Imidazole-based Synthetic Lipidoids for In Vivo mRNA Delivery into Primary T Lymphocytes

Purpose: In vivo engineering of T lymphocytes is limited by poor transfection efficiency and reliance on ex vivo electroporation. A non-viral mRNA delivery platform that can target primary T cells in vivo is needed for CAR T-cell therapy, vaccines, and immunotherapy.

Hypothesis: Lipidoids containing an imidazole head group, identified through rough-to-detailed combinatorial screening, can efficiently deliver mRNA into primary T lymphocytes ex vivo and in vivo, enabling functional gene recombination.

Aims: Screen a broad lipidoid library for mRNA delivery to primary human CD8+ T cells. - Identify key structural features, especially imidazole-containing amine heads. - Construct and screen a detailed library of imidazole analogues and carbon tails to optimize delivery. - Evaluate in vivo biodistribution and Cre mRNA-mediated gene recombination in mouse T lymphocytes after intravenous administration.

Delivery system: Platform: Synthetic lipid-like molecules (lipidoids) formulated into lipid nanoparticles (LNPs). - Chemistry: Combinatorial Michael addition of amine heads and carbon tails; imidazole or imidazole-analogue head groups. - LNP excipients: Lipidoid, cholesterol, DOPE, DSPE-PEG at 16:4:1:1 weight ratio. - Lead formulations: 93-O17S and 9322-O17S. - Payloads: Firefly luciferase mRNA, EGFP mRNA, Cre recombinase mRNA. - Targeting ligand: None; spleen accumulation was observed. - Route: Intravenous injection in mice. - Physicochemical properties: 93-O17S LNP/mRNA complex ~154 ± 2.52 nm, PDI 0.16, mRNA encapsulation ~90%.

Approach: In vitro: Primary human CD8+ T cells; FLuc and EGFP mRNA delivery; viability by CCK-8. - In vivo: BALB/c mice injected IV with FLuc mRNA-LNPs at 0.6 mg/kg; IVIS imaging at 6 h. - Gene recombination: Ai14 Cre-reporter mice injected IV with Cre mRNA-LNPs twice, every 5 days; spleen analyzed 10 days after injection. - Controls: Untreated cells/mice; ineffective LNP 9313-O18S-S as negative control. - Group sizes: Typically three separate experiments in vitro; four mice for in vivo flow cytometry.

Key methods: ESI-MS for lipid purity. - Luminescence assays for FLuc mRNA translation. - Flow cytometry for EGFP and tdTomato expression in T cell subsets. - CCK-8 for cytotoxicity. - IVIS imaging for in vivo bioluminescence and organ distribution. - Confocal microscopy for tdTomato co-localization with CD3ε, CD8a, and F4/80.

Key results: Rough screening identified imidazole head 93 with O17O, O17S, and O17Se tails as effective for mRNA delivery to primary T cells; Lipofectamine 2000 was poor. - Detailed screening identified 93-O17S and 9322-O17S as top performers. - EGFP mRNA transfection in primary human CD8+ T cells: 7.1% with 93-O17S and 11.1% with 9322-O17S. - In vivo FLuc mRNA: 93-O17S showed strong spleen-specific expression; 9322-O17S showed spleen signal 1.6-fold higher than liver. - Cre mRNA in Ai14 mice: 93-O17S achieved ~8.2% gene recombination in splenic CD4+ T cells and ~6.5% in CD8+ T cells. - Delivery was also observed in B cells, macrophages, and dendritic cells, indicating incomplete T cell specificity. - Heteroatom substitution (O or S) or disulfide bond in the tail was essential; O17C, amide-linked, and epoxide tails were ineffective.

Interpretation: Structure-based rough-to-detailed screening identified imidazole-containing lipidoids capable of delivering mRNA to primary T lymphocytes in vitro and in vivo. The lead LNP 93-O17S enabled ~8.2% gene recombination in mouse CD4+ T cells, demonstrating potential for in situ T cell engineering. Further refinement is needed to improve T cell specificity.

Limitations: Delivery was not exclusive to T cells; B cells, macrophages, and dendritic cells were also transfected. - In vivo T cell recombination efficiency remained modest (~8.2% CD4+, ~6.5% CD8+). - No therapeutic efficacy or disease model; only reporter and Cre recombination readouts. - No large-animal validation. - Long-term safety, repeated-dose toxicity, and immunogenicity were not evaluated. - Accepted Article version; final peer-reviewed version may differ. - Mechanism of delivery, pKa, and membrane disruption did not fully explain efficacy differences.