Froelich, B. | bioRxiv Science

bioRxiv2025

PEG-free, Triphosphate-Stabilized LNPs Enable Potent RNA Delivery

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/695515v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@d919d8org.highwire.dtl.DTLVardef@12fe9aforg.highwire.dtl.DTLVardef@96e2d2org.highwire.dtl.DTLVardef@54f73_HPS_FORMAT_FIGEXP M_FIG C_FIG Polyethylene glycol (PEG)-lipids have long enabled lipid nanoparticle (LNP) formulations by providing steric stabilization and prolonged circulation. However, anti-PEG immune responses may impede repeated systemic dosing in mRNA therapeutics due to the phenomenon known as accelerated blood clearance (ABC). Here, we introduce a PEG-free, polyanionic alternative in which sodium triphosphate (3P) electrostatically associates with ionizable surface lipids, conferring long-term colloidal stability through charge repulsion rather than steric shielding. 3P-LNPs maintained size uniformity, morphology, and mRNA integrity for over nine months at 4 {degrees}C. In a proof-of-concept study involving a single intravenous injection of LNPs in mice carrying a luciferase and a GFP transgene, early expression was approximately two-fold higher compared to PEG-LNPs, while expression levels at 24 hours and hepatic tolerability remained comparable. Serum enzyme and cytokine profiles indicated no necrosis or inflammation. These findings establish 3P-LNPs as a stable, biocompatible platform free of PEG, with potential for repeat systemic mRNA administration while avoiding PEG-related effects.

biochemistry↗

bioRxiv2024

Insights into the Contribution of Type VI Secretion Towards AHPND Pathogenesis

The Type VI Secretion System (T6SS) is a contractile injection apparatus which delivers effector toxins in a contact-dependent manner, and has drawn considerable attention as a determinant of pathogenicity and environmental fitness in diverse bacterial taxa. Past reports have noted that many strains of Vibrio parahaemolyticus which cause Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp harbor two functional T6SSs (T6SS1 and T6SS2), leading to speculation that these systems may contribute to virulence. Here, we evaluate the involvement of Type VI Secretion (T6S) as a pathogenicity factor during shrimp infection by two geographically distinct isolates of AHPND-causing V. parahaemolyticus (VpAHPND). Combining in vitro contact-killing assays with comparative genomic analysis, we demonstrate that these strains -- 13-306/D4 (Mexico) and 12-297/B (Vietnam) -- leverage overlapping yet distinct effector arsenals to antagonize bacterial and fungal prey under warm, high-salinity conditions. Moreover, our in silico analyses reveal a predicted T6SS effector/immunity (E/I) module encoded in proximity to the pirAB-Tn903 composite transposon on pVA1-type plasmids. We demonstrate that acquisition of a plasmid expressing this module allows the non-AHPND isolate V. parahaemolyticus BB22OP to antagonize its parental strain in a T6SS1-dependent fashion. In turn, prey equipped with a plasmid expressing only the immunity gene gain protection against BB22OP attackers armed with the full E/I module. Employing an immersion bioassay, we show that both D4 and 297B require T6S functionality for full virulence against whiteleg shrimp (Litopenaeus vannamei) postlarvae under warm, marine-like conditions, and provide preliminary evidence that T6SS-dependent interactions with host-associated bacteria may contribute to virulence. Our findings offer empirical evidence supporting the involvement of T6S in AHPND pathogenesis under conditions relevant to commercial aquaculture, inviting further studies to decipher the role/s of T6S as a virulence determinant.

microbiology↗