Hydroxycholesterol substitution in ionizable lipid nanoparticles for mRNA delivery to T cells

Purpose: mRNA delivery to T cells could enable ex vivo and in vivo T cell engineering, but LNPs still face poor extrahepatic delivery, endosomal recycling, and limited T cell transfection. Cholesterol analogs such as hydroxycholesterols may alter NPC1 recognition and endosomal trafficking, providing a route to improve T cell mRNA delivery.

Hypothesis: Substituting cholesterol in LNPs with specific hydroxycholesterols at certain percentages will reduce NPC1-mediated recycling, increase late endosome retention, and improve functional mRNA delivery to T cells without increasing toxicity.

Aims: Engineer a library of LNPs containing six hydroxycholesterols at 12.5%, 25%, 50%, and 100% cholesterol substitution. - Characterize LNP size, PDI, zeta potential, pKa, encapsulation, and stability. - Screen the library for luciferase mRNA delivery and viability in Jurkat T cells and primary human T cells. - Investigate endosomal trafficking using Rab5, Rab7, and Rab11 markers and colocalization with acidic organelles.

Delivery system: Platform: Ionizable lipid nanoparticles (LNPs) for mRNA delivery. - Base formulation (S2): 35% C14-4 ionizable lipid, 16% DOPE, 46.5% total sterol, 2.5% PEG. - Sterol substitutions: 7α-hydroxycholesterol (A1), 7β-hydroxycholesterol (A2), 19-hydroxycholesterol (A3), 20(S)-hydroxycholesterol (B1), 24(S)-hydroxycholesterol (B2), 25-hydroxycholesterol (B3). - Payload: Nucleoside-modified luciferase mRNA. - Preparation: Microfluidic mixing; no targeting ligand. - Target cells: Jurkat immortalized T cells and primary human CD3+ T cells.

Approach: In vitro: Jurkat cells treated at 60 ng mRNA per 60,000 cells for 24 h; luciferase expression and viability measured. - Ex vivo: Primary human T cells activated with CD3/CD28 Dynabeads; selected LNPs tested at 60–400 ng mRNA per 60,000 cells. - Endosomal trafficking: Jurkat cells treated at 60 or 150 ng mRNA for 4 h; Rab5/Rab7/Rab11 immunofluorescence and Lysotracker/DiO colocalization. - No in vivo studies were performed.

Key methods: DLS for size and PDI; zeta potential; TNS assay for pKa; RiboGreen assay for encapsulation efficiency. - Luciferase luminescence assay for functional mRNA delivery; CellTiter-Glo for viability. - Confocal microscopy for LNP–acidic organelle colocalization. - Immunofluorescence for Rab5, Rab7, and Rab11; Fiji image analysis with Spearman’s rank correlation and integrated density.

Key results: In primary human T cells, A1-25 and A1-50 improved mRNA delivery by 1.8-fold and 2.0-fold, respectively, vs S2. - In Jurkat cells, A1-25, A1-50, and B1-50 increased luciferase expression 2.1-fold, 1.9-fold, and 1.7-fold, respectively. - No hydroxycholesterol LNP caused significant cytotoxicity; viability remained similar to S2. - Most LNPs remained stable over 28 days; body-modified hydroxycholesterols were generally stable, while tail-modified B2/B3 showed larger size and higher PDI. - A1-25 increased colocalization with acidic organelles and late endosomes (Rab7) at low dose, and reduced recycling endosomes (Rab11). At high dose, A1-25 and A1-50 increased Rab5 and decreased Rab11.

Interpretation: Hydroxycholesterol substitution, especially 7α-hydroxycholesterol at 25–50%, improves mRNA delivery to T cells without increasing toxicity. The mechanism involves increased late endosome production and reduced endosomal recycling, supporting further development for T cell immunotherapies such as CAR T cell engineering and vaccines.

Limitations: No in vivo delivery, biodistribution, or safety data. - Only one base LNP formulation was tested. - No disease model or functional CAR T cell efficacy endpoint. - Primary human T cell data showed patient-to-patient variability. - Mechanism of improved delivery is not fully resolved; LNP morphology was not characterized. - Long-term stability was assessed for only 28 days. - No large-animal validation.