Optimization and Characterization of the Intelligent Phagemid-Assembled Gene Expression (iPhAGE) Miniphagemid Gene Delivery Platform

Abstract

The Intelligent Phagemid-Assembled Gene Expression (iPhAGE) system is an M13 bacteriophage-derived, non-viral gene delivery platform. It produces miniaturized phagemid particles (miniphagemids): non-replicative, non-infectious particles that package circular single-stranded DNA (cssDNA) carrying a gene cassette with a gene of interest (GOI), such as a reporter or therapeutic gene. Unlike conventional viral vectors, miniphagemids package cssDNA cassettes devoid of bacterial backbone sequences, thereby reducing immunogenicity and addressing some regulatory concerns. Because miniphagemids cannot infect cells autonomously, traditional plaque-based quantification methods are inapplicable. Furthermore, key upstream production parameters, including genetic modifications to improve yield, growth media selection, and long-term storage stability, have not been systematically evaluated, yet are essential for the system's further development. This thesis addresses four key research gaps. First, ssDNA was identified as the most appropriate reference standard for quantifying the encapsidated genome in miniphagemids because it best reflects its single-stranded conformation and avoids topology-dependent amplification bias under the tested conditions. Second, in-house-developed SW8 helper plasmids achieve comparable yields to commercially available counterparts while exhibiting lower backbone contamination and superior packaging fidelity. Modification of the ribosome-binding site (RBS) in gV of the helper plasmid also improves yield; however, current findings suggest that this alteration alone is insufficient to enhance the output of pV-mediated ssDNA. Third, comparison of LB and TB growth media showed that, contrary to the expectation that nutrient-rich TB would increase titers, LB supported comparable or higher titers with lower backbone contamination and at a reduced cost under the conditions tested. Fourth, long-term stability was assessed over 120 days at various storage temperatures for the best-performing miniphagemids: DNA content was monitored by qPCR and NanoDrop; particle morphology and aggregation were evaluated by AFM and DLS; DNase protection assays assessed encapsidation integrity; and a luciferase-based transfection assay measured functional activity in the host mammalian cell line. Miniphagemids retained detectable DNA and functional transfection activity across all storage temperatures, though qPCR-based titers were best maintained at -20°C and 4°C, identifying these as optimal storage conditions. AFM and DLS revealed aggregation over time, and DNase protection assays confirmed that a second DNase treatment prior to quantification improves accuracy by removing residual non-encapsidated DNA. The SW8-EGF miniphagemid produced measurable luminescence without a transfection reagent after four months, consistent with receptor-mediated uptake. Taken together, these findings advance the characterization and process understanding of the iPhAGE system, providing a methodological framework for quantification, quality assessment, and production optimization of phagemid-derived particles.