Multiple phage capsid proteins suitable for peptide display have been identified in both lambda and M13. Despite this, simultaneous display from two or more protein platforms has rarely been described, even in the more widely studied filamentous phage display system (37–40
). Recently, a bifunctional filamentous phage intended for delivering biological agents was described (40
). The phage combined an integrin-targeting moiety at pIII with a streptavidin-binding sequence at pVIII. The authors tested their phage in vitro
for receptor specific cell binding and internalization. In addition, they complexed their phage with quantum dots and then demonstrated tumor-specific accumulation after intravenous injection in mice. Despite these few successes, the design of bifunctional phage remains underexplored and underutilized. In 1996, Dunn proposed a bifunctional lambda phage combining gpD and gpV display, yet, until now, a bifunctional lambda phage has never been described (8
The experiments reported here resulted in the simultaneous co-display of two different peptide modifications on the head and tail of lambda phage particles, as translational fusions to gpD and gpV, respectively. In both cases, a high copy number of the displayed peptide was achieved (full replacement, or roughly 400 copies/phage particle in the case of the gpD fusion protein and partial replacement, or roughly 100 copies/phage particle in the case of the gpV fusion protein). Moreover, because the gpD and gpV expression plasmids described here are compatible with the previously described, CDF-origin-based plasmid, pTrcCDF:gpD-Fusion (17
), it should be possible to introduce up to three modifications to a single phage particle in the future (two to gpD, and one to gpV).
The long-term goal of our experiments is to develop a bacteriophage lambda vector system that is capable of mediating efficient gene transfer into mammalian cells. As a first step towards this goal, we evaluated phage-mediated gene transfer in a murine macrophage cell line using recombinant lambda phage particles that encoded a luciferase reporter gene. These experiments showed that the surface display of an ubiquitinylation motif resulted in a profound enhancement of phage-mediated gene transfer.
The ubiquitinylation motif may enhance phage-mediated gene transfer as a result of proteasome-mediated uncoating of the phage particle, or because of effects on the intracellular trafficking of internalized phage particles (30
), and possible localization of internalized phage particles to multi-vesicular bodies (31
), or due to effects on other intracellular pathways (29
). It is noteworthy that certain mammalian viruses such as Vesicular Stomatitis Virus (VSV) rely on multi-vesicular bodies for endosomal escape and efficient infection of host cells (41
). Attempts to resolve the mechanism by which the displayed ubiquitinylation motif enhances phage-mediated gene transfer were inconclusive, since the well-characterized proteasome inhibitors MG-132 and lactacystin proved toxic to the RAW 264.7 cells, even at the minimum effective dose. Thus, future studies will be needed to investigate the mechanism by which the ubiquitinylation motif enhances phage-mediated gene transfer.
Our proof-of-principle experiments also evaluated whether the simultaneous display of two different modifications on the phage surface may further enhance phage-mediated gene transfer. To do this, we generated luciferase-encoding phage particles that displayed a receptor (CD40)-binding peptide on their major tail protein (gpV) in addition to the ubiquitinylation motif on their major head protein (gpD). These bifunctional phage particles were able to mediate an enhanced efficiency of gene transfer into a cultured murine macrophage cell line, when compared to phage particles that displayed only a single peptide moiety on their surface. This effect was dose-dependent, with higher levels of gene transfer being detected when larger amounts of phage were added to cells. Furthermore, co-display of both motifs on the same phage particle was required for the observed enhancement of phage-mediated gene transfer; gene transfer efficiency was significantly improved when compared to a simple mixture of gpD-UBHA phage plus gpV-CD40 phage. Thus, the enhanced gene transfer effect by the dual-display construct cannot be attributed to a trans-effect.
Overall, the results reported provide strong support for the notion that it may be possible to rationally improve the efficiency of phage-mediated gene transfer by displaying several different peptides on the phage surface. In the future it should be possible to introduce and test other modifications with the intention of eventually designing a highly efficient phage-based gene delivery vector.