Construction and Characterization of a Targeting M13-Based Phagemid Carrying an Anti-Angiogenic DNA-Encoded Virus-Like Particle

Abstract

Over the past several years, molecular targeted therapy has emerged as a promising strategy for cancer treatment. Unlike broad-spectrum cytotoxic drugs used in conventional chemotherapy, targeted therapy aims to address specific molecular alterations unique to cancer cells. To develop effective targeted therapies, numerous delivery platforms have been investigated to optimize safety, specificity, and efficiency. The work presented here investigates the construction and characterization of a miniphagemid-mediated cancer therapy delivering anti-angiogenetic DNA-encoded virus-like particles (VLPs). VLPs have shown a robust ability to stimulate potent immune responses and overcome the immunosuppressive state of the tumour microenvironment (TME). Additionally, the filamentous bacteriophage (phage) M13 has been explored as a safe and efficient vehicle for delivering therapeutic genes and drugs. Phage-based vectors (phagemids) can be engineered to transfer exogenous genetic material to mammalian cells safely, as they possess no natural tropism. The present study aims to combine the advantages of both VLPs and phagemids to construct a hybrid biological platform for the specific delivery of DNA encoding VLP-displaying anti-tumour peptides, specifically VGB4, to tumour cells via M13 – a filamentous phage capable of phagemid production as well as phage display. The VGB4 peptide has demonstrated potent ability to inhibit angiogenesis in the tumour vasculature by blocking the downstream signalling pathways of vascular endothelial growth factor receptor (VEGFR). The human papillomavirus (HPV) type 16 L1 capsid gene with an inserted VGB4 peptide sequence was cloned into a miniaturized phagemid (miniphagemid) engineered by our lab. This genetically engineered miniphagemid was produced in Escherichia coli using a novel non-packaging M13 helper plasmid. The helper plasmid not only complements phagemid packaging but also enables the display of a cell-specific targeting ligand, the epidermal growth factor (EGF), which promotes receptor-mediated endocytosis for specific phage uptake by tumour cells overexpressing epidermal growth factor receptors (EGFRs). This thesis project investigated the formation of VGB4-displaying HPV VLPs within HEK 293T and HeLa cells. Our results demonstrated that the EGF-displaying miniphagemid improves gene delivery to cells compared to non-displaying miniphagemids. Furthermore, the VGB4-displaying HPV VLPs do not form in cells treated with miniphagemids, but these VLPs are successfully formed in cells treated with the precursor phagemids encoding the same gene cassette. Overall, this study highlights the necessity for further investigation and optimization to enhance miniphagemid-mediated gene transfer by overcoming cellular barriers, paving the way for its application as a novel targeted gene therapy for cancer.