BACKGROUND: The ability to direct efficiently and specifically carriers toward target cells and express the transgene of interest is a critical step in gene therapy trails. The display of targeting molecules on the surface of phage particles might represent a potent solution. In the present study, we evaluated a chemical coupling strategy for displaying human holotransferrin as a targeting molecule on the surface of phage lambda particles for specifically delivering green fluorescent protein (GFP) encoding gene into a human cell line. METHODS: Human holotransferrin was coupled on the phage lambda particles bearing a GFP-expression cassette by a chemical coupling strategy to formulate transferrin-targeted lambda-GFP (Tf-targeted-λ-GFP) gene nanocarrier. The carrier was then characterized by phage-enzyme-linked immunosorbent assay experiments and used for transfection of the human 293T cell line. Particle internalization into the cells was evaluated by immunocytochemical staining and transfection efficacy was studied using fluorescence-activated cell sorting (FACS) analysis. RESULTS: Characterization of the nanocarrier showed a rather high copy number (274 molecules) of transferrin molecules coupled per phage particle. Immunocytochemical staining revealed efficient internalization of the Tf-targeted-λ-GFP compared to wild lambda-GFP (λ-GFP) particles. FACS analysis showed 6.72% GFP positive cells for transfections mediated by Tf-targeted-λ-GFP, whereas the value was 0.61% for wild lambda-GFP particles. CONCLUSIONS: Our findings highlight chemical coupling as an efficient and straightforward strategy for displaying a targeting molecule at high density on the phage surface, which, in turn, may improve the efficiency of phage-mediated gene transfer and expression.
BACKGROUND: The ability to direct efficiently and specifically carriers toward target cells and express the transgene of interest is a critical step in gene therapy trails. The display of targeting molecules on the surface of phage particles might represent a potent solution. In the present study, we evaluated a chemical coupling strategy for displaying human holotransferrin as a targeting molecule on the surface of phage lambda particles for specifically delivering green fluorescent protein (GFP) encoding gene into a human cell line. METHODS:Human holotransferrin was coupled on the phage lambda particles bearing a GFP-expression cassette by a chemical coupling strategy to formulate transferrin-targeted lambda-GFP (Tf-targeted-λ-GFP) gene nanocarrier. The carrier was then characterized by phage-enzyme-linked immunosorbent assay experiments and used for transfection of the human 293T cell line. Particle internalization into the cells was evaluated by immunocytochemical staining and transfection efficacy was studied using fluorescence-activated cell sorting (FACS) analysis. RESULTS: Characterization of the nanocarrier showed a rather high copy number (274 molecules) of transferrin molecules coupled per phage particle. Immunocytochemical staining revealed efficient internalization of the Tf-targeted-λ-GFP compared to wild lambda-GFP (λ-GFP) particles. FACS analysis showed 6.72% GFP positive cells for transfections mediated by Tf-targeted-λ-GFP, whereas the value was 0.61% for wild lambda-GFP particles. CONCLUSIONS: Our findings highlight chemical coupling as an efficient and straightforward strategy for displaying a targeting molecule at high density on the phage surface, which, in turn, may improve the efficiency of phage-mediated gene transfer and expression.
Authors: Rolando Paciello; Richard A Urbanowicz; Gennaro Riccio; Emanuele Sasso; C Patrick McClure; Nicola Zambrano; Jonathan K Ball; Riccardo Cortese; Alfredo Nicosia; Claudia De Lorenzo Journal: J Gen Virol Date: 2015-10-29 Impact factor: 3.891