Investigating Miniphagemid Mediated Anti-Angiogenic VLPs for Targeted Therapeutic Applications

Overview

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.

We investigated the construction and characterization of a targeted miniphagemid-mediated anti-angiogenic DNA-encoded virus-like particle (VLPs) delivery system to determine if these VLPs may be an effective therapeutic avenue. M13 is a filamentous phage capable of targeted ligand display, and it has been explored as a safe and efficient vehicle for delivering therapeutic genes and drugs. Phage-based vectors (phagemids or miniphagemids) can be engineered to transfer exogenous genetic material to mammalian cells safely, as they possess no natural tropism. VLPs have shown a robust ability to stimulate potent immune responses and overcome the immunosuppressive state of the tumour microenvironment (TME). We aimed to combine the advantages of both VLPs and phagemids to construct a hybrid biological platform for the specific delivery of DNA via engineered M13 miniphagemids encoding HPV-VLPs displaying anti-tumour peptides (specifically VGB4) to tumour cells, to then be produced by the tumour cells themselves.

Leveraging the existing M13 iPhAGE System we designed genetically engineered miniphagemids, produced by E. coli, to specifically target tumour cells. The VGB4 peptide has demonstrated a potent ability to inhibit the angiogenesis of tumour vasculature by blocking the downstream signalling pathways of vascular endothelial growth factor receptor (VEGFR). We cloned the VGB4 peptide sequence inserted into the human papillomavirus (HPV) type 16 L1 capsid gene into a miniphagemid engineered by our lab. The HPV type 16 L1 capsid is a key component for the creation of VLPs and was chosen for it’s high infectivity and self-assembly process. The helper plasmid not only complements phagemid packaging but also enabled the display of a cell-specific targeting ligand, epidermal growth factor IV (EGF), which promotes receptor-mediated endocytosis for phage uptake by tumour cells that over-express epidermal growth factor receptors (EGFRs).

This project investigated the formation of VGB4-displaying HPV VLPs within HEK 293T and HeLa cells. We hypothesized that EGF-displaying miniphagemids would enable targeted gene transfer to EGFR overexpressing tumour cells. The DNA sequence encoding the VGB4-displaying HPV VLPs would then be expressed in the targeted cells and produce HPV VLPs displaying VGB4 with the ability to bind to VEGFR. Our results demonstrated that the EGF-displaying miniphagemid improved gene delivery to cells compared to non-displaying miniphagemids. The VGB4-displaying HPV VLPs did not form in cells treated with miniphagemids, but these VLPs were successfully formed in cells treated with the precursor phagemids encoding the same gene cassette.

Overall, this study highlighted 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 and other diseases.

Learn more about this project here.