Formulation optimisation of an mRNA-lipid nanoparticle platform sustains in vivo production of the SARS-CoV-2-neutralizing nanobody VHH 72

Neutralizing antibodies act rapidly against SARS-CoV-2, but monoclonal antibodies are costly to manufacture and single-domain antibodies are cleared rapidly. Here we evaluated an mRNA-lipid nanoparticle (LNP) platform encoding the neutralizing nanobody VHH 72 and optimized the carrier by raising DSPC from 10 to 40 mol% and the citrate buffer from 25 to 300 mM. Relative to 10%PC-25mM, 40%PC-300mM produced larger particles (approximately 193 versus 75 nm), lower polydispersity, high encapsulation efficiency and better mRNA preservation in 50% serum (approximately 80% versus 43% at 24 h). Molecular dynamics simulations of the two lipid compositions gave structural descriptors consistent with tighter packing at 40 mol% DSPC, but did not address free energy or the citrate effect. In HEK293T cells, 40%PC-300mM increased VHH 72-eGFP-positive cells at 24, 48 and 72 h and cell-associated fusion protein at 72 h. In CD-1 mice, DiD-associated fluorescence declined more slowly in blood and was higher in liver through 72 h and spleen through 48 h; a luciferase reporter showed higher organ expression and a whole-body advantage through day 5. Serum VHH 72 peaked later and higher with 40%PC-300mM, and neutralizing activity persisted through day 7. In K18-hACE2 mice challenged intraperitoneally or intratracheally 1, 3 or 7 days after treatment, 40%PC-300mM gave the lowest blood and lung pseudovirus reporter signals at day 3 and was the only treatment significantly below PBS at day 7. These data support formulation optimization as a way to improve mRNA protection, reporter expression and the duration of functional VHH 72 exposure. Because the study used reporter constructs, a lipophilic dye, pseudovirus and mice, it does not establish intact-LNP biodistribution or protection against authentic SARS-CoV-2 infection.

Abstract from PubMed (PMID 42962583). This entry was added automatically by our daily literature monitor because it matches the topics we follow; read the full paper at the original source.