A new peptide ligand that targets particles and heterologous proteins to hepatocytes in vivo
Authors: Alex V Sokoloff, So C Wong, James J Ludtke, Magdolna G Sebestyén, Vladimir M Subbotin, Guofeng Zhang, Tatyana Budker, Marcus Bachhuber, Yoshihiro Sumita, Jon A Wolff
Journal: Molecular Therapy (2003)

Hepatocytes are an important target in the treatment of various metabolic and infectious disorders [1Wu J. Nantz M.H. Zern M.A. Targeting hepatocytes for drug and gene delivery emerging novel approaches and applications.Front. Biosci. 2002; 7: d717-725Crossref PubMed Google Scholar]. Most approaches for targeting hepatocytes have relied upon ligands reacting with the asialoglycoprotein receptor (ASGPr) [1Wu J. Nantz M.H. Zern M.A. Targeting hepatocytes for drug and gene delivery emerging novel approaches and applications.Front. Biosci. 2002; 7: d717-725Crossref PubMed Google Scholar, 2Groman E.V. Enriquez P.M. Josephson L. Arabinogalactan for hepatic drug delivery.Bioconj. Chem. 1994; 5: 547-556Crossref PubMed Scopus (101) Google Scholar, 3Lee Y.C. et al.Binding of synthetic oligosaccharides to the hepatic gal/GalNAc lectin.J. Biol. Chem. 1983; 258: 199-202PubMed Google Scholar]. Such ligands have allowed hepatocyte targeting with small molecules and proteins [4Rogers J.C. Kornfeld S. Hepatic uptake of proteins coupled to fetuin glycoprotein.Biochem. Biophys. Res. Commun. 1971; 45: 622-629Crossref PubMed Scopus (121) Google Scholar, 5Fiume L. et al.Inhibition of hepatitis viral replication by vidarabine monophosphate conjugated with lactasaminated serum albumin.Lancet. 1988; 2: 13-15Abstract PubMed Scopus (68) Google Scholar]. However, the utility of ASGPr ligands for targeting particles is less clear, as judged from a number of often conflicting reports concerning the size limitation of ASGPr-reactive particles and liposomes. A wide variety of size limits for ASGPr-reactive particles, ranging from 10 to 70 nm, has been reported [1Wu J. Nantz M.H. Zern M.A. Targeting hepatocytes for drug and gene delivery emerging novel approaches and applications.Front. Biosci. 2002; 7: d717-725Crossref PubMed Google Scholar, 6Rensen P.C. et al.Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo.J. Biol. Chem. 2001; 276: 37577-37584Crossref PubMed Scopus (172) Google Scholar, 7Schlepper-Schafer J. et al.Endocytosis via galactose receptors in vivo ligand size directs uptake by hepatocytes and/or liver macrophages.Exp. Cell Res. 1986; 165: 494-506Crossref Scopus (71) Google Scholar, 8Furumoto K. et al.Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver.Biochim. Biophys. Acta. 2001; 1526: 221-226Crossref Scopus (40) Google Scholar]. Overall, the complexity of effects observed with particles and liposomes in hepatocyte delivery experiments suggests that the finding of new targeting ligands remains an important goal for creating robust delivery systems. New hepatocyte ligands of proteinaceous origin would also be beneficial in that they could be incorporated genetically into proteins and viral vectors. In our previous studies, we attempted to select organ-targeting peptides by using peptide libraries displayed on icosahedral T7 phage (∼60 nm in diameter) that mimics the size and shape of liposomes and DNA polyplexes (Fig. 1A). However, a T7 library displaying random peptides at the carboxy-terminus of the coat protein 10B (p10; 415 copies per phage particle) was rapidly inactivated by complement when injected into rats at a ratio of 109–1010 pfu per animal [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The activation of complement was mediated by a large population of natural IgM antibodies reacting with phage-displayed peptides in a sequence-specific manner [10Sokoloff A.V. et al.Specific recognition of protein carboxy-terminal sequences by natural IgM antibodies in normal serum.Mol. Ther. 2001; 3: 821-830Abstract Full Text Full Text PDF Scopus (15) Google Scholar]. A portion of the injected phage library was taken up by liver [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar], presumably by nonparenchymal liver cells (NPCs) expressing complement receptors [11Ahearn J.M. Rosengard A.M. Complement receptors.in: Volanakis J.E. Frank M.M. The Human Complement System in Health and