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

A description for the construction of the bioengineered P4-EKORhE as a means of producing transducing particles and a comprehensive method for the production of very high yields, of up to 10 ¹² particles per milliliter, enables the possibility of using virus-like particles for the transduction of genetically encoded antimicrobials using a combination of synthetic biology and upstream and downstream processing optimization. The final product, a gene-delivered antimicrobial in the form of the multi-lysins cassette, is shown to be fully functional before and after being packaged within P4-EKORhE particles. The antimi-crobial activity of the multi-lysins cassette is characterized by the activity of its contributor lysis proteins and tested in vivo in both pure bacterial Escherichia coli cultures and in a model of infection using A549 immortalized human epithelial tissue cell cultures. This work exemplifies several bioproduction methods and demonstrates how the virology of the P4 and P2 bacterio-phages can be harnessed to establish a bioprocess for the production of transducing particles with very high yields, avoiding contamination of the natural virus and maintaining the antimi-crobial effectiveness of the final product.