembled in vitro, then introduced into host bacteria via electroporation, thus rebooting the phages. The group succeeded in rebooting multiple phages that infect a variety of gram-negative bacteria, multiple phages that infect acid-fast mycobacteria, which have a special membrane structure, and phages modified from these. Using this method, they were able to create an acid-fast mycobacteria phage from chemically synthesized DNA based on sequence data. This phage has a genome size of 52,797 base pairs, making it the longest virus created from chemically synthesized DNA to date. The phage genome was recreated in accordance with its original genome, with no difference in any bases.

The group also succeeded in creating a functional phage by assembling the genomes of a phage that infects E. coli in vitro and adding this to a cell-free transcription and translation (TXTL) system formed from extract from E. coli. The outcome demonstrated that the whole process, from phage genome assembly to rebooting, could be done entirely in vitro rather than in vivo.

In addition, the group developed two methods for biological containment using a phage synthetic engineering technique. One of these involves packaging plasmid DNA rather than phage genomes into phage head particles. When the resulting phage infects the target bacteria, it injects the plasmid DNA into the host cells and expresses its function. The other method is to delete virion genes from the phage genome. This is no different to the original phage in terms of its appearance, infectiousness, and bactericidal properties, but it cannot produce progeny phages. It could be called a nonproliferable biologically contained phage that can infect and kill bacteria once only. A nonproliferable phage can proliferate through the host bacteria, which expresses the missing virion genes.

When the group carried out phage therapy on mice with lethal sepsis using nonproliferable phages, it had a clear therapeutic effect. Moreover, they did not find any proliferable phages inside the bodies of the cured mice. Based on these results, it is possible to use nonproliferable phages in phage therapy, and 100% of these were successfully contained.

Ando commented, "We have developed innovative technology that enables us to modify various phages, and methods to ensure that the genetically modified phages are biologically contained. In the future, I want to extend our research to modified phage therapy, looking to also use wild-type phages and anti-bacterial agents, with the aim of social implementation."

■ Electroporation: A method that uses short-pulse electric currents to introduce DNA and RNA to bacteria and cells.

Journal Information
Publication: Proceedings of the National Academy of Sciences (PNAS)
Title: Synthetic engineering and biological containment of bacteriophages
DOI: 10.1073/pnas.2206739119