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 ( 340RGD 342). 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.
|Number of pages||10|
|Journal||Journal of Molecular Medicine|
|Publication status||Published - Jul 2004|
- Endosomal escape
- Gene therapy
ASJC Scopus subject areas