Targeted Lysosome Disruptive Elements for Improvement of Parenchymal Liver Cell-specific Gene Delivery
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m203510200
ISSN1083-351X
AutoresSabine M.W. van Rossenberg, Karen Sliedregt‐Bol, Nico J. Meeuwenoord, Theo J.C. van Berkel, Jacques H. van Boom, Gijs A. van der Marel, Erik A.L. Biessen,
Tópico(s)Cell death mechanisms and regulation
ResumoThe transfection ability of nonviral gene therapy vehicles is generally hampered by untimely lysosomal degradation of internalized DNA. In this study we describe the development of a targeted lysosome disruptive element to facilitate the escape of DNA from the lysosomal compartment, thus enhancing the transfection efficacy, in a cell-specific fashion. Two peptides (INF7 and JTS-1) were tested for their capacity to disrupt liposomes. In contrast to JTS-1, INF7 induced rapid cholesterol-independent leakage (EC50, 1.3 μm). INF7 was therefore selected for coupling to a high affinity ligand for the asialoglycoprotein receptor (ASGPr), K(GalNAc)2, to im- prove its uptake by parenchymal liver cells. Although the parent peptide disrupted both cholesterol-rich and -poor liposomes, the conjugate, INF7-K(GalNAc)2, only induced leakage of cholesterol-poor liposomes. Given that endosomal membranes of eukaryotic cells contain <5% cholesterol, this implies that the conjugate will display a higher selectivity toward endosomal membranes. Although both INF7 and INF7-K(GalNAc)2 were found to increase the transfection efficiency on polyplex-mediated gene transfer to parenchymal liver cells by 30-fold, only INF7-K(GalNAc)2appeared to do so in an ASGPr-specific manner. In mice, INF7-K(GalNAc)2 was specifically targeted to the liver, whereas INF7 was distributed evenly over various organs. In summary, we have prepared a nontoxic cell-specific lysosome disruptive element that improves gene delivery to parenchymal liver cells via the ASGPr. Its high cell specificity and preference to lyse intracellular membranes make this conjugate a promising lead in hepatocyte-specific drug/gene delivery protocols. The transfection ability of nonviral gene therapy vehicles is generally hampered by untimely lysosomal degradation of internalized DNA. In this study we describe the development of a targeted lysosome disruptive element to facilitate the escape of DNA from the lysosomal compartment, thus enhancing the transfection efficacy, in a cell-specific fashion. Two peptides (INF7 and JTS-1) were tested for their capacity to disrupt liposomes. In contrast to JTS-1, INF7 induced rapid cholesterol-independent leakage (EC50, 1.3 μm). INF7 was therefore selected for coupling to a high affinity ligand for the asialoglycoprotein receptor (ASGPr), K(GalNAc)2, to im- prove its uptake by parenchymal liver cells. Although the parent peptide disrupted both cholesterol-rich and -poor liposomes, the conjugate, INF7-K(GalNAc)2, only induced leakage of cholesterol-poor liposomes. Given that endosomal membranes of eukaryotic cells contain <5% cholesterol, this implies that the conjugate will display a higher selectivity toward endosomal membranes. Although both INF7 and INF7-K(GalNAc)2 were found to increase the transfection efficiency on polyplex-mediated gene transfer to parenchymal liver cells by 30-fold, only INF7-K(GalNAc)2appeared to do so in an ASGPr-specific manner. In mice, INF7-K(GalNAc)2 was specifically targeted to the liver, whereas INF7 was distributed evenly over various organs. In summary, we have prepared a nontoxic cell-specific lysosome disruptive element that improves gene delivery to parenchymal liver cells via the ASGPr. Its high cell specificity and preference to lyse intracellular membranes make this conjugate a promising lead in hepatocyte-specific drug/gene delivery protocols. The development of a viable nonviral gene delivery system continues to be an important theme in gene therapy (1Niidome T. Urakawa M. Sato H. Takahara Y. Anai T. Hatakayama T. Wada A. Hirayama T. Aoyagi H. Biomaterials. 