Background

Hepatocellular carcinoma (HCC) is a cancer with poor prognosis due to its molecular heterogeneity and frequent recurrence and drug resistance after treatment. It is estimated that approximately 850,000 new cases were diagnosed worldwide in 2024, with over 730,000 deaths. Although treatment options such as surgery, local therapy, immune checkpoint inhibitors, and targeted therapies have expanded, achieving sustained efficacy in advanced patients remains challenging.

Gene therapy, which delivers therapeutic genes into tumor cells, is considered a promising alternative, but the delivery vector remains a major obstacle. Eukaryotic viral vectors such as adenovirus and adeno-associated virus (AAV) have high cell penetration capabilities but are limited by pre-existing immunity, non-target tissue exposure, and constraints in cargo capacity and production. Non-viral nanoparticles are relatively safer but often lack sufficient cellular uptake and gene expression efficiency.

Bacteriophages, viruses that infect bacteria, are inherently non-infectious to mammalian cells but can be relatively easily modified in surface and genome structure. The research team reviewed prior studies on the application of phage-based vectors for targeted gene delivery to liver cancer and summarized the barriers to clinical translation. This paper is a narrative review that critically analyzes existing literature, not a new animal experiment or clinical trial.

Key Findings

The fundamental strategy proposed by the researchers is phage display. By expressing tumor-targeting peptides or antibody fragments on the surface coat proteins of phages such as M13, T4, T7, and lambda phage, the phages can be designed to selectively recognize receptors on liver cancer cells, such as glypican-3 (GPC3) and integrin. Candidates include the GPC3-targeting L5 peptide and integrin-binding arginine-glycine-aspartic acid (RGD) ligands.

After phage entry via receptor-mediated endocytosis, the process of endosomal escape, cytoplasmic cargo release, nuclear translocation, and therapeutic gene expression must occur sequentially. In this context, AAVP vectors, which combine phage and AAV elements, have attracted attention. These vectors incorporate a mammalian expression cassette between AAV2 inverted terminal repeats and display tumor-targeting ligands on the phage coat.

A representative cargo is the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. Recombinant TRAIL protein has a short half-life in the bloodstream and low tumor accumulation, but delivering the TRAIL gene into liver cancer cells allows for intracellular TRAIL production. Expressed TRAIL activates death receptors and caspase-8, triggering downstream executioner caspases and mitochondrial pathways to induce apoptosis. In the reviewed cell culture studies, no TRAIL gene delivery was observed in normal liver cells.

The concept of loading CRISPR-Cas9 to cleave or correct mutant oncogenes was also discussed. Single-guide RNA recognizes a 20-nucleotide target sequence, and Cas9 cuts double-stranded DNA three nucleotides upstream of the protospacer adjacent motif (PAM). However, the phage-CRISPR combination remains at an early developmental stage and is not yet a clinically validated therapeutic technology for liver cancer.

Implications and Outlook

Bacteriophages offer the advantage of combining targeting ligands and therapeutic cargo into a single particle and can be amplified through bacterial culture. Their lack of natural affinity for mammalian cells can also be a design element to reduce non-target delivery. The platform can be extended to integrate anti-cancer genes, gene-editing tools, and imaging agents, enabling combined diagnostic and therapeutic applications.

However, surface binding does not necessarily equate to efficient gene expression. Phages are prone to degradation in endosomes and lysosomes, and the mechanisms for cytoplasmic escape remain insufficiently understood. After intravenous administration, the mononuclear phagocyte system in the liver and spleen rapidly clears the particles, and neutralizing antibodies may develop with repeated administration. Strategies such as polyethylene glycol conjugation or lipid vesicle encapsulation are potential candidates to prolong in vivo retention, but the balance between tumor binding and immune evasion must be validated.

At the large-scale production stage, removal of bacterial endotoxins and residual DNA, as well as particle purity, titer, and stability, must be managed. Standardized good manufacturing practice (GMP) processes and regulatory frameworks are still under development. In particular, since this review does not provide independent efficacy data, preclinical validation of biodistribution, toxicity, and repeated administration effects in liver cancer organoids and immunocompetent animal models must be conducted first. The clinical potential of phage-based vectors can only be assessed after such preclinical evidence is accumulated.