Frohns, F. | Search bioRxiv

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↗

bioRxiv2025

Nek family members regulate Rad54 during homologous recombination in developing mice

Homologous recombination (HR) represents an important pathway for repairing DNA double-strand breaks (DSBs) but HR factors, including RAD51, also serve to protect and restart stalled replication forks. RAD54 functions during DSB repair where it removes RAD51 from duplex DNA including heteroduplex DNA which is formed during D-loop formation. This allows subsequent DNA repair synthesis and completion of the HR process. We have previously suggested that RAD54s activity is regulated by never-in-mitosis-gene A (NIMA) related kinase 1 (NEK1) in a cell cycle-specific manner to promote RAD51 removal and HR in G2 phase without interfering with RAD51s fork stabilization role during S phase. Here, we establish that Nek1 regulates the phosphorylation of Rad54 at Ser572 (S572) to promote HR in vivo in adult mice and in vitro in fibroblasts derived from such mice. In contrast, embryonic mice and fibroblasts derived from them do not require Nek1 for HR. We further show that HR requires Rad54 phosphorylation at S572 both in embryonic and adult fibroblasts and that this is mediated in embryonic fibroblasts by Nek3 and Nek5 instead of Nek1. Thus, our work identifies a developmental change in the regulation of HR and uncovers two new factors involved in this process.

cell biology↗