Nanoparticles for nucleic acid delivery: Applications in cancer immunotherapy

Purpose: Nucleic acid therapeutics—DNA, mRNA, and CRISPR/Cas9 genome editing systems—hold significant potential for cancer immunotherapy but face major delivery barriers, including rapid in vivo degradation, poor cellular uptake, required nuclear entry, and toxicity in healthy tissues. Nanoparticle delivery systems are being engineered to safely and effectively deliver these therapeutics to immune cells.

Hypothesis: No formal experimental hypothesis. Central thesis: nanoparticle platforms can overcome extracellular and intracellular barriers to nucleic acid delivery—protecting cargo, promoting cellular/nuclear uptake, enabling endosomal escape, and reducing off-target toxicity—thereby enhancing the potency and safety of DNA, mRNA, and genome-editing therapeutics for cancer immunotherapy.

Aims: Review applications of nucleic acid therapeutics in cancer immunotherapy. - Detail how nanoparticle platforms are designed to deliver mRNA, DNA, and genome editing systems. - Discuss DNA delivery for cancer vaccines and immunotherapy. - Discuss mRNA delivery for cancer vaccination and adoptive T-cell therapy. - Discuss nanoparticle-mediated CRISPR/Cas9 gene editing for immune cell engineering. - Outline future directions and challenges for clinical translation.

Delivery system: Nanoparticle types: lipid nanoparticles (liposomes, ionizable LNPs, polymer-lipid NPs), polymeric nanoparticles (poly(L-lysine), polyethyleneimine, poly(beta-amino esters), chitosan, polyplexes), zwitterionic amino lipids, cationic lipid-assisted polymeric nanoparticles, PEG-PLGA/PLGA/BHEM-Chol/DOTAP NPs, gold NPs, silica NPs, magnetic NPs. - Lipid components: DOTMA, DOTAP, DOPE, DSPC, DOTC, POPC, cholesterol, lipid-anchored PEG, DC-cholesterol, C12-200. - Payloads: plasmid DNA, CpG oligodeoxynucleotides, mRNA (OVA, TRP2, gp100, MART1, luciferase, erythropoietin, Foxo1ΔA, megaTAL nuclease, CAR, TCR), CRISPR/Cas9 mRNA, sgRNA, Cas9 ribonucleoprotein, cyclic dinucleotide STING agonist, siRNA/miRNA. - Targeting/functionalization: anti-CD3ε F(ab′)2, anti-CD3, anti-CD8 antibodies, PGA surface coating, DC-targeting ligands, PD-1-targeting NPs. - Routes: systemic intravenous, subcutaneous, intranasal, aerosol, ex vivo transfection. - Applications: cancer vaccines, adoptive T-cell therapy, CAR/TCR engineering, checkpoint blockade, allogeneic CAR T cells, in vivo CAR T generation, gene regulation, treatment of solid tumors.

Approach: Review of preclinical and clinical literature, not primary experiments. In vitro systems include dendritic cells, T cells, macrophages, B cells, and tumor cells. In vivo models include B16F10 melanoma, EG7-OVA thymoma, Nalm6 leukemia, orthotopic colorectal tumors, hereditary tyrosinemia type 1, type 2 diabetes, and rheumatoid arthritis. Clinical context includes LNP-mRNA melanoma trial NCT02410733, Curevac NCT03291002, and FDA-approved Onpattro (lipid-siRNA NP, 2018). Doses and controls vary across cited studies.

Key methods: Review-level synthesis of: - Nanoparticle characterization: size, surface chemistry, material composition, encapsulation/binding. - Transfection and expression: luciferase, erythropoietin, antigen expression. - Gene editing: indel rates by deep sequencing, knockdown percentages, homology-directed repair. - Immune response: antigen-specific T-cell responses, IFN-α, cytokine/chemokine responses, DC maturation, CD8+ T-cell activation. - Antitumor efficacy: tumor volume, tumor rejection, lung metastasis, survival. - Biodistribution and targeting: lymph node accumulation, spleen, liver, lung expression. - Safety/toxicity: tolerability, off-target editing, immune responses.

Key results: Ionizable LNPs with CpG ODN co-administered with tumor antigens showed preferential immune cell uptake in lymph nodes and greater tumor rejection in a murine EG7-OVA model. - PLL-coated polystyrene NPs delivering OVA pDNA inhibited EG7 tumor growth after two vaccinations. - Aerosolized PEI-IL-12 pDNA in a SAOS-LM7 osteosarcoma lung metastasis model produced IL-12 expression in lungs and significantly fewer lung metastases versus untreated controls. - Multilamellar ionizable LNPs delivering TRP2 and gp100 mRNA reduced tumor volume and extended survival in B16F10 melanoma; they transfected neutrophils, macrophages, and dendritic cells. - PBAE terpolymer/PEG-lipid NPs achieved selective luciferase expression in mouse lungs after IV injection and improved serum stability. - Histidylated lipopolysplexes with MART1 mRNA produced ~10-fold reduction in tumor volume and 75% reduction in lung metastases versus control in B16/F10 melanoma. - Antibody-coated PBAE NPs improved ex vivo T-cell transfection 10-fold versus non-targeted NPs. - Clinical LNP-mRNA vaccine (NY-ESO-1, MAGE-A3, tyrosinase, TPTE) was well tolerated, with dose-dependent IFN-α and antigen-specific T-cell responses in three melanoma patients. - CRISPR/Cas9 LNP + AAV achieved 6% hepatocyte gene editing and 24.1% indel rate in total liver genomic DNA. - Ionizable LNP co-delivering Cas9 mRNA and modified sgRNA achieved 70% gene editing and >97% knockdown in hepatocytes after a single IV injection in mice. - ZALs co-delivering Cas9 mRNA and sgRNA enabled stable gene editing two months after administration. - CLANs with CD68 promoter achieved macrophage/monocyte-specific Ntn1 knockout in vivo with little editing in off-target immune cells. - Triple knockout (TCR-, HLA-1-, PD-1-) CAR T cells showed quicker and complete elimination of tumor cells versus double-knockout CAR T cells in an aggressive Nalm6-PDL1 leukemia model.

Interpretation: Nanoparticle platforms can overcome key nucleic acid delivery barriers and enable DNA vaccines, mRNA vaccines, CAR/TCR engineering, and CRISPR/Cas9 gene editing for cancer immunotherapy. Early clinical translation is underway, including FDA approval of Onpattro and LNP-mRNA vaccine trials. The authors argue that NPs can shift cancer therapy toward potent, biocompatible nucleic acid delivery systems and may enable in vivo T-cell engineering and improved solid tumor immunotherapy.

Limitations: Review article; no primary data. - Most immunotherapies and NP delivery systems have been primarily effective against hematological cancers or melanoma; solid tumor application remains challenging. - Many materials used have not yet entered clinical trials or received FDA approval. - Cationic lipids and polymers can cause toxicity, immune responses, and clot formation; ionizable LNPs can have difficulty encapsulating large cargo such as pDNA. - Polymeric NPs often have poor serum stability and limited in vivo success. - CRISPR/Cas9 delivery requires precise timing and co-delivery of guide RNA and Cas9 mRNA/protein; sufficient in vivo editing remains difficult. - Targeting ligands are often needed to enhance binding, biodistribution, and uptake in vivo. - Clinical translation is still limited; long-term safety, manufacturing, and regulatory pathways remain to be established.