Mammalian cell transduction and internalization properties of λ phages displaying the full-length adenoviral penton base or its central domain
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In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage λ is a promising backbone for the development of new vectors: its genome can host large inserts, DNA is protected from degradation by the capsid and the ligand-exposed D and V proteins can be extensively modified. Current phage-based vectors are inefficient and/or receptor-independent transducers. To produce new, receptor-selective and transduction-efficient vectors for mammalian cells we engineered λ by inserting into its genome a GFP expression cassette, and by displaying the penton base (Pb) of adenovirus or its central region (amino acids 286–393). The Pb mediates attachment, entry and endosomal escape of adenovirus in mammalian cells, and its central region (amino acids 286–393) includes the principal receptor-binding motif (340RGD342). Both the phage chimerae λ Pb and λ Pb (286–393) were able to transduce cell lines and primary cultures of human fibroblasts. Competition experiments showed that the transduction pathway was receptor-dependent. We also describe the different trafficking properties of λ Pb and λ Pb (286–393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of λ Pb (286–393), but not that of λ Pb. The proteasome inhibitor MG-132 improved the efficiency of λ Pb (286–393)-mediated transduction, but not that of λ Pb. In summary, this work shows the feasibility of using λ phage as an efficient vector for gene transfer into mammalian cells. We show that λ Pb and λ Pb (286–393) can both mediate receptor-dependent transduction; while only λ Pb is able to promote endosomal escape and proteasome resistance of phage particles.
KeywordsBacteriophage Adenovirus Gene therapy Endosomal escape
This work was supported by contributions from CNR, Progetto Finalizzato Biotecnologie, Istituto Pasteur Cenci Bolognetti, Università di Roma La Sapienza, MIUR, and Consorzio Interuniversitario Biotecnologie. We are grateful to P. Boulanger, A. Orecchia, and M. Nakanishi for their gifts of materials. We thank G. Ragone, F. Nasorri, M.C. Bonaccorsi, V. Velotta and P. Orlando for technical assistance, and L. Cordier, P. Bianco and C.M. Failla for fruitful scientific discussion.
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