Disease. Dekker, New York1998: 167-202Crossref Google Scholar]. In agreement with this model, the phage selected for long-term circulation in rats (clone 20-6) lost its p10 peptide display region (truncated after Q343) and, along with it, the ability to bind natural IgM, activate complement, and accumulate in liver [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The truncation after Q343 was the only structural change in the p10 of selected phage. Later on, we found that mice were a better model than rats for studying the interaction of IgM-reactive T7 phage with liver. The inactivation of phage by complement in mice was slower than in rats, and a majority of liver sequestered phage could be detected in an infectious state (data not shown). When we introduced the p10 truncation mutation into nonselected phage, the resultant phage, when injected into mice, unexpectedly disappeared from the circulation and rapidly accumulated in liver. Apparently, there was a complement-independent mechanism of phage uptake that was mediated by a phage determinant absent in the selected phage. Two-dimensional electrophoresis and DNA sequencing revealed that phage 20-6 also expressed a mutated tail fiber protein, p17, with residue K211 substituted with an E (Table 1). We confirmed the suspected role of the p17 mutated region in liver targeting by reverting the E211 in phage 20-6 back to a K present in this position in the wild-type phage. While phage 20-6 did not target liver to a noticeable degree, the reverted phage 20-6F (F stands for "fixed") accumulated in liver with an average efficiency of ∼63% 5 min after injection and ∼75% (up to 90% in individual animals) 60 min after injection (Table 1). We evaluated accumulation of phage 20-6F in other organs (lung, heart, brain, muscle, spleen, kidney, pancreas, testes or ovaries, thyroid, adrenal, skin, bone marrow, intestine, stomach, uterus, and eye) in a similar fashion, by injecting 1011 pfu of phage into the tail vein and, 5 min later, perfusing the whole animal with PBS/heparin. In contrast to the liver, these organs accumulated individually and together <0.5% and <4.5% of injected phage, respectively. The results from immunohistochemical experiments with different tissues also indicated that a predominant majority of injected 20-6F phage was targeted to the liver (Figs. 2A2 and 2A3 ; data for nonliver tissues are not shown). Thus, a determinant that apparently included the K211 residue of p17 was necessary for selective phage targeting to liver.TABLE 1T7 phage uptake by liverPhage typeP17 sequenceaThe critical amino acid residues in p17 position 211 are shown in bold. Both clones had a truncated p10 coat protein ending in sequence VVFQ343.Live phage in liver,b109 pfu of T7 phage was injected into the tail vein of mice, and the amount of infectious phage sequestered by liver was evaluated at 60 min after injection. The results are presented as mean ± standard deviation for 6 mice.%Phage per hepatocytec1011 pfu of T7 phage was injected into the tail vein of mice, and the percentage of phage-containing hepatocytes was determined at 5 or 30 min after phage injection. The amount of intracellular material reactive with anti-T7 antibody was semi-quantitated by confocal fluorescence microscopy (described in the figrue legend for Fig. 2). Five randomly chosen cells in each group were examined in 4-fold enlarged reconstructed images (3 μm thick) acquired at a 630-fold magnification. At this magnification, the smallest "phage particle" appeared as a red pixel. The average amount of phage in an individual hepatocyte was estimated as a number of stained pixels normalized to the intensity of staining. The number of bright red pixels was multiplied by 4, the number of pixels with an intermediate brightness was multiplied by 2, and the number of weakly stained pixels was multiplied by 1.5 min30 min20-6R207DEAE211RF3.7 ± 1.661–5 in 20% cells3–7 in 50% cells20-6FdT7-phage 20-6F was prepared by amplifying the wild-type gene 17 region of T7 DNA with 5′-gagtacttggagcGTGCACtggacccaac-3′ and 5′-ccatcagtGGCCacaacGGCCtgacctac-3′ primers containing unique ApaL I and Sfi I sites, respectively. Amplified DNA was digested with Sfi I and ApaL I and ligated to the genomic DNA