2000; 21: 1811-1819Crossref PubMed Scopus (50) Google Scholar). The packaging of DNA into compact particles, the cellular uptake, the endosomal escape, and unpacking of these particles as well as the subsequent transfer of DNA to the nucleus are considered important steps in this regard (2Kichler A. Mechtler K. Behr J.P. Wagner E. Bioconj. Chem. 1997; 8: 213-221Crossref PubMed Scopus (89) Google Scholar). A number of DNA-packaging compounds have been reported, including cationic lipids, polymers and/or peptides that were designed to self-assemble with DNA to form intermolecular complexes (3Simoes S. Slepushkin V. Pires P. Gaspar R. de Lima M.C.P. Duzgunes N. Gene Ther. 1999; 65: 1798-1807Crossref Scopus (162) Google Scholar, 4Watabe A. Yamaguchi T. Kawanishi T. Uchida E. Eguchi A. Mizuguchi H. Mayumi T. Nakanishi M. Hayakawa T. Biochim. Biophys. Acta Biomembr. 1999; 12: 1-2Google Scholar, 5Zanta M.A. Belguise Valladier P. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 91-96Crossref PubMed Scopus (669) Google Scholar, 6Lim D.W. Yeom Y.I. Park T.G. Bioconj. Chem. 2000; 11: 688-695Crossref PubMed Scopus (84) Google Scholar, 7Morris M.C. Vidal P. Chaloin L. Heitz F. Divita G. Nucleic Acids Res. 1997; 25: 2730-2736Crossref PubMed Scopus (458) Google Scholar, 8Torchilin V.P. Rammohan R. Weissig V. Levchenko T.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8786-8791Crossref PubMed Scopus (700) Google Scholar). Upon internalization, the packaged DNA is transported to the lysosome, which subsequently degrades its content, making lysosomal escape a key step in gene delivery. To facilitate the intracellular transport of the packaged DNA to the nucleus and thus to enhance the transfection capacity of the nonviral gene delivery vehicles, lysosome disruptive elements (LDEs) 1The abbreviations used for: LDE(s), lysosome disruptive element(s); ASGPr, asialoglycoprotein receptor; BHK, baby hamster kidney; Boc, t-butoxycarbonyl; BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate; BSA, bovine serum albumin; CMV, cytomegalovirus; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HA, hemagglutinin; HOBt, hydroxybenzotriazole; HPLC, high performance liquid chromatography; K(GalNAc)2, di-N α,N ε-(5-(2-acetamido-2-deoxy-β-d-galactopyranosyloxy)pentanomido) lysine; LC-MS, liquid chromatography-mass spectrometry; Luc, luciferase; MALDI, matrix-assisted laser desorption/ionization; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TOF, time-of-flight. 1The abbreviations used for: LDE(s), lysosome disruptive element(s); ASGPr, asialoglycoprotein receptor; BHK, baby hamster kidney; Boc, t-butoxycarbonyl; BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate; BSA, bovine serum albumin; CMV, cytomegalovirus; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HA, hemagglutinin; HOBt, hydroxybenzotriazole; HPLC, high performance liquid chromatography; K(GalNAc)2, di-N α,N ε-(5-(2-acetamido-2-deoxy-β-d-galactopyranosyloxy)pentanomido) lysine; LC-MS, liquid chromatography-mass spectrometry; Luc, luciferase; MALDI, matrix-assisted laser desorption/ionization; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TOF, time-of-flight. have been successfully applied, including amphipathic peptides (9Wolfert M.A. Seymour L.W. Gene Ther. 1998; 5: 409-414Crossref PubMed Scopus (92) Google Scholar, 10Gottschalk S. Sparrow J.T. Hauer J. Mims M.P. Leland F.E. Woo S.L.C. Smith L.C. Gene Ther. 1996; 3: 448-457PubMed Google Scholar, 11Wyman T.B. Nicol F. Zelphati O. Scaria P.V. Plank C. Szoka F.C. Biochemistry. 1997; 36: 3008-3017Crossref PubMed Scopus (421) Google Scholar, 12Martin I. Pecheur E.I. Ruysschaert J.M. Hoekstra D. Biochemistry. 