Approaches towards Biomaterial-Mediated Gene Editing for Cancer Immunotherapy

Purpose: Gene therapies are transforming treatment for many diseases, but clinical efficacy and safety depend on both the delivery material and the cargo. Non-viral delivery to immune cells remains especially challenging, and no clinical translation breakthrough has yet been achieved for non-viral gene editing. This mini-review addresses that gap by surveying biomaterial-based delivery to immune cells, CRISPR/Cas9 cargo options, and how the two fields can be integrated for cancer immunotherapy. ---

Hypothesis: The central thesis is: if non-viral biomaterial carriers can overcome extracellular and intracellular delivery barriers to immune cells, then they can enable efficient, safe, and scalable CRISPR/Cas9-mediated gene editing of immune cells in situ for cancer immunotherapy. ---

Aims: - Review notable examples of biomaterial-based gene delivery to immune cells, with emphasis on recent in vivo successes. - Review CRISPR technology and its promise for immune cell gene editing. - Describe how leading non-viral delivery materials and CRISPR technology can be integrated to advance clinical potential for therapeutic gene transfer to immune cells to treat cancer. - Discuss barriers and design considerations related to extracellular delivery, intracellular transport, and gene editing cargo formats. ---

Delivery system: Polymeric carriers: - PEI and derivatives, PLL, chitosan, PLGA, poly(β-amino ester) (PBAE), charge-altering releasable transporters (CART; poly(carbonate)-b-(α-amino esters)), pHEMA-g-pDMAEMA comb/sunflower polymers. - Payloads: pDNA, mRNA, siRNA, CRISPR/Cas9 components (RNP, plasmid DNA, mRNA + sgRNA). - Targeting/functionalization: anti-CD3ε Fab2, anti-CD8 antibodies, MTAS/NLS peptides, polyglutamic acid (PGA), Di-mannose. Lipid-based carriers: - Ionizable lipid nanoparticles (LNPs): DODAP, DODMA, C14-4, OF-Deg-Lin, 306O10, B-11, CKK-E12, A-11, 93-O17S, 9322-O17S. - Helper lipids: DOPE; PEGylated LNPs; selective organ targeting (SORT) technology. - Payloads: mRNA, pDNA, CRISPR/Cas9 components. Inorganic carriers: - Porous metal–organic frameworks (e.g., Ni-IRMOF-74-II to -V). - Cationic arginine-coated gold nanoparticles. - Payloads: ssDNA, Cas9 RNPs. Biomaterial-based genetic vaccines: - mRNA-1273 and BNT162b2 (lipid nanoparticle delivery of SARS-CoV-2 spike mRNA). ---

Approach: Mini-review; no primary experimental design. Synthesizes in vitro and in vivo literature on immune cell gene delivery and editing. Model systems discussed include: - In vitro: Jurkat T cells, primary human T cells, RAW264.7 murine macrophages, D1 dendritic cells, NK-92 cells. - In vivo: Murine cancer models, murine GvHD model, non-small-cell lung cancer clinical trial. - Disease context: Cancer immunotherapy, with brief coverage of COVID-19 vaccines. ---

Key methods: Techniques highlighted across cited studies: - Flow cytometry for transfection efficiency, biodistribution, and immune cell subset analysis. - Confocal microscopy for nanoparticle internalization. - Bioimaging/IVIS for luciferase reporter expression. - Indel assays for CRISPR editing efficiency. - Phagocytosis assays for macrophage function. - Cytotoxicity assays for cancer cell killing. - Reporter expression (GFP, luciferase) for gene delivery. ---

Key results: - PBAE nanoparticles with anti-CD3ε Fab2, MTAS, and NLS delivered DNA encoding leukemia-specific CARs to T cells at 34% efficiency in vivo, programming functional antigen recognition and anti-tumor effects. - PBAE mRNA CAR nanoparticles (anti-CD8, PGA-coated) transiently transfected T cells at 10% efficiency in vivo. - CART polymers: ~80% mRNA transfection in Jurkat T cells in vitro, but only ~1.5% in vivo; mixed hydrophobic blocks outperformed single-lipid CARTs (~1%). - Lipid LNPs: C14-4 delivered CAR mRNA to primary human T cells ex vivo with reduced cytotoxicity and similar cancer cell killing vs electroporation/lentivirus. OF-Deg-Lin achieved 85% of LNP-induced protein production in splenic immune cells. B-11 transfected 4.6% DCs, 1.2% macrophages, 3.3% neutrophils, 0.06% B cells. 93-O17S showed 8.2% and 6.5% gene recombination in splenic CD4+ and CD8+ T cells. 306O10 achieved 86–88% Kupffer cell transfection. - CRISPR editing: Lu et al. PD-1 knockout in T cells via plasmid electroporation gave 5.81% editing efficiency; RNPs improved editing. Stadtmauer et al. reported average on-target editing efficiencies of 99.4 ± 0.8% (PDCD1), 98.6 ± 1.3% (TRAC), and 95.8 ± 1.6% (TRBC). - Macrophage SIRP-α knockout using cationic arginine-coated gold nanoparticles enhanced phagocytosis of U2OS cancer cells (~20% vs ~5%). - NK cells: CRISPR-Cas9 RNP modification of primary human NK cells (CIS knockout enhanced IL-15 responsiveness; ADAM17/PDCD1 knockout increased potency; CD16/DNAM-1 knock-in into NK-92 enhanced cytotoxicity). ---

Interpretation: The authors claim that the convergence of immunotherapy and gene therapy holds great promise not only for new paradigms of cancer treatment but also for infectious diseases, autoimmune diseases, and other human diseases. Non-viral biomaterials can enable safe, efficient, and targeted delivery of CRISPR/Cas9 to immune cells for in situ genetic engineering, and biomaterial-based systems have the potential to be low cost and broadly accessible, unlike viral- and cell-based systems. ---

Limitations: - Non-viral delivery to immune cells remains challenging due to limited endocytosis, low protein production, meager lymphocyte viability, inherent immune protective functions, and nucleic acid sensors (e.g., TLRs). - Weak correlation between in vitro/ex vivo and in vivo transfection performance. - In vivo transfection efficiencies are often low (e.g., 1.5–10% for T cells; ~2% for pulmonary immune cells). - CRISPR delivery adds complexity: multiple components, cargo format selection, co-encapsulation of Cas9 mRNA and sgRNA, and manufacturing at scale. - Most early gene editing research is ex vivo using viral vectors and/or electroporation; clinical translation of non-viral gene editing is still lacking. - As a mini-review, the coverage is selective rather than systematic.