Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering

Purpose: CAR T cell therapy currently relies on viral vectors or electroporation for T cell engineering. Viral vectors cause permanent CAR expression and carry safety/manufacturing limitations, while electroporation is cytotoxic and requires specialized equipment. A safer, efficient non-viral mRNA delivery platform for human T cells is needed.

Hypothesis: Ionizable lipid nanoparticles (LNPs) can deliver mRNA to human T cells ex vivo, induce functional CAR expression at levels comparable to electroporation, and cause substantially lower cytotoxicity.

Aims: Synthesize a library of 24 ionizable lipids and formulate them into LNPs. - Screen LNPs for luciferase mRNA delivery to Jurkat T cells and identify top formulations. - Optimize the lead LNP (C14–4) by purifying the ionizable lipid. - Deliver CAR mRNA to primary human T cells and compare CAR expression, viability, and cancer-killing function against electroporation.

Delivery system: Platform: Ionizable lipid nanoparticles (LNPs) for mRNA delivery. - Lead ionizable lipid: C14–4; also screened C12, C14, and C16 alkyl-chain variants. - LNP excipients: DOPE, cholesterol, PEG-lipid; typical molar ratio 35% ionizable lipid, 16% DOPE, 46.5% cholesterol, 2.5% PEG. - Payload: Luciferase mRNA or CD19-targeting CAR mRNA; mRNA contained N1-methylpseudouridine and 5-methylcytosine modifications. - Formulation: Microfluidic mixing of aqueous mRNA and ethanolic lipid phases. - Targeting ligand: None. - Application: Ex vivo human T cell engineering; no in vivo delivery tested.

Approach: In vitro screen: Jurkat immortalized human T cells; 24 LNPs screened with luciferase mRNA at 30 ng/60,000 cells; compared to lipofectamine. - Primary human T cells: CD4+ and CD8+ T cells isolated from healthy donors, combined 1:1, activated with CD3/CD28 Dynabeads. - Lead optimization: Crude vs purified C14–4 LNPs compared for luciferase and CAR mRNA delivery. - Functional assay: CAR T cells cocultured with luciferase-expressing Nalm-6 acute lymphoblastic leukemia cells at varying effector-to-target ratios for 48 h. - Comparison: Electroporation and lentiviral transduction used as benchmarks. - Replicates: Typically n = 3–4 biological replicates.

Key methods: Dynamic light scattering (DLS) for LNP size and PDI. - RiboGreen assay for mRNA encapsulation efficiency. - TNS assay for LNP pKa. - Luciferase luminescence assay for functional mRNA delivery. - CellTiter-Glo assay for viability. - Flow cytometry for surface CAR expression. - Coculture killing assay with luciferase-expressing Nalm-6 cells.

Key results: Seven LNP formulations enhanced luciferase mRNA delivery to Jurkat cells over lipofectamine; C14–4 was the top performer. - Purified C14–4 LNPs: diameter ~65 nm, mRNA encapsulation 86.3%, pKa 6.505; crude C14–4: encapsulation 92.5%, pKa 6.143. - In primary human T cells, purified C14–4 LNPs produced a 9.4-fold increase in CAR surface MFI vs untreated cells; electroporation produced a 10-fold increase. - Viability: purified C14–4 LNPs ~76–78% vs electroporation ~31%. - CAR T cells generated with purified C14–4 LNPs killed Nalm-6 ALL cells in coculture as effectively as electroporated CAR T cells. - Luciferase expression was transient, decreasing ~23% after 48 h and declining further over 96 h.

Interpretation: LNPs can deliver mRNA to primary human T cells ex vivo and generate functional CAR T cells with potency comparable to electroporation but much lower cytotoxicity. This supports LNPs as a promising non-viral platform for mRNA-based CAR T cell engineering and potentially broader T cell engineering applications.

Limitations: Ex vivo study only; no in vivo delivery, biodistribution, or antitumor efficacy. - mRNA CAR expression is transient and would require repeated dosing for sustained effect. - Primary T cells required CD3/CD28 activation before LNP transfection. - Only CD19 CAR and Nalm-6 coculture were tested; no animal tumor model. - No long-term persistence, memory, or exhaustion analysis. - No large-scale manufacturing or clinical translation validation. - No active targeting ligand; T cell delivery was non-specific in the ex vivo setting. - Long-term safety and immunogenicity of repeated LNP dosing were not evaluated.