Purpose: CAR T cell immunotherapy requires efficient ex vivo gene delivery to primary human T cells, but viral vectors have limitations including cost, cargo size, insertional mutagenesis risk, and manufacturing complexity. Non-viral cationic polymers are attractive alternatives but historically show low gene transfer efficiency in T cells. There is a need to identify polymer architectures and transfection protocols that work effectively in cultured and primary human T cells.
Hypothesis: A panel of cationic polymers—particularly comb- and sunflower-shaped pHEMA-g-pDMAEMA architectures—can mediate efficient gene delivery to cultured and primary human T cells with minimal toxicity, and optimized transfection conditions will enable delivery of both plasmid DNA and mRNA to CD4⁺ and CD8⁺ primary T cells.
Aims: Synthesize and screen a panel of cationic polymers for transfection efficiency and cytotoxicity in the Jurkat human T cell line. - Identify optimal polymer architectures for T cell gene delivery. - Optimize primary human T cell transfection conditions, including activation time, cell density, DNA dose, culture media, and cytokine treatment. - Demonstrate delivery of mRNA and plasmid DNA to CD4⁺ and CD8⁺ primary human T cells.
Delivery system:
Component: Polymer panel; Details: Branched PEI (bPEI); VIPER (virus-inspired polymer for endosomal release); linear pDMAEMA (LP-290); comb pHEMA-g-pDMAEMA (CP-25-12, CP-25-16, CP-25-21, CP-15-28, CP-15-50); sunflower pHEMA-g-pDMAEMA (SP-25-11, SP-25-16, SP-25-22)
Component: Lead polymer; Details: Comb polymer CP-25-16 (pHEMA₂₅-g-pDMAEMA₁₆)
Component: Payload; Details: pmaxGFP plasmid DNA; eGFP mRNA
Component: Complexation; Details: Electrostatic self-assembly into polyplexes; N/P ratios 3, 5, 7
Component: Targeting ligand; Details: None
Component: Transfection media; Details: OptiMEM, Jurkat culture medium, or T cell culture medium
Component: Primary T cell activation; Details: CD3/CD28 Dynabeads; cytokines IL-2 (200 IU/mL) or IL-21 (10 ng/mL)
Approach: In vitro only. No in vivo studies. - Cell models: Jurkat human T lymphocyte line; primary human T lymphocytes from healthy donors (CD4⁺ and CD8⁺). - Jurkat transfection: 250K cells/well; 1.5 µg nucleic acid; 48 h; flow cytometry for GFP and viability. - Primary T cell transfection: 500K–750K cells/well; 2 µg DNA (optimal); 48 h post-activation; OptiMEM; IL-21 supplement; flow cytometry for GFP, CD4, CD8, and viability. - Controls: Cells only, DNA only, bPEI, and untransfected controls. - Optimization: DOE-style screening of cell density, DNA dose, transfection medium, cytokine, and activation time.
Key methods: Polymer characterization: GPC and ¹H NMR. - Polyplex characterization: Dynamic light scattering (DLS) and zeta potential. - Transfection efficiency: Flow cytometry for GFP⁺ live cells. - Viability: Zombie Violet fixable live/dead stain. - T cell subset analysis: Anti-human CD4 and CD8 antibody staining. - Statistical analysis: Multiple t tests or one-way ANOVA with Tukey’s or Dunnett’s post hoc tests.
Key results: Jurkat T cell line: Comb and sunflower polymers mediated transfection up to 50% in serum-free medium with >90% viability. Linear pDMAEMA and VIPER performed poorly in T cells despite high efficacy in HeLa cells. - Lead polymer: CP-25-16 achieved the highest transfection in Jurkat cells. In serum-free medium at N/P 5, CP-25-16 reached ~32% transfection; SP-25-16 reached ~12%. - Primary human T cells: Maximal plasmid DNA transfection was 18%; mRNA transfection reached 25%, with >75% viability. - CD4⁺ vs CD8⁺: Similar transfection efficiencies for pDNA; mRNA delivery was slightly higher in CD4⁺ T cells. - Optimal conditions: 500–750K cells/well, 2 µg DNA, 48 h post-activation, OptiMEM, IL-21 supplement. Higher DNA dose (3 µg) reduced viability and did not improve efficiency. - Activation time: Maximum transfection at 48 h post-activation; quiescent/unactivated T cells showed negligible expression.
Interpretation: The authors claim that comb pHEMA-g-pDMAEMA polymers, especially CP-25-16, are promising non-viral vectors for ex vivo genetic reprogramming of primary human T cells for CAR T cell manufacturing. They suggest these polymers could reduce cost and improve safety compared with viral vectors, and may be useful for screening new CAR constructs in Jurkat cells. Further polymer engineering is needed for in vivo delivery.
Limitations: In vitro only: No in vivo validation or CAR T functional studies. - Modest efficiency: Primary T cell transfection (18% pDNA, 25% mRNA) remains below viral vector efficiency. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions. - No stable integration or gene editing demonstrated. - Requires ex vivo activation and manipulation, which may affect T cell phenotype. - No long-term expression or safety data. - Patent application filed on sunflower and comb polymers by the University of Washington.