isolated from T7 phage 20-6 and digested with Sfi I and ApaL I. The ligation product was packaged (Novagen [21]) and the resultant infectious phage was amplified.R207DEAK211RF74.8 ± 5.7050–250 in 100% cells500–2000 in 100% cellsNote. T7 phage (109 pfu) was injected into the tail vein of anesthetized male ICR mice (18–22 g) in 1 ml of PBS under low pressure (over 60 sec). Animals were preinjected with 10 U of heparin and the liver was perfused with PBS containing 2 U/ml heparin through the portal vein, with the right atrium cut. Livers were homogenized in PBS containing 2% Triton X-100 and 1 M NaCl and the amount of liver-associated phage was evaluated by a plaque assay 9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar.a The critical amino acid residues in p17 position 211 are shown in bold. Both clones had a truncated p10 coat protein ending in sequence VVFQ343.b 109 pfu of T7 phage was injected into the tail vein of mice, and the amount of infectious phage sequestered by liver was evaluated at 60 min after injection. The results are presented as mean ± standard deviation for 6 mice.c 1011 pfu of T7 phage was injected into the tail vein of mice, and the percentage of phage-containing hepatocytes was determined at 5 or 30 min after phage injection. The amount of intracellular material reactive with anti-T7 antibody was semi-quantitated by confocal fluorescence microscopy (described in the figrue legend for Fig. 2). Five randomly chosen cells in each group were examined in 4-fold enlarged reconstructed images (3 μm thick) acquired at a 630-fold magnification. At this magnification, the smallest "phage particle" appeared as a red pixel. The average amount of phage in an individual hepatocyte was estimated as a number of stained pixels normalized to the intensity of staining. The number of bright red pixels was multiplied by 4, the number of pixels with an intermediate brightness was multiplied by 2, and the number of weakly stained pixels was multiplied by 1.d T7-phage 20-6F was prepared by amplifying the wild-type gene 17 region of T7 DNA with 5′-gagtacttggagcGTGCACtggacccaac-3′ and 5′-ccatcagtGGCCacaacGGCCtgacctac-3′ primers containing unique ApaL I and Sfi I sites, respectively. Amplified DNA was digested with Sfi I and ApaL I and ligated to the genomic DNA isolated from T7 phage 20-6 and digested with Sfi I and ApaL I. The ligation product was packaged (Novagen 21Novagen (1999). T7Select System ManualGoogle Scholar) and the resultant infectious phage was amplified. Open table in a new tab Note. T7 phage (109 pfu) was injected into the tail vein of anesthetized male ICR mice (18–22 g) in 1 ml of PBS under low pressure (over 60 sec). Animals were preinjected with 10 U of heparin and the liver was perfused with PBS containing 2 U/ml heparin through the portal vein, with the right atrium cut. Livers were homogenized in PBS containing 2% Triton X-100 and 1 M NaCl and the amount of liver-associated phage was evaluated by a plaque assay 9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar. All clones found in the phage population selected for long-term circulation in rats and analyzed soon after the selection contained the p17-E211 [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. We found less common p17 mutations after a prolonged storage of selected phage at −80°C, apparently due to preferential inactivation of p17-E211 clones. These mutations included R207 substituted with an L, R200 substituted with a C, and R200 substituted with an S. The T7 phage tail fiber is composed of three parallel p17 chains that form proximal (residues 1 to ∼267) and distal (residues ∼268–553) half-fibers separated by a kink (Fig. 1C). Residues 151–267 of the proximal half-fiber form a triple-stranded coiled coil, known as the rod domain (Fig. 1B; [17]). All mutations disrupting the liver targeting were within the rod domain and occupied the exterior positions, b and e, of two adjacent coiled-coil heptad repeats. Thus, the mutations delineated a compact area on the coiled-coil surface that presumably served as a liver-targeting determinant or constituted a part of this determinant. We analyzed the association of phage sequestered by liver with hepatocytes and NPCs (Kupffer, endothelial, and