1999; 38: 9337-9347Crossref PubMed Scopus (21) Google Scholar, 13Chowdhury N.R. Hays R.M. Bommineni V.R. Franki N. Chowdhury J.R. Wu C.H. Wu G.Y. J. Biol. Chem. 1996; 271: 2341-2346Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 14Plourde R. Phillips A.T. Wu C.H. Hays R.M. Chowdhury J.R. Chowdhury N.R. Wu G.Y. Bioconj. Chem. 1996; 7: 131-137Crossref PubMed Scopus (8) Google Scholar, 15Arar K. Aubertin A.M. Roche A.C. Monsigny M. Mayer R. Bioconj. Chem. 1995; 6: 573-577Crossref PubMed Scopus (77) Google Scholar, 16Derossi D. Chassaing G. Prochiantz A. Trends Cell Biol. 1998; 8: 84-87Abstract Full Text PDF PubMed Scopus (653) Google Scholar).The majority of the amphipathic peptides are derived from viral elements that promote cellular entry and correct intracellular handling. Their membrane permeabilizing capacity generally depends on the lipid composition and the pH. Although they are random coil at pH 7.0, these peptides undergo a conformational change into an amphipathic α-helix at pH 5.0 and aggregate into multimeric clusters (11Wyman T.B. Nicol F. Zelphati O. Scaria P.V. Plank C. Szoka F.C. Biochemistry. 1997; 36: 3008-3017Crossref PubMed Scopus (421) Google Scholar, 12Martin I. Pecheur E.I. Ruysschaert J.M. Hoekstra D. Biochemistry. 1999; 38: 9337-9347Crossref PubMed Scopus (21) Google Scholar). Subsequently, the clustered helical peptides associate with and/or penetrate endosomal membranes, thereby destabilizing the membrane. Apart from complete virus capsids and purified capsid proteins, hemagglutinin (HA)-derived peptides and synthetic analogs have also been shown to induce pH-sensitive membrane disruption, leading to improved transfection of DNA-polycation complexes in vitro(9Wolfert M.A. Seymour L.W. Gene Ther. 1998; 5: 409-414Crossref PubMed Scopus (92) Google Scholar, 17Schoen P. Chonn A. Cullis P.R. Wilschut J. Scherrer P. Gene Ther. 1999; 6: 823-832Crossref PubMed Scopus (85) Google Scholar). Although several groups have studied the stimulatory effect of LDEs on nonviral gene transfer (18Decout A. Labeur C. Goethals M. Brasseur R. Vandekerckhove J. Rosseneu M. Biochim. Biophys. Biomembr. 1998; 1372: 102-116Crossref PubMed Scopus (15) Google Scholar, 19Plank C. Oberhauser B. Mechtler K. Koch C. Wagner E. J. Biol. Chem. 1994; 269: 12918-12924Abstract Full Text PDF PubMed Google Scholar, 20Wagner E. Plank C. Zatloukal K. Cotten M. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7934-7938Crossref PubMed Scopus (656) Google Scholar), the use of targeted LDEs, which concomitantly improve the cellular delivery of DNA and its translocation to the nucleus, is rather unexplored.Previous studies have shown that coupling of a homing device to liposomes or a universal carrier leads to a higher uptake of liposome-encapsulated drugs by the target cell (6Lim D.W. Yeom Y.I. Park T.G. Bioconj. Chem. 2000; 11: 688-695Crossref PubMed Scopus (84) Google Scholar, 23Zhang X. Simmons C.G. Corey D.R. Bioorg. Med. Chem. Lett. 2001; 11: 1269-1272Crossref PubMed Scopus (45) Google Scholar, 24Sliedregt L. Rensen P.C.N. Rump E.T. van Santbrink P.J. Bijsterbosch M.K. Valentijn A. van der Marel G.A. van Boom J.H. van Berkel T.J.C. Biessen E.A.L. J. Med. Chem. 1999; 42: 609-618Crossref PubMed Scopus (131) Google Scholar, 25Nishikawa M. Yamauchi M. Morimoto K. Ishida E. Takakura Y. Hashida M. Gene Ther. 2000; 7: 548-555Crossref PubMed Scopus (153) Google Scholar). The same strategy was applied to generate a targeted LDE. Two fusogenic peptides, INF7, a 23-mer peptide from HA, and JTS-1, an artificial INF7 mimic designed for pH-sensitive helix formation, were studied (10Gottschalk S. Sparrow J.T. Hauer J. Mims M.P. Leland F.E. Woo S.L.C. Smith L.C. Gene Ther. 1996; 3: 448-457PubMed Google Scholar, 21Smith L.C. Duguid J. Wadhwa M.S. Logan M.J. Tung C.H. Edwards V. Sparrow J.T. Adv. Drug Del. Rev. 1998; 30: 115-131Crossref PubMed Scopus (100) Google Scholar,22Vaysse L. Burgelin I. Merlio J.P. Arveiler B. Biochim. Biophys. Acta. 2000; 26: 369-376Crossref Scopus (26) Google Scholar). INF7, the most promising peptide in terms of cholesterol dependence and disruption kinetics, was equipped with a homing device for the asialoglycoprotein receptor (ASGPr), K(GalNAc)2 (1Niidome T. Urakawa M. Sato H. Takahara Y. Anai T. Hatakayama T. Wada A. Hirayama T. Aoyagi H. Biomaterials. 