Badarau, A.; Bauer, B.; Visram, Z.; Qiao, R.; Majoros-Hashempour, A.; Söllner, J.; Durica-Mitic, S.; Dunne, O. M.; Kluj, R.; Kestemont, D.; Kieninger, A.-K.; Kulig, M.; Berdaguer, R.; Schwebs, T.; Zerbs, M.; Czermak, P.; von Freyberg, M.; Schmidt, J.; Schmidberger, L.; Mayer, D.; Protano, M.; Miller, H. A.; Palowitch, G. M.; Dulberger, C. L.; Massaro, M.; Ivanisenko, N.; Jacomet, H.; Ngatcha Bakoue, D.; Oteri, F.; Sela, G.; Corsini, L.

Bacterial lysins are promising novel antimicrobials but are limited by poor pharmacokinetics and challenging manufacturability. We developed a lysin discovery platform tailored for lysin delivery via mRNA: staphylococcal LysM-CHAP (cysteine, histidine-dependent amidohydrolases/peptidase) autolysin was selected and its serum half-life extended via Immunoglobulin G1-Fc-fusion. The Fc-induced drop in lysin potency was rescued by the concerted optimization of linkers, binding kinetics and catalytic activity, using a combination of rational and AI-guided approaches. The engineered Fc-LysM-CHAP was active against planktonic bacteria (minimum inhibitory concentration of 1-2 ug/mL) and simulated endocardial vegetations and synergized strongly (Fractional eradication concentration index FECI = 0.06) with cell wall active antibiotics in vitro. In mouse models of Staphylococcus aureus sepsis, the recombinant Fc-LysM-CHAP - antibiotic combination was superior to single agent treatments and mRNA-delivered Fc-LysM-CHAP showed single agent activity at a mRNA-lipid nanoparticle dose as low as 0.2 mg/kg.