Mammalian cell transduction and internalization properties of λ phages displaying the full-length adenoviral penton base or its central domain

Abstract

In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage lambda 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 lambda 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 ((340)RGD(342)). Both the phage chimerae lambda Pb and lambda 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 lambda Pb and lambda Pb (286-393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of lambda Pb (286-393), but not that of lambda Pb. The proteasome inhibitor MG-132 improved the efficiency of lambda Pb (286-393)-mediated transduction, but not that of lambda Pb. In summary, this work shows the feasibility of using lambda phage as an efficient vector for gene transfer into mammalian cells. We show that lambda Pb and lambda Pb (286-393) can both mediate receptor-dependent transduction; while only lambda Pb is able to promote endosomal escape and proteasome resistance of phage particles.

A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery

“…Most attempts to enhance lambda phage-mediated gene transfer to mammalian cells have concentrated on optimizing the binding of phage particles to mammalian cells (7,8,13,14). However, the eukaryotic cell poses numerous barriers to phage-mediated gene transfer.…”

“…Thus, the ultimate success of phage-mediated gene transfer depends on the ability to overcome multiple intracellular barriers. This is likely to require the use of combination strategies that increase the efficiency of each step involved in phage-mediated gene transfer, including cell attachment, cytoplasmic entry, endosomal escape, uncoating and nuclear import (7,15,16). …”

A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda

“…This technology was developed and explored for the most part with filamentous phage and it has been shown that surface expression of receptor-binding peptides or proteins can result in phage binding to mammalian cells and, in some cases, in phage internalization (14–16,23–25,35,36,52,53). When such phage contain mammalian expression cassettes, this can result in expression of encoded genes within the target cells of interest (14,15,23–25,35,37). These advances have led to the proposed use of bacteriophage as gene delivery vehicles.…”

“…Bacteriophage λ has been shown to be able to accommodate the display of foreign peptides and proteins on the gpD major coat protein (3,4,15,20,28,35,38). However, in the course of our studies, we have encountered certain proteins that are poorly tolerated when displayed on the bacteriophage λ capsid at high copy number (405–420 copies per capsid), resulting in an inability to generate recombinant phage particles that display the desired molecule.…”

Phage display and its application in vaccine design

“…Dickerson et al (2005) reported that engineered filamentous bacteriophage displaying cocainesequestering antibodies delivered into the central nervous system are capable of treating cocaine addiction. Receptortargeted phage might have important implications for the use of phage as a DNA vaccine or therapeutic gene delivery vehicle directed to specific cell types (Barry et al 1996;Dunn 1996;Larocca et al 1998Larocca et al , 1999Piersanti et al 2004). …”