2000; 21: 1811-1819Crossref PubMed Scopus (50) Google Scholar,9Wolfert M.A. Seymour L.W. Gene Ther. 1998; 5: 409-414Crossref PubMed Scopus (92) Google Scholar, 23Zhang X. Simmons C.G. Corey D.R. Bioorg. Med. Chem. Lett. 2001; 11: 1269-1272Crossref PubMed Scopus (45) Google Scholar, 24Sliedregt L. Rensen P.C.N. Rump E.T. van Santbrink P.J. Bijsterbosch M.K. Valentijn A. van der Marel G.A. van Boom J.H. van Berkel T.J.C. Biessen E.A.L. J. Med. Chem. 1999; 42: 609-618Crossref PubMed Scopus (131) Google Scholar, 25Nishikawa M. Yamauchi M. Morimoto K. Ishida E. Takakura Y. Hashida M. Gene Ther. 2000; 7: 548-555Crossref PubMed Scopus (153) Google Scholar, 26Valentijn R.A. van der Marel G.A. Sliedregt L.A.J.M. van Berkel T.J.C. Biessen E.A.L. van Boom J.H. Tetrahedron. 1997; 53: 759-770Crossref Scopus (48) Google Scholar) on parenchymal liver cells.In this report, we show that the glycoconjugated peptide, INF7-K(GalNAc)2, displays a high affinity for the ASGPr and possesses high lytic activity in cholesterol-poor liposomes only, making it eminently suitable for targeted fusogenic activity in parenchymal cells. Moreover, INF7-K(GalNAc)2, unlike the parental INF7, accumulates efficiently in the liver after in vivo administration and strongly improves the transfer of polyplexed genes to parenchymal liver cells in an ASGPr-dependent fashion.DISCUSSIONThe main objective of this study was to improve nonviral gene delivery by the use of targeted LDEs. As a first step we have tested two HA-derived fusogenic peptides, JTS-1 and INF7, for their lysosome disruptive capacity in calcein-laden liposomes (10Gottschalk S. Sparrow J.T. Hauer J. Mims M.P. Leland F.E. Woo S.L.C. Smith L.C. Gene Ther. 1996; 3: 448-457PubMed Google Scholar). INF7 was found to induce a rapid and cholesterol-independent leakage of liposomes, whereas JTS-1-induced leakage was much slower and cholesterol-dependent. Moreover, JTS-1 was unable to disrupt cholesterol-poor (<20%) liposomes completely. These findings concur with the studies of Gottschalk et al. (10Gottschalk S. Sparrow J.T. Hauer J. Mims M.P. Leland F.E. Woo S.L.C. Smith L.C. Gene Ther. 1996; 3: 448-457PubMed Google Scholar), who showed complete leakage of cholesterol-poor liposomes by INF7 compared with only partial leakage by JTS-1 (35%). The differential fusogenic profile of JTS-1 and INF7 is not the result of differences in net negative charge or hydrophobicity of the peptides because the peptides are very similar in that respect. Possibly, INF7 and JTS-1 have a different orientation in liposomal membranes (12Martin I. Pecheur E.I. Ruysschaert J.M. Hoekstra D. Biochemistry. 1999; 38: 9337-9347Crossref PubMed Scopus (21) Google Scholar, 37Pecheur E.I. Martin I. Ruysschaert J.M. Bienvenue A. Hoekstra D. Biochemistry. 1998; 37: 2361-2371Crossref PubMed Scopus (30) Google Scholar, 38Pecheur E.I. Sainte Marie J. Bienvenue A. Hoekstra D. Biochemistry. 1999; 38: 364-373Crossref PubMed Scopus (28) Google Scholar, 39Pecheur E.I. Sainte Marie J. Bienvenue A. Hoekstra D. J. Membr. Biol. 1999; 167: 1-17Crossref PubMed Scopus (121) Google Scholar, 40Pecheur E.I. Martin I. Bienvenue A. Ruysschaert J.M. Hoekstra D. J. Biol. Chem. 2000; 275: 3936-3942Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Further experiments will be imperative to confirm this hypothesis.In the second step, the most promising peptide, INF-7, was equipped with a homing device to render the peptide specific for parenchymal liver cells. As a homing device we used K(GalNAc)2, which was shown previously to be able to redirect liposomes, lipoproteins, and drugs to liver parenchymal cells (26Valentijn R.A. van der Marel G.A. Sliedregt L.A.J.M. van Berkel T.J.C. Biessen E.A.L. van Boom J.H. Tetrahedron. 