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We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.

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.

Although growth factor- and antibody-targeted filamentous phage have recently been demonstrated to transduce mammalian cells, there is a significant need to increase transduction efficiency so as to improve the usefulness of targeted phage vectors for gene therapy and ligand discovery. Here, we describe the use of multivalent phagemid vectors that are specifically designed for ligand-targeted mammalian cell transduction. This phagemid system has certain advantages over phage vectors, such as larger insert size and vector stability, and it retains the multivalent display necessary for efficient cell binding and internalization. Immunoblotting revealed that the most efficient multivalent display (exceeding that of a phage vector) was achieved in the phagemid system when epidermal growth factor (EGF) was fused to the C-terminal domain of the pIII coat protein. We compared phagemid particles displaying EGF at high or low valence by rescuing the vector with R408d3 (pIII deleted) or wild-type R408 helper phage, respectively. More efficient display of EGF correlated with increased internalization, vector potency, and transduction efficiency ( approximately 9%). The findings described here support our original hypothesis that phage-based vectors can be modified for more efficient gene transfer and suggest that directed evolution may be applied to increase their potential even further.

Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.

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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% 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 multivalent phagemid-based vectors with transduction efficiencies of up to 45% 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 with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

Human embryonic stem cells offer a scalable and renewable source of all somatic cell types. Human embryonic progenitor (hEP) cells are partially differentiated endodermal, mesodermal and ectodermal cell types that have not undergone terminal differentiation and express an embryonic pattern of gene expression. Here, we describe a large-scale and reproducible method of isolating a diverse library of clonally purified hEP cell lines, many of which are capable of extended propagation in vitro. Initial microarray and non-negative matrix factorization gene-expression profiling suggests that the library consists of at least 140 distinct clones and contains many previously uncharacterized cell types derived from all germ layers that display diverse embryo- and site-specific homeobox gene expression. Despite the expression of many oncofetal genes, none of the hEP cell lines tested led to tumor formation when transplanted into immunocompromised mice. All hEP lines studied appear to have a finite replicative lifespan but have longer telomeres than most fetal- or adult-derived cells, thereby facilitating their use in the manufacture of purified lineages for research and human therapy.