stellate cells) by immunohistochemistry (Figs. 2A1–2A3 and 2B1). Hepatocytes were typically seen as large polygonal cells with round-shaped nuclei. Many hepatocytes contained double nuclei, which is a common morphological marker of these cells [12Severin E. Willers R. Bettecken T. Flow cytometric analysis of mouse hepatocyte ploidy. II. The development of polyploidy pattern in four mice strains with different life spans.Cell Tissue Res. 1984; 238: 649-652Crossref PubMed Scopus (30) Google Scholar]. Another distinctive marker of hepatocytes was the presence of highly counterstained F-actin along bile capillaries [13Benkoel L. et al.Effect of ischemia–reperfusion on bile canalicular F-actin microfilaments in hepatocytes of human liver allograft image analysis by confocal laser scanning microscopy.Dig. Dis. Sci. 2001; 46: 1663-1667Crossref PubMed Scopus (31) Google Scholar]. NPCs typically had less cytoplasm and contained single, often elongated, relatively small nuclei. The morphological differences between NPCs and hepatocytes correlated with selective staining of NPCs by antibodies specifically reacting with macrophages or endothelial cells (data not shown). Immunohistochemical experiments showed that phage 20-6, expressing p17-E211, was present in liver almost exclusively in sinusoidal areas occupied by NPCs and blood (Fig. 2A1). We observed only occasional particles of this phage in hepatocytes. In stark contrast, phage 20-6F, expressing the wild-type p17-K211, was largely associated with hepatocytes (Figs. 2A2, 2A3, and 2B1). A large amount of phage 20-6F was associated with every hepatocyte as early as 5 min after injection (Fig. 2A2). We observed a significant portion of this phage as small clusters inside hepatocytes. The rapid accumulation of phage in hepatocytes presumably was due to the separation of hepatocytes and the bloodstream by sinusoid fenestrae (∼100 nm in diameter) that are large enough to allow phage passage [7Schlepper-Schafer J. et al.Endocytosis via galactose receptors in vivo ligand size directs uptake by hepatocytes and/or liver macrophages.Exp. Cell Res. 1986; 165: 494-506Crossref Scopus (71) Google Scholar]. The amount of phage inside hepatocytes increased by 30 min after injection (Fig. 2A3). Importantly, as mentioned above, ∼75% of liver-sequestered phage remained infectious at 60 min after infection, suggesting that the phage observed in hepatocytes was largely represented by intact phage particles. The amounts of phage 20-6 and phage 20-6F present in sinusoidal areas at 30 min after injection were similar (Figs. 2A1 and 2A2, respectively). Preinjection of mice with 1 mg of asialofetuin did not affect the amount of 20-6F phage accumulated in hepatocytes (data not shown), suggesting that the ASGPr was not involved in phage uptake [4Rogers J.C. Kornfeld S. Hepatic uptake of proteins coupled to fetuin glycoprotein.Biochem. Biophys. Res. Commun. 1971; 45: 622-629Crossref PubMed Scopus (121) Google Scholar]. A portion of phage 20F observed inside hepatocytes was in the same optical section as hepatocyte nuclei (Fig. 2B1). The analysis of consecutive 0.4-μm optical sections through a 7-μm span of the hepatocyte body indicated that the phage clusters in the nuclear optical plane represented discrete structures that did not extend toward the periphery of the cells (data not shown). We ruled out the possibility of extracellular phage being relocated into the hepatocyte interior by the liver sectioning procedure by injecting highly concentrated phage 20-6 into a bile duct and observing that the phage in the liver sections remained exclusively in the bile duct lumen (data not shown). Thus, the phage seen inside hepatocytes was truly internalized. To determine the cellular distribution of infectious 20-6F phage in the mouse liver, we isolated mouse liver cells 60 min after injecting 109 pfu of phage into the tail vein and subjected them to FACS as described by Weglarz et al. [14Weglarz T.C. Degen J.L. Sandgren E.P. Hepatocyte transplantation into diseased mouse liver. Kinetics of parenchymal repopulation and identification of the proliferative capacity of tetraploid and octaploid hepatocytes.Am. J. Pathol. 