Display of two distinct modifications on the phage capsid results in a dose-dependent enhancement of phage-mediated gene transfer in RAW 264

<p><b>Copyright information:</b></p><p>Taken from "A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery"</p><p></p><p>Nucleic Acids Research 2007;35(8):e59-e59.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1885665.</p><p>© 2007 The Author(s)</p>7 cells. RAW 264.7 cells were incubated with phage particles at the indicated MOIs (high, intermediate and low doses corresponding respectively to 1 × 10, 3 × 10 or 1 × 10 p.f.u./cell). After an initial centrifugal enhancement step (1200 , 1 h), cells were incubated with the phage for 48 h at 37°C. Cells were then washed, lysed, and analyzed for luciferase expression. Luciferase assays were performed using equal total protein quantities for each sample. Unmodified phage particles (gpD) and particles which displayed only the CD40-binding peptide (gpV-CD40) did not mediate levels of luciferase gene expression that were significantly above background, when compared to control cell lysates; a modest but non-statistically significant trend towards increased gene transduction was observed in the case of the gpV-CD40 phage at the high dose level. In contrast, surface display of gpD-UBHA produced a 10-fold increase in gene expression above baseline at the high dose level ( < 0.01, when compared to unmodified phage) and a modest (1.7-fold) but non-statistically significant increase at the intermediate dose level. Gene transfer efficiency was also tested in cells that were exposed to a simple mixture of phage particles that displayed either gpV-CD40 or gpD-UBHA on their surface (designated as ‘gpD-UBHA + gpV-CD40’). In this case, the full-indicated dose of phage was added to the cells (i.e. at the high dose levels, cells received 1 × 10 p.f.u. of gpV-CD40 phage 1 × 10 p.f.u. of gpD-UBHA phage). Exposure of cells to this mixture of phage particles resulted in levels of gene transfer that were very similar to those elicited by the gpD-UBHA phage alone. There was a statistically significant 7-fold increase in gene transfer efficiency at the high dose group level ( < 0.01, compared to unmodified phage) and a modest (2-fold) but non-statistically significant increase at the intermediate dose level. Finally, cells were exposed to the bi-functional phage, which displayed both gpV-CD40 with gpD-UBHA on its surface (designated gpD-UBHA/gpV-CD40). This resulted in a further increase in phage-mediated gene expression (by ∼2-fold) when compared to the gpD-UBHA head modification alone, or to the simple mixture of gpD-UBHA and gpV-CD40 phage, at both the high and intermediate dose levels. At the intermediate dose level, this result achieved statistical significance ( < 0.01, compared to cells exposed either to gpD-UBHA phage alone or to the mixture of gpD-UBHA + gpV-CD40 phage). At the high dose level, the result achieved statistical significance when compared to the mixture of gpD-UBHA + gpV-CD40 phage ( < 0.05), but not when compared to the gpD-UBHA phage alone. Statistical analysis was performed using one-way ANOVA with Tukey's post-test; significance was taken as < 0.05