The iPhAGE Platform

 

Overview

Despite the significant promise of gene therapy, the clinical translation of many approaches is hampered by limitations in current gene delivery systems. These limitations often include issues with safety, efficiency, gene cargo capacity, and the presence of inflammatory components within the delivery vector — not to mention practical limitations including purity of the final product, production yield, and overall production cost.

We engineered an advanced gene delivery system based on bacteriophages, termed iPhAGE (intelligent Phage-Assembled Gene Expression) to solve these issues.

The iPhAGE system represents a significant advancement in gene delivery technology by leveraging E. coli to produce a minimized DNA vector (“miniphagemid”), enhancing the efficacy and safety of gene transfer while minimizing cost of production. By eliminating the prokaryotic backbone, immunogenic DNA content is significantly reduced, offering a highly pure, scalable, and efficient platform, combining the benefits of targeted ligand display with the enhanced performance of minimized DNA vectors. The result is a highly efficient, modifiable, scalable, and cost effective gene therapy delivery platform that holds considerable potential for a wide range of applications.

 
 

The iPhAGE System at a Glance:

The iPhAGE System: Next-Generation Targeted Gene Delivery

iPhAGEs is an innovative platform designed to overcome critical limitations in nonviral gene therapy. Building on NSERC-funded work, the iPhAGE system offers a robust, scalable, and safe approach to gene delivery, ensuring universal and accessible treatment modalities.

We exploited both lytic (λ) and filamentous (M13) bacteriophages, combining their phage display capabilities with genomic engineering. This enabled targeted delivery of therapeutic transgenic cassettes and facilitated diverse applications. The M13 system was engineered for highly pure and efficient extrusion of miniphagemids, while our novel tunable phage display (TPD) approach with λ phage enhanced cellular penetration and gene expression. Both phagemid vectors demonstrated the ability to target and penetrate multiple tissue types, revealing unique mechanisms of phage tissue translocation for targeted gene delivery in immuno-oncology and DNA vaccine production.

Localization of iPhAGES miniphagemids with and without EGF display over time in HeLa cells

Key features:

  • Elimination of Bacterial Backbone: Our split-origin miniphagemid design ensures that upon induction in E. coli, only the therapeutic gene cassette undergoes site-specific excision and circularization. This process completely removes the bacterial backbone, preventing the inclusion of antibiotic resistance genes or CpG motifs. This not only enhances biosafety and regulatory compliance but also reduces immunostimulation and significantly enhances gene expression.

  • Enhanced Targeted Uptake: The display of an EGF ligand enables receptor-mediated uptake, which dramatically improves targeted cellular internalization and can lead to enhanced in gene expression (e.g., 700x increase in HeLa cells).

  • Ensured Vector Purity: Packaging of the excised minivector is enabled by a specialized helper phage system. The iPhAGE platform utilizes M13SW8, a proprietary helper phage specifically engineered to be self-packaging-deficient. This inability to encapsidate its own genome means M13SW8 supports packaging only for miniphagemids containing the functional packaging origin, minimizing contamination with full-length phagemids and helper DNA.

These features provide a scalable, regulatory-aligned, and clinically suitable gene delivery solution that addresses the long-standing limitations of both plasmid DNA and traditional phage-based vectors. Through advanced genetic engineering, we've enabled the production of miniphagemid-packaged DNA minivectors with unparalleled purity, efficiency, and consistency.

The M13 iPhAGE Production System:

M13 is a nonlytic phage that specifically infects F+ E. coli.​ It is genetically and structurally simple; as a result is very amenable to genetic modifications both to its encapsidated genome and for the display of peptides on its virion coat​. Due to it’s capsid assembly and structure, M13 miniphagemid vectors have a theoretically limitless gene cargo capacity.

The M13 iPhAGE system packages and delivers both single-stranded circular (ssDNA) and linear covalently closed (LCC) DNA minivectors in a targeted manner. These represent unique inventions in phagemid design.

  1. Cell uptake of infecting phage/precursor plasmid

  2. Synthesis of double-stranded (ds) replicative factor (RF) and expression of phage gene products

    • Helper phage provides phage proteins in trans

    • Interrupted origin → preferential packaging of target phagemid

    • Split origin precursor: transgene cassette between separated START & STOP

    • Rolling circle amplification of intracellular RF

    • Predicated new recombinant factor (RFx) →  transgene cassette without plasmid backbone

  3. Sequestration of ssDNA intermediates for assembly

  4. Extrusion of progeny phage, purified from environment