Delivery Technologies for T Cell Gene Editing: Applications in Cancer Immunotherapy

Purpose: Adoptive T cell therapy has been revolutionized by genetic engineering, but viral transduction—the standard delivery method—has limitations including small cargo capacity, high immunogenicity, high manufacturing cost, and risks of genotoxicity/insertional mutagenesis. There is a need for improved non-viral delivery technologies to enable precise gene editing in T cells for cancer immunotherapy, especially for solid tumours and allogeneic applications. ---

Hypothesis: The review’s central thesis is: if gene editing technologies (transposons, ZFN, TALEN, CRISPR/Cas9) are paired with advanced non-viral delivery systems (electroporation, cell squeezing, nanoparticles), then T cells can be engineered with improved antigen responses, enhanced antitumour activity, and potential for universal allogeneic applications—broadening the scope and efficacy of cancer immunotherapies. ---

Aims: - Provide an overview of gene editing strategies used in T cell therapy, including transposons, designer nucleases (ZFN, TALEN), and CRISPR/Cas9. - Review delivery technologies utilized to genetically engineer T cells, including viral, electroporation, cell squeezing, and nanoparticle platforms. - Discuss recent investigations and clinical trials that have used gene editing to enhance T cell efficacy and broaden cancer immunotherapy applications. - Highlight future directions for improving T cell immunotherapies, including novel delivery systems and new gene targets. ---

Delivery system: Gene editing technologies: - Transposons: Sleeping Beauty (SB), piggyBac; transposase binds terminal inverted repeats (TIRs) to mobilize DNA. - Designer nucleases: Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN); both use FokI endonuclease for DNA cleavage. - CRISPR/Cas9: sgRNA + Cas9 endonuclease; delivers plasmid DNA, mRNA, or ribonucleoprotein (RNP) complexes. Delivery methods: - Viral: Lentivirus, gamma-retrovirus (stable integration); adenovirus, AAV (transient expression). - Electroporation/Nucleofection: Pulsed high-voltage electrical currents to create transient membrane pores. - Cell squeezing: Microfluidic mechanical membrane disruption. - Nanoparticles: Lipid, polymer, and gold nanoparticles for DNA, mRNA, siRNA, miRNA, and RNP delivery; surface modifications for T cell targeting and controlled release. Payloads: CAR transgenes, TCR genes, CRISPR/Cas9 components (mRNA, sgRNA, RNPs), transposon plasmids, ZFN/TALEN mRNA, donor DNA templates, cytokines (IL-12, IL-15). Targets for knockout: TRAC, TRBC, PD-1, CTLA-4, HLA-A, B2M, CD52, GM-CSF, LAG-3, TGFBR2, DGK, CD7, HPK1. ---

Approach: Narrative review of preclinical and clinical literature. Model systems discussed include: - In vitro: Primary human T cells, T cell lines, various cancer cell lines. - In vivo: Mouse models of leukaemia, lymphoma, melanoma, glioblastoma, breast cancer, colorectal carcinoma, bladder cancer, hepatocellular carcinoma, pancreatic carcinoma, ovarian cancer. - Clinical trials: Phase I/II trials using SB, TALEN, and CRISPR/Cas9-engineered T cells (Table 2 lists trial numbers including NCT00968760, NCT02746952, NCT02793856, NCT03166878, NCT03399448, etc.). - Disease context: Haematological malignancies (ALL, CLL, NHL, AML, multiple myeloma) and solid tumours (melanoma, glioblastoma, breast, colorectal, bladder, hepatocellular, pancreatic). ---

Key methods: Techniques and endpoints highlighted across cited studies: - Flow cytometry for CAR/TCR expression, T cell phenotype, cytokine production. - In vitro cytotoxicity assays (IFN-γ secretion, target cell killing). - In vivo tumour growth inhibition and survival analysis. - Indel analysis for gene editing efficiency. - Integration site analysis for genotoxicity assessment. - T cell expansion and persistence assays. - Clinical response assessment (molecular remission, GVHD incidence, toxicity). ---

Key results: - Five FDA-approved CAR T cell therapies: Kymriah, Yescarta, Tecartus, Breyanzi, and Abecma. - Sleeping Beauty clinical trials: Patients with advanced NHL and ALL undergoing HSCT and CAR T cell infusion showed no acute or latent toxicities and no exacerbation of GVHD. - CRISPR/Cas9 first-in-human trial: T cells from three patients with refractory cancer were edited to remove endogenous TCRs and PD-1, achieving highly specific editing at targeted loci without clinical toxicity; edited T cells effectively targeted tumour cells. - Universal CAR T cells: TALEN-mediated disruption of TRAC and CD52 in donor cells enabled molecular remission in two infants with relapsed/refractory ALL within 28 days. - Enhanced potency: CRISPR/Cas9-mediated insertion of CD19-specific CAR into the TRAC locus enhanced potency and delayed effector differentiation and exhaustion. - T cell expansion: Scaffolds mimicking antigen-presenting cells enabled 2- to 10-fold greater polyclonal expansion of primary mouse and human T cells than commercial expansion beads. ---

Interpretation: The authors conclude that gene editing technology has transformed adoptive T cell therapies by enabling precise genetic modification to generate T cells with improved antigen responses, enhanced antitumour activity, and potential for allogeneic applications. While T cell immunotherapies have shown success in B cell malignancies, challenges remain for solid tumours. Novel delivery systems (cell squeezing, nanoparticles) and identification of new gene targets through genome-wide CRISPR screens could broaden the application of T cell immunotherapies to treat other haematological malignancies and solid tumours. ---

Limitations: - Viral delivery limitations: Risks of genotoxicity, insertional mutagenesis, immunogenicity, small cargo capacity, and high manufacturing cost. - Electroporation limitations: Cytotoxicity from high voltage, limited in vivo penetration, scalability challenges. - Cell squeezing limitations: Requires cell isolation, limited to ex vivo engineering, not widely explored in T cells. - Nanoparticle limitations: Lower transfection efficiencies, accumulation in liver and spleen (toxicity concerns), not widely explored for T cell gene editing. - CRISPR/Cas9 limitations: Less specific than ZFN/TALEN, risks of off-target mutagenesis and immunogenicity, inefficient in vivo delivery. - ZFN/TALEN limitations: Substantial protein engineering required for new targets; TALEN difficult to deliver due to large size. - Solid tumour challenges: Immunosuppressive tumour microenvironment, T cell exhaustion, poor infiltration. - Clinical translation: Manufacturing complexity, cost, and scalability remain major barriers. - As a review: Not a systematic review or meta-analysis; no primary data.