1997; 53: 759-770Crossref Scopus (48) Google Scholar, 32Rensen P.C.N. Sliedregt L. Ferns A. Kieviet E. van Rossenberg S.M.W. van Leeuwen S.H. van Berkel T.J.C. Biessen E.A.L. J. Biol. Chem. 2001; 276: 37577-37584Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 41Biessen E.A.L. Sliedregt-Bol K.M. 'T Hoen P.A. Prince P. Van der Bilt E. Valentijn A.R. Meeuwenoord N.J. Princen H. Bijsterbosch M.K. Van der Marel G.A. Van Boom J.H. van Berkel T.J. Bioconj. Chem. 2002; 13: 295-302Crossref PubMed Scopus (32) Google Scholar). The conjugate appeared to be slightly less fusogenic than the parent peptide (1.3 μm versus 6.1 μm). However, the lytic activity of the conjugate was cholesterol-dependent in that it only disrupted cholesterol-poor liposomes. This may be an advantage as K(GalNAc)2-conjugated INF7 is designed to act specifically on the cholesterol-deficient lysosomal membranes of ASGPr-expressing cells. INF7, by contrast, may also be fusogenic at the level of the plasma membrane.The intriguing observation that INF7-K(GalNAc)2-induced leakage depends on the liposome cholesterol content, in contrast to INF7 alone, prompted further study. Lysosome disruptive peptides may facilitate leakage, through pore formation and through a detergent-like solubilization of the membrane (36Parente R.A. Nir S. Szoka Jr., F.C. Biochemistry. 1990; 29: 8720-8728Crossref PubMed Scopus (302) Google Scholar). The differential lytic profile of INF7 and its glycoconjugate suggests that INF7 disrupts membranes via both pathways, one of which predominates in cholesterol-rich membranes and is blocked by the presence of the bulky glycoside moiety. However, the lack of leakage of 125I-trypsin inhibitor from INF7 (glycoconjugate)-treated liposomes points to pore formation as the major pathway of liposome disruption for both INF7 and the glycoconjugated INF7. Rather, steric hindrance of the exposed glycoside group might interfere with the ability of the fusogenic peptide to form pores. This also explains the reduced lytic activity of INF7-K(GalNAc)2 in cholesterol-rich liposomes because the liposomal cholesterol may hamper pore formation by INF7-K(GalNAc)2 caused by steric hindrance of the glycoside group. To confirm this hypothesis, the orientation and distribution of the peptides in the liposomal membrane need to be addressed.The glycoconjugated peptide was designed for ASGPr-directed delivery. Indeed, glycoconjugated INF7 bound to the ASGPr with an affinity of 87 nm, which is about two times lower than K(GalNAc)2 itself. Earlier studies have shown that an affinity of 87 nm for the ASGPr should be sufficient for effective targeting of INF7-K(GalNAc)2 (32Rensen P.C.N. Sliedregt L. Ferns A. Kieviet E. van Rossenberg S.M.W. van Leeuwen S.H. van Berkel T.J.C. Biessen E.A.L. J. Biol. Chem. 2001; 276: 37577-37584Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 42Biessen E.A.L. Vietsch H. Rump E.T. Fluiter K. Kuiper J. Bijsterbosch M.K. van Berkel T.J.C. Biochem. J. 1999; 3: 783-792Crossref Google Scholar).The final goal of this study was to elaborate a targeted LDE for improving nonviral gene transfer. To this end, we have evaluated the effect of the fusogenic peptides on the gene transfer efficiency of an established nonviral gene delivery protocol based on the cationic peptide K8 in mouse parenchymal liver cells (10Gottschalk S. Sparrow J.T. Hauer J. Mims M.P. Leland F.E. Woo S.L.C. Smith L.C. Gene Ther. 