Evolving Phage Vectors for Cell Targeted Gene Delivery

David      Larocca; Michael   A.   Brug; Kristen      Jensen-pergakes; Edward   Prenn   Ravey; Ana   Maria   Gonzalez; Andrew      Baird

Abstract

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2 percent of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create mu ltivalent phagemid-based vectors with transduction efficiencies of up to 45 percent in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors

Keywords: Phage Vectors, Gene Delivery, single-chain antibody, adenoassociated virus (AAV)

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