First mRNA-based approach to endolysin therapy reported
Recombinant endolysins represent a promising solution to the antibiotic resistance crisis since they can lyse bacterial cells without an associated loss of sensitivity over time. However, challenges related to organ targeting and bioavailability have restricted their therapeutic utility. To address these limitations, researchers at University Medicine Rostock in Germany used synthetic mRNA to drive production of the Cpl-1 endolysin in human cells. Their data, published in Molecular Therapy: Nucleic Acids, show the encoded protein to have activity against Streptococcus pneumoniae, and that a secretory variant containing a point mutation to avoid N-linked glycosylation is active against nine clinically relevant pneumococcal strains. This study is thought to represent the first mRNA-based approach to endolysin therapy and has potential for targeting many other bacterial pathogens.
Cpl-1-encoding mRNA is translated into the active endolysin in human cell lines
Cpl-1 mRNA was produced from codon-optimized DNA templates using the HiScribe T7 mRNA Kit (New England Biolabs) with TriLink’s CleanCap® Reagent AG, and its uridines were completely substituted by N1-methylpseudouridines. Varying amounts of Cpl-1 mRNA were used to transfect HEK293T, A549, and HepG2 cells, with CleanCap® EGFP mRNA serving as a positive control. Western blot analysis of cell lysates generated 24 hours post-transfection showed bands corresponding to Cpl-1, which were absent in non-transfected control samples. Application of the lysates to blood agar plates inoculated with S. pneumoniae produced clear lysis zones, indicating Cpl-1 activity.
Introducing a signal peptide leads to Cpl-1 secretion
To promote Cpl-1 secretion, the University Medicine Rostock researchers created mRNA constructs featuring various signal peptides. Following cell transfection, and confirmation of signal peptide expression by SDS-PAGE, a series of turbidity reduction experiments was conducted by adding the supernatants to S. pneumoniae suspension cultures and measuring the optical density (OD600) over time. This enabled the selection of an mRNA construct employing the human lysozyme signal peptide for further analysis (hlySP-sCpl-1).
Pneumococcal killing is enhanced with a point mutation preventing N-linked glycosylation
N-linked glycosylation, a common post-translational modification in the secretory pathway of eukaryotic cells, was found to limit the pneumococcal killing effect of Cpl-1. To address this, the University Medicine Rostock researchers generated two hlySP-sCpl-1 mRNA constructs featuring point mutations at the N-linked glycosylation consensus sequence: hlySP-sCpl-1S217A and hlySP-sCpl-1N215D. Of these, hlySP-sCpl-1N215D was found to have a more pronounced pneumococcal killing effect than hlySP-sCpl-1S217A, with additional investigation showing it to be effective against nine clinically relevant pneumococcal strains.
Conclusion
Innovative solutions are urgently needed to tackle the antibiotic resistance crisis. This study shows that an mRNA-based approach to endolysin therapy could be successful in treating pneumococcal disease, as well as could be adapted for targeting many other bacterial pathogens.
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Article reference: Jansson MK, Nguyen DT, Mikkat S, et al. Synthetic mRNA delivered to human cells leads to expression of Cpl-1 bacteriophage-endolysin with activity against Streptococcus pneumoniae. Mol Ther Nucleic Acids. 2024 Feb 8;35(1):102145. https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(24)00032-5