Abstract by Simon Pápai

RNA-based therapeutics represents a versatile and transformative modality for treating a wide range of diseases. The clinical success of messenger RNA (mRNA) vaccines during the coronavirus disease 2019 pandemic demonstrated the safety and efficacy of lipid nanoparticle (LNP)-mediated mRNA delivery, paving the way for RNA-loaded LNPs (mRNA-LNPs) in a broad range of applications, e.g., protein replacement therapy, vaccination, immunotherapy, and gene editing.

Advancing the LNP design to achieve precise organ tropism and efficient cellular delivery is essential to unlock the full therapeutic potential of mRNA, particularly for extrahepatic applications. Optimizing the composition of the four LNP components, i.e., the ionizable lipid, helper lipid, sterol, and PEG-lipid, enable modulation of the delivery efficiency and tissue specificity. The LNP biodistribution is governed by passive, endogenous, and active targeting mechanisms that can be exploited to enhance organ- and cell-specific delivery. The particle size influences passive extravasation, while surface charge-mediated plasma protein binding determines the biological fate via receptor interactions. Targeting ligands can enhance tissue- and cell-specific uptake and reduce off-target delivery.

This thesis investigated strategies to rationally modify mRNA-LNPs to enhance organ-specific mRNA delivery, providing insights for increasing the therapeutic index. I hypothesized that modifications of safe and clinically approved heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102)-based LNPs can enhance tissue- and cell-specific mRNA delivery to: (i) the liver through systematic replacement of cholesterol and the phospholipid component, and (ii) the lungs via surface functionalization with cationic peptides.

Substituting cholesterol with the plant-derived cholesterol analogue β-sitosterol in clinically advanced SM-102- and [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315)-based LNPs enhanced the mRNA transfection efficiency 43-fold in a murine hepatocyte cell line without compromising cell viability. In vivo, β-sitosterol-modified SM-102 LNPs loaded with firefly luciferase (fluc) mRNA increased hepatic expression by 5.3-fold and reduced splenic uptake, achieving a 30-fold higher liver-to-spleen radiance ratio. Comparable effects were observed with ALC-0315 LNPs, which exhibited an 8.1-fold increase in hepatic bioluminescence. β-sitosterol-modified SM-102 LNPs loaded with erythropoietin (epo) mRNA mediated a 4.15-fold increase in circulating EPO levels, highlighting the potential of rational lipid substitution to repurpose clinically approved LNPs for therapeutic application. Replacing the helper lipid distearoylphosphatidylcholine with 9A1P9, which is a phospholipid that enhances endosomal escape, increased hepatic FLuc expression by 6.4-fold and improved the liver-to-spleen radiance ratio by 6.6-fold, demonstrating an additional strategy to enhance the protein expression and organ specificity of SM-102 LNPs in vivo.

Delivery of mRNA to the liver is efficient due to the inherent liver tropism of LNPs, but selective targeting of extrahepatic tissues remains a major challenge. Surface functionalization of SM-102 LNPs with cationic peptides, including an ApoE-derived peptide (ApoE-P) and scrambled variants, enhanced in vitro mRNA transfection in multiple cell lines, reaching over a 100-fold increase in bioluminescence. These cationic peptide-functionalized LNPs redirected the in vivo tropism, enabling efficient targeting of lung and brain tissue in a peptide density-dependent manner. Further optimization of the lipid composition resulted in a lung-to-liver radiance ratio of 13:1. Both ApoE-P and a scrambled variant mediated similar organ biodistribution and FLuc expression levels, which were shown, at least partially, to arise from charge-dependent adsorption of vitronectin in the protein corona, rather than sequence-specific mechanisms. Peptide-functionalized LNPs mediated cre mRNA delivery to pulmonary endothelial and epithelial cells, as well as brain endothelial cells, and delivery of cas9 mRNA resulted in up to 57 % gene editing in lung endothelial cells. These findings demonstrate that surface peptide engineering is a simple, modular, and safe strategy to tailor LNP biodistribution for extrahepatic mRNA delivery.

This thesis demonstrates that rational modification of clinically approved LNPs enhances mRNA delivery and modulates organ specificity. Substitution of cholesterol with β-sitosterol increased hepatic expression and reduced splenic uptake, and cationic peptide functionalization redirected LNPs to the lungs and brain. These strategies lay the groundwork for therapeutic applications, expanding the potential of mRNA-LNPs for diseases such as cancer.