Naturally-Occurring Cholesterol Analogues in Lipid Nanoparticles Induce Polymorphic Shape and Enhance Intracellular Delivery of mRNA

Purpose: Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery, with only <2% of LNPs reaching the cytosol. The role of cholesterol structure in LNP-mediated mRNA delivery and endosomal escape was poorly understood. There is a need to decode the structural characteristics of cholesterol that are crucial for efficient intracellular delivery and improved gene transfection.

Hypothesis: If cholesterol is replaced with naturally-occurring C-24 alkyl phytosterol analogs (e.g., β-sitosterol) in LNPs (creating eLNPs), then the resulting nanoparticles will have altered surface morphology (polyhedral shape), enhanced cellular uptake and retention, increased intracellular diffusivity, and improved endosomal escape, leading to enhanced mRNA transfection. The C-24 alkyl tail, flexible sterol ring, and free C-3 hydroxyl group are required for this enhancement.

Aims: Screen a library of natural cholesterol analogs (Groups I–III) for mRNA encapsulation and transfection efficiency. - Identify structural features of cholesterol analogs critical for enhanced gene delivery (head, body, tail). - Characterize the structural and morphological differences between LNPs and eLNPs using Cryo-TEM and SAXS. - Investigate intracellular trafficking, uptake, retention, and endosomal escape of eLNPs vs LNPs. - Evaluate the role of NPC1/NPC2 cholesterol transporters in eLNP retention and transfection.

Approach: In vitro only. No in vivo animal studies. - Cell lines: HeLa cells; five human patient-derived lysosomal storage disease cell lines; J774A.1 and RAW264.7 mouse macrophages; ex vivo human peripheral blood macrophages. - Transfection: Luciferase mRNA at 10–400 ng mRNA per well; Cas9 mRNA for gene editing; siRNA for silencing. - Uptake/trafficking: High-content imaging, confocal microscopy, smFISH, 3D-DyPLoT single-particle tracking. - NPC studies: Cell lines with loss-of-function mutations in NPC1 or NPC2. - Statistics: Multiple t-test, unpaired t-test, nonlinear regression with ANOVA; p ≤ 0.05.

Key methods: Screening: Luciferase assay, encapsulation efficiency (RiboGreen), DLS for size. - Morphology: Cryo-TEM for core-shell structure and surface faceting; SAXS for internal lamellar organization. - Cellular uptake: High-content imaging with Cy5-EGFR mRNA; rhodamine-DOPE for uptake kinetics. - Endosomal escape: smFISH with Stellaris probes; ratio of cytosolic mRNA to internalized LNPs. - Intracellular transport: 3D-Dynamic Photon Localization Tracking (3D-DyPLoT) for real-time 3D single-particle tracking; linear efficiency analysis. - Gene editing: FACS for frameshift mutations (Cas9); luciferase silencing (siRNA). - NPC studies: Transfection in NPC1/NPC2-deficient cell lines.

Key results: Screening: β-sitosterol LNPs (eLNPs) showed up to 211-fold improvement in transfection vs cholesterol LNPs, with comparable size (~100 nm) and encapsulation (>90%). Group I (Vitamin D analogs) and Group III (5th ring analogs) failed. - Structural requirements: C-24 alkyl (ethyl/methyl) group, flexible body/tail, and free C-3 -OH head group are required for enhanced transfection. Rigid analogs (brassicasterol, ergosterol, 9,11-dehydroergosterol) showed moderate to no transfection; head modifications (polar/non-polar conjugates) abolished activity. - Morphology: Cryo-TEM showed eLNPs have a polyhedral/faceted shape vs spherical LNPs. SAXS showed similar internal lamellar structure but smaller d-spacing for eLNPs (6.64 nm vs 7.22 nm for MC3). - Uptake/retention: eLNPs showed higher rate of uptake and retention over 24 h compared to LNPs. At 400 ng, eLNPs showed significantly higher cellular uptake at 24 h. - Intracellular transport: 3D-DyPLoT showed eLNPs have significantly more mobile/linear trajectories (mobile fraction p = 0.0009) vs LNPs, suggesting directed transport to productive pathways. - NPC studies: NPC1 deficiency enhanced transfection for both LNPs and eLNPs; NPC2 deficiency showed lower relative enhancement for eLNPs, indicating differential interaction with endosomal efflux machinery. - Generalizability: eLNPs enhanced transfection across eight additional cell types, with DODMA (48-fold) and Lipid 9 (32-fold at 6 h) ionizable lipids, and for Cas9 mRNA (2.5-fold gene editing) and siRNA (6.5-fold silencing).

Interpretation: The authors claim that C-24 alkyl phytosterols (e.g., β-sitosterol) in LNPs induce a polyhedral surface morphology with packing defects that enhance fusogenic properties, cellular uptake, retention, and intracellular trafficking to productive pathways, leading to improved endosomal escape and mRNA delivery. The findings emphasize the importance of cholesterol structure in subcellular transport and LNP surface composition/structure for designing improved endosomal escape.

Limitations: In vitro only: No in vivo validation, biodistribution, or therapeutic efficacy. - Mechanism not fully resolved: Exact role of surface morphology, lipid composition, and NPC interactions remains unclear. - Variability: Fold-differences varied across experiments due to scale-up, batch quality of phytosterols, purification, and cell types. - Ionizable lipid dominates: Lipid 9-eLNP showed higher transfection than MC3-eLNP, suggesting the ionizable lipid remains the main driver of transfection efficiency. - No long-term safety or toxicity data. - No comparison with clinically approved LNP formulations (e.g., Onpattro). - Single time points for some assays (e.g., 24 h uptake); longer-term retention not assessed. - No in vivo gene editing or therapeutic protein expression data. - Patent/licensing interests: Several authors are Moderna Therapeutics employees/stockholders; OSU and Moderna have a licensing agreement.