2000; 157: 1963-1974Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar]. We lysed 5000 sorted cells with an equal volume of PBS containing 2% Triton X-100 and 1 M NaCl and evaluated the amount of cell-associated phage by a plaque assay [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The sorting of liver cells into diploid cells, including both NPCs and hepatocytes, and tetraploid cells, containing almost exclusively hepatocytes [12Severin E. Willers R. Bettecken T. Flow cytometric analysis of mouse hepatocyte ploidy. II. The development of polyploidy pattern in four mice strains with different life spans.Cell Tissue Res. 1984; 238: 649-652Crossref PubMed Scopus (30) Google Scholar], showed that a single tetraploid cell contained on average ∼2.5 times more 20-6F phage particles than a single diploid cell (data not shown). Assuming that all sorted diploid cells were represented by NPCs and that hepatocytes constituted ∼60% of liver cells [15Alpini, G. P., ??, J. O., Larusso, N. F. (1994). The biology of biliary epithelia. In The Liver: Biology and Pathobiology (I. M. Arias, Ed.), 3rd ed., pp. 623–664. Raven Press, New YorkGoogle Scholar] (61% in our experiments), the ratio between the amounts of phage sequestered by hepatocytes and NPCs in the whole liver was (2.5/1) × (6/4) = 3.75/1. This ratio corresponds to ∼79% of liver-sequestered phage being in hepatocytes, which is a conservative estimate based on the assumption that all diploid cells are represented by NPCs. We also discerned the presence in liver of two different populations of sequestered phage, one apparently internalized by hepatocytes and another by macrophages, by comparing the degradation rate for phage mostly associated with hepatocytes or NPCs. A phage clone, IL-1M, expressing p17-E211 and a natural IgM-reactive peptide on p10 (modified clone IL-1 [9Sokoloff A.V. Bock I. Zhang G. Sebestyen M.G. Wolff J.A. The interactions of peptides with the innate immune system studied with use of T7 phage peptide display.Mol. Ther. 2000; 2: 131-139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]), fixed complement and hence accumulated almost exclusively in NPCs (data not shown) [11Ahearn J.M. Rosengard A.M. Complement receptors.in: Volanakis J.E. Frank M.M. The Human Complement System in Health and Disease. Dekker, New York1998: 167-202Crossref Google Scholar]. Approximately 35% of phage IL-1M was present in an infectious state in the total liver extract prepared at 60 min after phage injection. However, only 0.35% of this phage was recovered in an infectious state at 4 h after injection. In contrast, the percentage of infectious phage 20-6F in the liver only decreased from ∼75% at 60 min (Table 1) to ∼11% at 4 h after phage injection. This suggested that phage 20-6F was degraded ∼15-fold more slowly than phage IL-1M and hence was located in a different liver compartment. Finally, mice depleted of macrophages by treatment with clodronate liposomes [16Schneider G.S. et al.Selective depletion or blockade of Kupffer cells leads to enhanced and prolonged hepatic transgene expression using high-capacity adenoviral vectors.Mol. Ther. 2003; 7: 35-43Abstract Full Text Full Text PDF Scopus (35) Google Scholar] exhibited no discernable decrease in phage 20-6F liver uptake, while phage IL-1M uptake was drastically reduced (data not shown). High as it was, the specificity of hepatocyte targeting exhibited by the p17 determinant might be somewhat underestimated. It appears that the uptake of 20-6F phage by nonparenchymal cells may be at least partly associated with phage that has sustained structural degradation. ELISA experiments have shown that prolonged storage of 20-6 phage at −80°C leads to the recognition of this phage by natural IgM antibodies (data not shown). Thus, the reaction of partially degraded 20-6F and 20-6 phage with natural IgM, likely to be followed by complement activation, could account for the phage uptake by liver macrophages. To determine whether p17 retained its ability to target hepatocytes when removed from its structural context within phage, we expressed the whole-length p17 in bacteria. The full-length gene 17 coding region (wild type) was generated by PCR amplification of gene 17 cloned into pAR1332 (generously provided by Dr. F. W. Studier, Brookhaven