In Vivo Selection and Validation of Liver-Specific Ligands Using a New T7 Phage Peptide Display System

Peptide-mediated targeting of hepatocytes via low density lipoprotein receptor-related protein (LRP)

“…The contribution of LDLr to the p17 targeting of hepatocytes is rather limited and is likely to be even smaller in normal animals as compared to LRP knock-outs, since the latter over-express LRP by at least 2-fold (Rohlmann et al, 1998). The limited role of LDLr in phage uptake is consistent with our previous observations that only the phage-displayed sequence derived from ApoE, which reacts with both LRP and LDLr, but not the sequence derived from ApoB, which mostly reacts with LDLr, mediates efficient phage internalization by hepatocytes (Ludtke et al, 2007).…”

“…The absence of LRP expression in these cells, which are readily accessible to blood-born LRP-targeting particles and proteins, is an important prerequisite for the therapeutic use of LRP-targeted bulky agents, as it essentially limits the targeting of blood-restricted agents to hepatocytes and macrophages. Macrophages can be differentially and reversibly suppressed, which would enhance the specificity of hepatocyte targeting (Sokoloff et al, 2003;Ludtke et al, 2007;Russell et al, 2007). Also, internalized phage is degraded by Kupffer cells much faster than by hepatocytes (Sokoloff et al, 2003), which suggests that the endosomal release and functional expression in Kupffer cells of genes, oligonuclotides, and siRNA is less likely than in hepatocytes.…”

“…Notably, the phage p17 targeting sequence also contains several positively charged residues that are critical for hepatocyte targeting (Sokoloff et al, 2003). Using our newly designed p17 display system, with the p17 targeting sequence replaced with a peptide display site, we have found that all p17-displayed random sequences selected for hepatocyte targeting are also positively charged (Ludtke et al, 2007).…”

“…Up to 95% of injected phage is taken up by liver, with ∼ up to 80% of the phage being associated with hepatocytes. The rest of the liver-sequestered phage is mostly associated with Kupffer cells (Sokoloff et al, 2003;Ludtke et al, 2007). The phage reaches hepatocytes within 5 min after injection.…”

“…Preinjection of asialofetuin has no effect on phage uptake, suggesting that the uptake is not mediated by asialoglycoprotein receptor (ASGPr) (Sokoloff et al, 2003). Immunohistochemistry studies have shown that a large portion of hepatocyte-associated phage is internalized (Sokoloff et al, 2003;Ludtke et al, 2007). A synthetic analogue of the p17 sequence imparts hepatocyte targeting onto fusion proteins, polymers, siRNA, liposomes, and DNA particles (Wong et al, 2006).…”