Non-Viral Gene Delivery to T Cells with Lipofectamine LTX

Purpose: Retroviral gene delivery for T cell therapies is expensive, semi-randomly integrating, and variable between patients. Non-viral alternatives are needed, but T cells are notoriously hard to transfect, especially primary T cells.

Hypothesis: If Lipofectamine LTX is used with optimized transfection variables—vehicle, promoter, media, and activation timing—then transfection efficiency in Jurkat and primary T cells can be improved. Low efficiency in primary T cells may be due to limited cellular uptake, low heparan sulfate proteoglycan (HSPG) expression, and/or cytoplasmic DNA sensing by PYHIN sensors.

Aims: Compare non-viral vehicles (cationic polymers vs. Lipofectamine LTX) for pDNA delivery to Jurkat T cells. - Optimize promoters, media, Lipofectamine dose, and activation timing for transfection. - Evaluate transfection efficiency in primary CD3+ T cells and track plasmid uptake/localization. - Analyze transcriptomes of PC-3, Jurkat, and primary T cells to identify mechanisms of transfection resistance.

Delivery system: Platform: Lipid-based lipoplex; Lipofectamine LTX + plasmid DNA. - Mechanism: Cationic liposomes complex with negatively charged pDNA; uptake via endocytosis; endosomal escape via proton-sponge effect. - Payloads: pEF-GFP, pEF-Luc, pGL4.50, fluorescein-labeled pDNA, EGFP nanoplasmid. - Comparator vehicles: Branched PEI, linear PEI, jetPEI, TurboFect. - Targeting ligand: None. - Cells: Jurkat T cell line, primary human CD3+ T cells, PC-3 prostate cancer cells as positive control. - Route/model: In vitro only; no in vivo studies.

Approach: Jurkat transfections: 24-well plates, 2 × 10⁵ cells/well, 1 µg pDNA/well; Lipofectamine LTX “Lipo-Hi” = 2.75 µL/well, “Lipo-Lo” = 1.25 µL/well. - Primary T cells: CD3+ T cells from three healthy donors; activated with CD3/CD28 Dynabeads + IL-2; transfected in X-VIVO 15 media. - Variables tested: vehicle, N:P ratio, promoter (EF1α, CAG, CMV), serum presence, media type, Lipofectamine dose, days post-activation. - Readouts: luciferase assay, flow cytometry for EGFP, MTT, Annexin V/PI, fluorescent pDNA tracking, confocal microscopy, mRNA sequencing. - Controls: untransfected cells, PC-3 positive control, no-plasmid controls.

Key methods: Luciferase assay for transgene expression. - Flow cytometry for %EGFP+ cells and mean fluorescence. - MTT assay for metabolic activity. - Annexin V/propidium iodide staining for viability/apoptosis. - Fluorescein-labeled pDNA transfection with trypsinization to distinguish surface-bound vs. internalized lipoplexes. - Confocal microscopy with Hoechst 33342 and CellBrite Red to visualize plasmid localization. - mRNA sequencing and FPKM analysis for HSPGs, syndecans, PYHIN DNA sensors, and interferon-related genes.

Key results: Lipofectamine LTX was the best vehicle in Jurkat cells; optimized conditions in X-VIVO media yielded 63.0 ± 10.9% EGFP+ Jurkat cells vs. 23.1 ± 5.5% in RPMI. - Primary CD3+ T cells reached only 8.1 ± 0.8% EGFP+ under the same optimized conditions. - PC-3 cells: 91 ± 1.1% EGFP+ and 96 ± 0.94% fluorescein+; Jurkat: 51 ± 9.7% EGFP+ and 74 ± 4.5% fluorescein+; primary T cells: 51 ± 11.2% fluorescein+ but only 8.1 ± 0.8% EGFP+. - Trypsinization significantly reduced fluorescein signal in Jurkat and primary T cells, but not PC-3 cells; confocal microscopy showed lipoplexes stuck on the primary T cell membrane. - EF1α promoter gave highest luciferase expression; CMV was significantly lower. Transfection was highest on Day 0/1 after activation and decreased on Days 2–3. - MTT showed 30–40% lower metabolic activity with vehicles; no significant differences among vehicles. - Transcriptome: PC-3 expressed HSPG2 and all syndecans; Jurkat and primary T cells mostly lacked them. Primary T cells constitutively expressed PYHIN DNA sensors AIM2, IFI16, and PYHIN1, which may induce apoptosis or repress transcription.

Interpretation: Lipofectamine LTX can transfect Jurkat T cells moderately well but primary T cells poorly. The authors propose that low primary T cell transfection is limited by poor cellular uptake due to low HSPG/syndecan expression and by high constitutive PYHIN DNA sensor expression, which may induce apoptosis or transcriptional repression. These factors should be considered in future non-viral and viral T cell gene delivery strategies.

Limitations: In vitro only; no in vivo delivery, biodistribution, or CAR-T functional efficacy. - Only three healthy donors for primary T cells; donor variability not extensively addressed. - Mechanistic roles of HSPGs and PYHIN sensors were inferred from transcriptomics, not confirmed by knockout, knockdown, or overexpression. - Limited vehicle comparison; no direct side-by-side electroporation or lentiviral transduction in the same experiments. - No long-term transgene expression, repeated transfection, or safety/toxicity studies. - Bulk mRNA sequencing, not single-cell; no protein-level validation of key resistance genes. - No targeted delivery or in vivo tumor model.