National Laboratory). We used forward and reverse primers containing NdeI and BamHI sites, 5′-AAGGAGGTCATATGGCTAACGTAATTAAAACCG-3′ and 5′-GATTGGATCCTTACTCGTTCTCCACCATGATTGCATTAG-3′, respectively. We subcloned the resultant PCR fragmentinto the pET28b plasmid (Novagen) in frame with the N-terminal His-tag coding sequence. We cloned the full-length p17 gene from mutated phage 20-6 into pET28b using the same strategy. We expressed fusion proteins using Rosetta DE3 Escherichia coli host cells (Novagen) and purified them using Ni-NTA agarose (Qiagen) affinity chromatography. Purified proteins were >95% pure by SDS–PAGE. Given that isolated p17 spontaneously forms trimers that are functionally equivalent to the phage tail fiber [17Steven A.C. et al.Molecular substructure of a viral receptor-recognition protein the gp17 tail-fiber of bacteriophage T7.J. Mol. Biol. 1988; 200: 351-365Crossref PubMed Scopus (163) Google Scholar], it was likely that cloned p17 would retain most of its structural determinants. Indeed, purified p17 injected into mice accumulated in liver in essentially the same manner as the phage from which they were derived. Specifically, a considerable amount of p17-K211 was associated with hepatocytes, while p17-E211 was mostly observed in sinusoidal areas (Figs. 2B2 and 2B3). We also observed a similar difference in hepatocyte targeting between p17 expressing K211 or E211 for similarly produced truncated p17, including the proximal half-fiber (residues 1–289), the rod domain combined with the distal half-fiber (residues 150 to 553), and the rod domain alone (residues 150 to 289) (data not shown). The hepatocyte targeting observed with the rod domain alone was consistent with the results of the mutation analysis, suggesting that this domain contained the entire hepatocyte-targeting determinant. We evaluated the utility of the hepatocyte targeting p17 determinant for protein delivery by genetically fusing the whole-length p17 to enhanced yellow fluorescent protein (EYFP). To this end, we amplified the coding region of EYFP from the pEYFP-Nuc plasmid (Clontech) using 5′-CGGTCGCCCATATGGTGAGCAAGGGCGAGGA-3′ and 5′-GATTATGATCATATGTCTAGATCCGGTGGATCCTAC-3′ primers with incorporated NdeI sites. We gel purified the PCR product, digested it with NdeI, and ligated it into the NdeI-digested pET28b plasmid encoding either the wild-type p17-K211 or the mutant p17-E211 full-length protein. We expressed and purified fusion proteins as above. EYFP/p17-K211 protein injected into the mouse tail vein accumulated in hepatocytes, while the EYFP/p17-E211 protein was mostly observed in sinusoidal areas (Figs. 2C1 and 2C2). We also observed hepatocyte targeting with a more therapeutically relevant fusion protein composed of interferon (IFN)-α fused to the p17 rod domain. We amplified the full-length human IFN-α2a coding region by PCR using the pRRB20IF-23 plasmid (ATCC) as a template, with NdeI sites incorporated into both forward and reverse primers, 5′-GCTGCTCTCATATGTGTGATCTGCCTCAAACCCACAGCCTGGG-3′ and 5′-TGAACCAGCATATGTTCCTTACTTCTTAAACTTTCTTGCAAGTTTGTTGACA-3′, respectively. We digested the amplified fragment with NdeI and subcloned it into the NdeI-digested pET28b encoding either the wild-type or the mutant p17 rod domain (residues 150–289). We expressed P17/IFN proteins as above and evaluated their distribution in liver by staining liver sections for either phage protein (Figs. 2C3 and 2D1) or IFN (Figs. 2D2 and 2D3) on separate tissue sections. We detected the IFN/p17-K211 protein mostly in hepatocytes (Figs. 2C3 and 2D2), while the IFN/p17-E211 protein, serving as a negative control, was occasionally seen in sinusoidal areas (Figs. 2D1 and 2D3). The weak signal observed for IFN/p17-E211 stained with anti-IFN antibody (Fig. 2D3) was apparently background staining, since we also observed similar staining in the absence of injected protein (Fig. 2E1). The ability of the p17 determinant to target hepatocytes was not restricted to rodent species. We also observed efficient targeting of hepatocytes by full-length p17-K211 when this protein was administered to rhesus monkeys (Fig. 2E2). In contrast, we detected only a small amount of p17-E211 in hepatocytes (Fig. 2E3). We observed the majority of liver-sequestered p17-E211 in association with nonparenchymal cells, resembling in this respect the distribution of p17-E211 in mice (Fig. 2B3). To summarize, the genetic attachment of the p17 targeting determinant could represent a simple way of converting viral particles and recombinant proteins into hepatocyte-targeting agents [18Okuno K. Nakamura K. Tanaka A. Yachi K. Yasutomi M. Hepatic immunopotentiation by galactose-entrapped liposomal IL-2 compound in the treatment of liver metastases.Surg. Today. 