# High-yield bioproduction of virus-free virus-like P4-EKORhE multi-lysin transducing particles as an antimicrobial gene therapeutic

## TL;DR

This paper describes a method to produce high-yield, virus-free particles that deliver antimicrobial genes to fight bacteria.

## Contribution

A novel bioproduction method for virus-like transducing particles with high yield and antimicrobial efficacy.

## Key findings

- P4-EKORhE particles can be produced at up to 10^12 particles per millilitre.
- The multi-lysin cassette remains functional before and after packaging.
- Antimicrobial activity was confirmed in E. coli and human cell co-cultures.

## Abstract

A description of the construction of the bioengineered P4-EKORhE and a comprehensive method for producing very high yields (up to 1012 particles per millilitre) enable the use of virus-like particles to transduce genetically encoded antimicrobials through a combination of synthetic biology and optimised upstream and downstream processing. The final product, a gene-delivered antimicrobial in the form of the multi-lysin cassette, is fully functional before and after packaging within P4-EKORhE particles. The antimicrobial activity of the multi-lysin cassette, characterised by its lysis proteins, was tested in vivo in both pure bacterial Escherichia coli cultures and a model of phage infection in co-culture with A549 immortalised human epithelial tissue cells. This work exemplifies several bioproduction methods and demonstrates how the virology of the P4 and P2 phages can be harnessed to establish a bioprocess for producing transducing particles at very high yields, avoiding contamination by the natural virus while maintaining the antimicrobial effectiveness of the final product.