Targeting Bacteriophage to Mammalian Cell Surface Receptors for Gene Delivery

Abstract

Filamentous bacteriophages represent one of nature's most elegant ways of packaging and delivering DNA. In an effort to develop novel methods for ligand discovery via phage gene delivery, we conferred mammalian cell tropism to filamentous bacteriophages by attaching basic fibroblast growth factor (FGF2), transferrin, or epidermal growth factor (EGF) to their coat proteins and measuring CMV promoter-driven reporter gene expression in target cells. In this system, FGF2 was a more effective targeting agent than transferrin or EGF. The detection of green fluorescent protein (GFP) or beta-galactosidase (beta-Gal) activity in cells required FGF2 targeting and was phage concentration dependent. Specificity of the targeting for high-affinity FGF receptors was demonstrated by competing the targeted phage with FGF2, by the failure of FGF2-targeted bacteriophage to transduce high-affinity FGF receptor-negative cells, and by their ability to transduce these same cells when stably transfected with FGFR1, a high-affinity FGF receptor. Long-term transgene expression was established by selecting colonies for G418 resistance, suggesting that with the appropriate targeted tropism, filamentous bacteriophage can serve as a vehicle for targeted gene delivery to mammalian cells.

Clathrin-mediated Endocytosis and Subsequent Endo-Lysosomal Trafficking of Adeno-associated Virus/Phage

“…However, these viruses are considered poor gene delivery vehicles due to inefficient transgene expression. Larocca et al described that filamentous phage displaying FGF was able to deliver GFP to COS-1 cells with 0.4% transduction efficiency (7). More recently, a filamentous phage displaying the full length adenovirus penton base was shown to transduce up to 4% of HeLa cells despite observations that 100% of cells internalized the virus (5).…”

Tumor vasculature‐targeted delivery of tumor necrosis factor‐α*

“…Larocca et al attached fibroblast growth factor 2 (FGF2) to the bacteriophage coat protein and observed long-term transgene expression in FGF receptor-expressing cells. 12,24 Bacteriophage that carries an antibody directed to the cell surface receptor ErbB2 can infect and express a transgene in receptor-positive cells. 25 However, the transduction efficiency for these vectors is very low (%1%-9%), although multivalent phagemid particles have been used.…”

“…25 However, the transduction efficiency for these vectors is very low (%1%-9%), although multivalent phagemid particles have been used. 12,16,25,26 Thus, the use of a native, singlestrand bacteriophage has very limited clinical utility as a therapeutic agent. In contrast, the use of targeted AAVP in our study was able to circumvent this problem, because AAVP-transduced cells generate functional AAV particles with reasonable transduction efficiency.…”

“…11 Bacteriophage vectors have been used to deliver mammalian genes in their native form; however, the transduction efficiency is extremely low. 12 Hence, there is a need for AAVP, which retains the advantageous features of bacteriophage vectors while increasing transduction efficiency through the use of elements of adeno-associated virus (AAV).…”

Development of efficient RNA interference system using EGF-displaying phagemid particles

“…In previous studies, scientists have successfully modified the filamentous bacteriophages, which are capable of delivering genes to mammalian cells using targeting ligands, such as growth factors [1][2][3][4][5] , antibodies [6] , and viral capsid proteins [7] . To improve the ligand display level on phages, we developed LMP cells (Escherichia coli bearing the ligandpIII-encoding helper phage) to make cell-targeted phagemid particles [8] .…”