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Updated: May 2, 2026
Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli
Published on: March 20, 2016
Synthetic mRNA delivered to human cells leads to expression of Cpl-1 bacteriophage-endolysin with activity against
Moritz K Jansson1, Dat Tien Nguyen1, Stefan Mikkat2
1Institute of Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Rostock, Germany.
Abstract:
Endolysins are bacteriophage-encoded hydrolases that show high antibacterial activity and a narrow substrate spectrum. We hypothesize that an mRNA-based approach to endolysin therapy can overcome some challenges of conventional endolysin therapy, namely organ targeting and bioavailability. We show that synthetic mRNA applied to three human cell lines (HEK293T, A549, HepG2 cells) leads to expression and cytosolic accumulation of the Cpl-1 endolysin with activity against Streptococcus pneumoniae. Addition of a human lysozyme signal peptide sequence translocates the Cpl-1 to the endoplasmic reticulum leading to secretion (hlySP-sCpl-1). The pneumococcal killing effect of hlySP-sCpl-1 was enhanced by introduction of a point mutation to avoid N-linked-glycosylation. hlySP-sCpl-1N215D, collected from the culture supernatant of A549 cells 6 h post-transfection showed a significant killing effect and was active against nine pneumococcal strains. mRNA-based cytosolic Cpl-1 and secretory hlySP-sCpl-1N215D show potential for innovative treatment strategies against pneumococcal disease and, to our best knowledge, represent the first approach to mRNA-based endolysin therapy. We assume that many other bacterial pathogens could be targeted with this novel approach.
Insights
Messenger RNA (mRNA) therapy enables endolysin expression for treating pneumococcal disease. This novel approach enhances endolysin delivery and antibacterial activity, offering a promising alternative to conventional methods.
Area of Science:
- Microbiology
- Biotechnology
- Molecular Biology
Background:
- Endolysins, derived from bacteriophages, possess potent antibacterial properties but face challenges in targeted delivery and bioavailability.
- Conventional endolysin therapies are limited by issues such as organ targeting and bioavailability.
Purpose of the Study:
- To investigate the potential of an mRNA-based approach for endolysin therapy to overcome limitations of conventional methods.
- To evaluate the expression, localization, and antibacterial efficacy of endolysins produced via mRNA delivery in human cell lines.
Main Methods:
- Synthetic mRNA encoding the Cpl-1 endolysin was introduced into human cell lines (HEK293T, A549, HepG2).
- A human lysozyme signal peptide was incorporated to facilitate secretion of the endolysin (hlySP-sCpl-1).
- A point mutation (N215D) was introduced to prevent N-linked glycosylation, enhancing the efficacy of the secreted endolysin (hlySP-sCpl-1N215D).
Main Results:
- Successful expression and cytosolic accumulation of Cpl-1 endolysin were observed in human cell lines.
- The modified, secreted endolysin (hlySP-sCpl-1N215D) demonstrated significant killing activity against multiple strains of *Streptococcus pneumoniae*.
- The mRNA-based endolysin therapy showed potential for treating pneumococcal infections.
Conclusions:
- mRNA-based endolysin therapy represents a novel strategy for combating bacterial pathogens, particularly *Streptococcus pneumoniae*.
- This approach offers enhanced bioavailability and targeted delivery, addressing key challenges in endolysin therapy.
- The methodology holds promise for developing innovative treatments against a wide range of bacterial infections.
