Construction and Characterization of intelligent Phage-Assembled Gene Expression systems (iPhAGEs).

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Background: ***Bacteriophages form the world's largest reservoir of genetic information, offering innumerable biotechnological applications. The proposed research program aims to exploit phages and phage genetic elements in the construction of systems to engineer highly efficient, efficacious, and safe therapeutic production platforms for transgene delivery applications. Current modalities of transgene delivery have been less successful than envisaged due to: cellular toxicity, poor transgene expression efficiency in target cells, extensive vector degradation, and inefficient/non-scale-able vector production. We have previously engineered a production platform for the generation of DNA minivectors that exploits phage-encoded genetic elements in their efficient production. Our technology employs a genetic system from phage PY54 to produce linear covalently-closed (LCC) DNA minivectors, or “DNA Ministrings”. These stable, linear, double-stranded DNA constructs carry only the eukaryotic promoter, gene of interest, intron and polyA sequence, and nuclear translocation enhancing sequences. DNA ministrings offer more efficient cellular trafficking and transfection compared to conventional plasmid vectors and integration of a DNA ministring into a cell's chromosome (bacterial or mammalian) is a highly lethal event that protects against vector integration events that may result in mutations or silencing of adjacent genes. This program proposes novel strategies for the construction and characterization of intelligent Phage Assembled Gene Expression systems (iPhAGEs) bacteriophage-based DNA delivery platforms that are efficient, inexpensive, scale-able, temperature-stable and easy to manipulate. To pioneer the iPhAGEs endeavour we aim to produce and package DNA ministrings using the single-stranded DNA (ssDNA) filamentous phage, M13, as well as a lytic Lambda phage, both of which offer the capacity for phage display and "intelligent" targeting. These platforms are expected to maximize transgene delivery efficiency, simplify purification, and provide the option of intelligent targeting.******Significance of the Research: This research program seeks to establish game-changing, robust, and powerful one-step genetic processes to generate stable and efficient production of novel DNA delivery vectors. The knowledge gained from this work will extend to maximize efficiency, efficacy, and safety of transgene delivery toward a number of downstream transgene delivery applications in agriculture and mammalian medicine. Furthermore, display phages are highly amenable to the surface expression of genetically fused peptides on the capsid, easily facilitating targeting of cell surface receptors, reporter tagging, or linkage with effector peptides. Such highly efficient systems will serve to greatly expand the genetic engineering and molecular biology toolbox.