1996; 3: 448-457PubMed Google Scholar). DNA polyplexed with small sized synthetic oligocations (like K8) may be better for systemic application because the derived condensates generally are smaller and less immunogenic, nonaggregating, and are readily unpacked intracellularly (43McKenzie D.L. Collard W.T. Rice K.G. J. Pept. Res. 1999; 54: 311-318Crossref PubMed Scopus (69) Google Scholar, 44Wadhwa M.S. Collard W.T. Adami R.C. McKenzie D.L. Rice K.G. Bioconj. Chem. 1997; 8: 81-88Crossref PubMed Scopus (200) Google Scholar). INF7, INF7-SGSC, and INF7-K(GalNAc)2 led to a substantial, 30-fold, increase in the transfection efficiency of K8·DNA complexes in freshly isolated parenchymal cells. This stimulatory effect was concentration-dependent. Even though in the leakage assay, INF7-K(GalNAc)2 appeared to be 6-fold less potent than INF7, the lower intrinsic activity is compensated for by the enhanced uptake of the targeted peptide, by parenchymal liver cells. However, it should also be kept in mind that liposomal and cellular assays are not fully comparable.The fusogenic activity of INF7-K(GalNAc)2 was abolished completely in the presence of excess GalNAc, which blocks ASGPr-mediated uptake, whereas GlcNAc had no effect on the transfection efficiency. Moreover, INF7-K(GalNAc)2 did not affect the transfection yield in ASGPr-deficient BHK cells, whereas INF7 and INF7-SGSC were equally potent in BHK and mouse parenchymal cells. This underlines that the stimulatory effect of glycoconjugated INF7 is mediated by the ASGPr and will have fewer side effects. In agreement with previous studies, primary parenchymal liver cells are more difficult to transfect than continuous cell lines; the intrinsic transfection efficacy in BHK cells was indeed found to be 10–100-fold higher than in parenchymal cells (45Beck N.B. Sidhu J.S. Omiecinski C.J. Gene Ther. 2000; 7: 1274-1283Crossref PubMed Scopus (54) Google Scholar).The observation that INF7-K(GalNAc)2 was completely inactive in BHK cells indicates that non-ASGPr-mediated uptake and the lytic activity of INF7-K(GalNAc)2 are reduced considerably compared with those of INF7 or INF7-SGSC. As our leakage data already showed that the glycoconjugate is less fusogenic, we propose that this is the major contributing factor underlying the reduced stimulatory effect of INF7-K(GalNAc)2 in BHK cells. The actual route of entry of INF7 and INF7-SGSC remains unclear. INF7 and INF7-SGSC may be cointernalized into the target cells, associated with the polyplexes. Because the peptides have only a very weak fusogenic activity at pH 7.4, it is unlikely that they may be stimulatory by facilitating polyplex/plasma membrane fusion. Cellular uptake of the cationic polyplexes may in turn implicate the use of receptor systems, possibly the scavenger receptor class B and CD36 (46Terpstra V. van Amersfoort E.S. van Velzen A.G. Kuiper J. van Berkel T.J.C. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1860-1872Crossref PubMed Scopus (118) Google Scholar), or alternative pathways such as pinocytosis. INF7-K(GalNAc)2, on the other hand, could be stimulatory by associating with the K8·DNA complex and subsequently promoting whole complex uptake via an ASGPr-mediated pathway. Intracellularly, INF7-K(GalNAc)2 will promote escape of the DNA from the lysosomal compartment.It has been reported that lysosome leakage and membrane disruption may lead to intracellular release of lytic enzymes including proteases, nucleases, and lipases. Although these enzymes are acid hydrolases, with a catalytic optimum near pH 5.0, leakage of these enzymes in the cytoplasm of the cell could promote apoptosis or necrosis (47Zang Y. Beard R.L. Chandraratna R.A.S. Kang J.X. Cell Death Differ. 