Purpose: T cells are an important target for ex vivo gene delivery and editing, but most commercial reagents cannot transfect them and cationic polymers show only moderate success. The specific intracellular barriers limiting non-viral gene delivery to human T cells are poorly understood, hindering rational polymer design.
Hypothesis: If cationic polymer gene delivery to T cells is limited by specific biological barriers—rather than only polymer design—then probing uptake, endosomal acidification, immune sensing (IFITM proteins), and autophagy will identify which barriers dominate in primary human T cells and inform the design of more effective T-cell-specific polymers.
Aims: Compare polyplex uptake and transfection efficiency in HeLa, Jurkat, and primary human T cells using two cationic polymers (Comb and VIPER). - Measure intracellular endosomal pH over time in these cell types. - Evaluate the role of interferon-induced transmembrane (IFITM) proteins in limiting polyplex transfection. - Test whether pharmacological modulation of autophagy (rapamycin, 3-methyladenine) improves transfection in T cells.
Delivery system:
Component: Polymers; Details: Comb: pHEMA₂₅-g-pDMAEMA₁₆ (comb-shaped, pDMAEMA branches); VIPER: virus-inspired polymer for endosomal release (linear di-block with pH-sensitive micelle containing melittin)
Component: Payload; Details: pCMV-Luc plasmid DNA (YOYO-1 labeled for uptake); pmaxGFP plasmid DNA for transfection
Component: Polyplex formulation; Details: N/P ratio 5; formed in water or 150 mM NaCl
Component: Targeting ligand; Details: None
Component: Cell models; Details: HeLa (adherent control), Jurkat (T-cell line), primary human T cells (activated with CD3/CD28 Dynabeads)
Component: Key feature; Details: Comparison of pH-triggered release (VIPER) vs. proton-sponge/comb architecture (Comb) in T cells
Approach: In vitro only. No in vivo studies. - Uptake: 4 h incubation with YOYO-1-labeled polyplexes; trypan blue quenching of extracellular fluorescence; flow cytometry. - Transfection: 4 h in OptiMEM, then 44 h in complete medium; GFP expression by flow cytometry. - Intracellular pH: pHrodo-labeled 10 kDa dextran; flow cytometry at 15, 30, 60, 120, 240 min; calibration with pH clamping buffers and ionophores. - IFITM studies: Jurkat lines overexpressing IFITM1, IFITM2, or IFITM3; western blot confirmation; transfection and viability. - Autophagy studies: Rapamycin (10, 100 nM) and 3-methyladenine (5, 10 mM); LC3B staining and confocal microscopy; transfection and live-cell counts. - Statistics: Two-way ANOVA with Dunnett’s or Tukey’s post hoc tests; n = 3.
Key methods: Flow cytometry: polyplex uptake, GFP transfection efficiency, viability, pHrodo dextran pH measurement. - Western blotting: IFITM1/2/3 protein expression. - Confocal microscopy: LC3B staining for autophagosomes. - Transfection and viability assays: GFP expression and live/dead staining. - Intracellular pH calibration: pH clamping buffers with valinomycin/nigericin.
Key results: Uptake: Polyplex uptake was significantly lower in primary human T cells than in HeLa or Jurkat cells. VIPER uptake was higher than Comb in Jurkat and HeLa, but uptake was not predictive of transfection in primary T cells. - Transfection: VIPER transfection was very low in Jurkat (1.5%) and primary T cells (3.5%), despite good uptake in Jurkat, indicating an intracellular trafficking barrier. - Endosomal acidification: HeLa intracellular pH dropped to ~6 within 30 min; Jurkat dropped below pH 6 within 1 h; primary T cells remained above pH 6 even after 4 h. This suggests pH-triggered endosomal release (VIPER, pKa 6.4) is poorly matched to primary T cells. - IFITM proteins: Overexpression of IFITM1/2/3 caused only a 5–10% reduction in transfection with Comb or VIPER. HeLa cells had the highest IFITM expression yet transfected efficiently, ruling out IFITM as a major barrier. - Autophagy modulation: Rapamycin reduced transfection and viability in Jurkat cells. 3-MA increased the percentage of GFP-positive cells but significantly reduced total live-cell and transfected-cell counts, negating the apparent benefit. Small-molecule autophagy regulators were not viable for improving T-cell transfection.
Interpretation: The authors conclude that poor polyplex uptake and slow/less severe endosomal acidification are key barriers to cationic polymer gene delivery in primary human T cells. Polymers relying on rapid pH-triggered endosomal escape (like VIPER) may be suboptimal for T cells. IFITM proteins and autophagy modulation are not dominant barriers. Future T-cell-specific polymers should use alternative endosomal release mechanisms rather than relying on low endosomal pH.
Limitations: In vitro only: No in vivo validation or therapeutic gene editing. - Limited polymer panel: Only two polymers (Comb and VIPER) were tested. - Cell model differences: Jurkat T-cell line does not fully recapitulate primary human T cells; primary T cells were activated and from healthy donors. - No direct measurement of endosomal escape: pH and trafficking were inferred from dextran and imaging, not direct cargo release. - Pleiotropic autophagy drugs: Rapamycin and 3-MA affect mTOR/PI3K and T-cell proliferation/survival, complicating interpretation. - No gene editing or therapeutic cargo: Only reporter genes (GFP, luciferase) were used. - No long-term expression or safety data. - Small sample size: n = 3 per condition.