1998; 28: 64-69Crossref Scopus (9) Google Scholar, 19Tomlinson E. Theory and practice of site-specific drug delivery.Adv. Drug Delivery Rev. 1987; 1: 87-198Crossref Scopus (129) Google Scholar, 20Fiume L. et al.The pathogenesis of vacuoles produced in rat and mouse liver cells by a conjugate of adenine arabinoside monophosphate with lactosaminated albumin.J. Hepatol. 1992; 15: 314-322Abstract Full Text PDF PubMed Scopus (19) Google Scholar]. The location of the p17 targeting determinant within a coiled-coil domain suggests that this determinant can be incorporated into viral vectors displaying triple-helix fibers, such as adenoviruses, in a "seamless" fashion [16Schneider G.S. et al.Selective depletion or blockade of Kupffer cells leads to enhanced and prolonged hepatic transgene expression using high-capacity adenoviral vectors.Mol. Ther. 2003; 7: 35-43Abstract Full Text Full Text PDF Scopus (35) Google Scholar]. Given the close location of the p17 mutations disrupting liver targeting, it is quite likely that the hepatocyte-targeting determinant is composed of just two or three heptad repeats. The internalization by hepatocytes of ∼60-nm phage particles suggests that the phage internalization pathway may be also useful for delivery to hepatocytes of such large structures as liposomes and nucleic acid complexes. This is particularly important given the fact that many particulate structures are excluded from internalization through the pathway mediated by the ASGPr [1Wu J. Nantz M.H. Zern M.A. Targeting hepatocytes for drug and gene delivery emerging novel approaches and applications.Front. Biosci. 2002; 7: d717-725Crossref PubMed Google Scholar, 6Rensen P.C. et al.Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo.J. Biol. Chem. 2001; 276: 37577-37584Crossref PubMed Scopus (172) Google Scholar, 7Schlepper-Schafer J. et al.Endocytosis via galactose receptors in vivo ligand size directs uptake by hepatocytes and/or liver macrophages.Exp. Cell Res. 1986; 165: 494-506Crossref Scopus (71) Google Scholar, 8Furumoto K. et al.Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver.Biochim. Biophys. Acta. 2001; 1526: 221-226Crossref Scopus (40) Google Scholar]. The p17 determinant also efficiently targets hepatocytes in nonhuman primates (Fig. 2E2), suggesting that it should be functional in humans as well. Elucidating the exact structure of the hepatocyte-targeting determinant will be the next important step toward potential implementation of this determinant in delivering therapeutic agents. Due to its origin, this determinant is likely to be immunogenic. Decreasing its immunogenicity by manipulating its primary structure and reducing its size may be required to enable its readministration. We have found in preliminary experiments that the hepatocyte-targeting determinant can be considerably modified without affecting its targeting properties, which provides plenty of room for such manipulations (data not shown). It is possible that the p17 internalization pathway is also operational with other ligands, such as chemical constructs or endogenous compounds reactive with the p17-binding receptor. Identification of such ligands could enable the design of nonimmunogenic delivery vehicles utilizing the same internalization pathway. Efforts are under way to identify the p17-binding receptor and its natural ligands. Finally, the utility of the hepatocyte-targeting determinant for therapeutic applications will also be determined by the degree of functional expression exhibited by the agents delivered to hepatocytes through the phage internalization pathway. The experiments aimed at assessing the functionality of protein and DNA molecules delivered through this pathway are currently in progress.