2001; 8: 477-485Crossref PubMed Scopus (113) Google Scholar). We show that the peptides are not cytotoxic in parenchymal liver cells.Another important issue in regard of potential in vivo use involves the pharmacokinetics of INF7 and its glycoconjugate. The biodistribution profile in mice shows that INF7-K(GalNAc)2is preferentially taken up by the liver, whereas INF7 is distributed evenly over various organs. In fact, liver uptake of the glycoconjugate is 6-fold higher than that of INF7, indicating that we have developed a hepatocyte-specific LDE for use in vivo.In conclusion, we present a targeted fusogenic peptide, INF7-K(GalNAc)2, which induces lysosomal escape in a receptor-dependent fashion. Its favorable pH- and cholesterol-dependent activity profile makes it even more lysosome-specific than the parental INF7. We envision that INF7-K(GalNAc)2 could be applied to improve the transfection efficacy of hepatic nonviral gene transfer vehicles (25Nishikawa M. Yamauchi M. Morimoto K. Ishida E. Takakura Y. Hashida M. Gene Ther. 2000; 7: 548-555Crossref PubMed Scopus (153) Google Scholar) and of antisense drugs for hepatic genes (42Biessen E.A.L. Vietsch H. Rump E.T. Fluiter K. Kuiper J. Bijsterbosch M.K. van Berkel T.J.C. Biochem. J. 1999; 3: 783-792Crossref Google Scholar, 41Biessen E.A.L. Sliedregt-Bol K.M. 'T Hoen P.A. Prince P. Van der Bilt E. Valentijn A.R. Meeuwenoord N.J. Princen H. Bijsterbosch M.K. Van der Marel G.A. Van Boom J.H. van Berkel T.J. Bioconj. Chem. 2002; 13: 295-302Crossref PubMed Scopus (32) Google Scholar, 48Sugano M. Makino N. J. Biol. Chem. 1996; 271: 19080-19083Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) by facilitating the escape from the lysosomal pathway, which appears to be a major drawback in both therapies (49Akhtar S. Hughes M.D. Khan A. Bibby M. Hussain M. Nawaz Q. Double J. Sayyed P. Adv. Drug Del. Rev. 2000; 44: 3-21Crossref PubMed Scopus (229) Google Scholar). Not only gene medicines, but also other drugs that accumulate in the lysosomal circuit might benefit from application of targeted LDEs. The development of a viable nonviral gene delivery system continues to be an important theme in gene therapy (1Niidome T. Urakawa M. Sato H. Takahara Y. Anai T. Hatakayama T. Wada A. Hirayama T. Aoyagi H. Biomaterials. 2000; 21: 1811-1819Crossref PubMed Scopus (50) Google Scholar). The packaging of DNA into compact particles, the cellular uptake, the endosomal escape, and unpacking of these particles as well as the subsequent transfer of DNA to the nucleus are considered important steps in this regard (2Kichler A. Mechtler K. Behr J.P. Wagner E. Bioconj. Chem. 1997; 8: 213-221Crossref PubMed Scopus (89) Google Scholar). A number of DNA-packaging compounds have been reported, including cationic lipids, polymers and/or peptides that were designed to self-assemble with DNA to form intermolecular complexes (3Simoes S. Slepushkin V. Pires P. Gaspar R. de Lima M.C.P. Duzgunes N. Gene Ther. 1999; 65: 1798-1807Crossref Scopus (162) Google Scholar, 4Watabe A. Yamaguchi T. Kawanishi T. Uchida E. Eguchi A. Mizuguchi H. Mayumi T. Nakanishi M. Hayakawa T. Biochim. Biophys. Acta Biomembr. 1999; 12: 1-2Google Scholar, 5Zanta M.A. Belguise Valladier P. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 91-96Crossref PubMed Scopus (669) Google Scholar, 6Lim D.W. Yeom Y.I. Park T.G. Bioconj. Chem. 2000; 11: 688-695Crossref PubMed Scopus (84) Google Scholar, 7Morris M.C. Vidal P. Chaloin L. Heitz F. Divita G. Nucleic Acids Res. 1997; 25: 2730-2736Crossref PubMed Scopus (458) Google Scholar, 8Torchilin V.P. Rammohan R. Weissig V. Levchenko T.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8786-8791Crossref PubMed Scopus (700) Google Scholar). Upon internalization, the packaged DNA is transported to the lysosome, which subsequently degrades its content, making lysosomal escape a key step in gene delivery. 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