Polymer- and lipid-based gene delivery technology for CAR T cell therapy

Purpose: CAR T cell therapy is FDA/EMA-approved for B cell malignancies and multiple myeloma, but manufacturing relies on viral vectors—associated with safety concerns, high cost, and production challenges—or electroporation, which can be highly cytotoxic. Nanosystems may offer a safer, cost-effective alternative, but T cells are difficult to transfect, so rational design of lipid- and polymer-based carriers is urgently needed.

Hypothesis: As a review, no formal experimental hypothesis is tested. The central premise is that polymer- and lipid-based nanoparticles can be engineered to overcome T-cell-specific barriers—cellular binding/uptake, endosomal escape, and nuclear translocation—and thereby enable efficient, safe, and cost-effective ex vivo or in vivo CAR T cell engineering.

Aims: Provide an overview from CAR constitution to transfection technologies used in T cells. - Highlight lipid- and polymer-based nanoparticles as potential delivery platforms for CAR T cell therapy. - Present examples, strengths, and weaknesses of nanosystem formulations. - Discuss advances in nanoparticle design to improve T cell transfection and guide next-generation CAR T therapeutics.

Delivery system: Polymer-based nanosystems: cationic polymers forming polyplexes with nucleic acids. Key materials include PEI (linear and branched), PDMAEMA, poly(β-amino esters) (PβAE), star-shaped polymers, dendrimers, and hybrid polymer systems. Lipid-based nanosystems: liposomes and lipid nanoparticles (LNPs) composed of cationic lipids (DOTMA, DOTAP, DC-Chol, DOGS), neutral helper lipids (DOPE, DOPC, cholesterol), ionizable lipids, and PEGylated lipids. Lipid–polymer hybrid nanosystems: hydrophobic polymeric core coated with a lipid layer and lipid-PEG surface. Targeting ligands: anti-CD3, anti-CD8, anti-CD4, anti-CD5, anti-β7 integrin, CD5-targeted LNPs, and others. Payloads: plasmid DNA, mRNA (e.g., CAR, FoxO1, megaTAL nuclease, FAP CAR), transposon systems (PiggyBac), and gene-editing components. Applications: ex vivo T cell engineering and in vivo T cell reprogramming.

Approach: Review article; no original experimental groups, n, doses, or controls. It synthesizes published in vitro studies (Jurkat cells, primary human T cells, primary murine T cells, PBMCs, HeLa, etc.) and in vivo mouse models (leukemia, B cell lymphoma, prostate tumor, cardiac injury/fibrosis). The review compares viral vectors, electroporation, and nanosystems for CAR T cell engineering.

Key methods: Summarizes techniques from cited studies: transfection efficiency, cell viability/cytotoxicity, luciferase reporter expression, CAR expression, flow cytometry, IVIS imaging, survival curves, tumor eradication, cytokine release, endocytosis pathway analysis, and physicochemical characterization of nanosystems.

Key results: Si-PDMAEMA-pDNA achieved 46% transfection in Jurkat cells and 44% in primary human T cells; pDNA/PEI-based systems reached 51% in Jurkat and 60% in primary human T cells. - pIAE + anti-CD3 + PiggyBac transposon eradicated tumors in 7 out of 10 mice with leukemia. - FoxO1 mRNA nanoparticles improved median survival in a B cell lymphoma mouse model (43 days vs 23 days in no-therapy controls). - CD5-targeted LNP delivering FAP CAR mRNA achieved 81% transfection efficiency and improved cardiac function/fibrosis in a murine hypertensive model. - Lipid–polymer hybrid charge-altering releasable transporters reached 80% transfection in Jurkat cells but only 1.5% in primary T lymphocytes, with minimal toxicity. - DOTAP/DC-Chol/DOPC/DOPE pDNA liposomes gave 38% transfection in Jurkat cells with 90% viability; DOTAP-HPC/DC-Chol/DOPE/DSPE-PEG gave 80% transfection but only 40% viability. - Lipofectamine LTX pDNA achieved 25% transfection in Jurkat cells with 70% viability. - EF1α promoter increased transgene expression by 63% compared with CMV promoter in T cells.

Interpretation: Polymer- and lipid-based nanosystems have demonstrated the ability to transfect T cells, enabling safe and cost-effective genetic engineering of functional T cells compared with other transfection technologies. With continued optimization—including targeting ligands, stimuli-responsive release, and nuclear localization strategies—these platforms can be used in clinical applications, improving current T-cell-based cancer treatments and leading to next-generation T cell therapeutics.

Limitations: Review only; no new primary data. T cells are inherently hard to transfect: primary T cells show lower endocytosis, slower division, and a higher nucleus-to-cytoplasm ratio than cell lines, so transfection values in cell lines are substantially higher than in primary cultures. Nanosystems still face risks of liver/spleen toxicity, limited in vivo applications, and few studies in CAR T cell engineering. Viral vectors remain the clinical standard. Long-term safety, large-scale manufacturing, and clinical translation remain unresolved.