- Email: [email protected]
Liver-directed gene delivery
Advanced Drug Delivery Reviews, 12 (1993) 159 167
159
© 1993 Elsevier Science Publishers B.V. All rights reserved. / 0169-409X/93/$24.00 ADR 00133
Liver-directed gene delivery G e o r g e Y. W u a n d C a t h e r i n e H. W u Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut School of Medicine, Farmington, CT, USA (Received April 27, 1992) (Accepted May 20, 1992)
Key words: Genes; Targeting; Asialoglycoproteins; Receptors
Contents Summary .................................................................................................................
159
I. Introduction ....................................................................................................
160
II. Targeted delivery of small molecules to hepatocytes .................................................
160
III. Targeted delivery and expression of genes in hepatocytes .......................................... 1. A targetable D N A carrier system .................................................................... 2. Receptor-mediated gene delivery and expression in hepatocytes in vitro ................... 3. Receptor-mediated gene delivery and expression in vivo ....................................... 4. Strategies for obtaining persistence of receptor-mediated gene transfection ...............
161 161 161 162 163
Acknowledgements .....................................................................................................
166
References ................................................................................................................
166
Summary Cell surface receptors can provide a convenient natural mechanism for delivery and internalization of substances to cells. We have utilized asialoglycoprotein receptors on hepatocytes to target DNA specifically to these cells. Studies initially began with marker genes in cell culture studies. More recently, normal genes in the form of targetable protein-DNA complexes have been introduced into animals Correspondence to: George Y. Wu, M.D., Ph.D., Department of Medicine, Division of GastroenterologyHepatology, University of Connecticut School of Medicine, 263 Farmington Avenue, AM-044, #1845, Farmington, CT 06030, USA.
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possessing inherited metabolic disorders. Partial, transient corrections of these diseases have been achieved by intravenous injection of the complexed genes. I. Introduction Normal hepatocytes possess cell-surface receptors that can recognize and internalize glycoproteins that possess clustered galactose residues [1]. Exposure of this class of proteins, known as 'asialoglycoproteins', to their receptor results in a complex series of cell biological events culminating in internalization of the ligand within membrane-limited endosomal vesicles [2]. The virtually unique presence of asialoglycoprotein receptors on normal hepatocytes has been to used to target substances specifically to these cells. II. Targeted delivery of small molecules to hepatocytes This concept has been studied for both therapeutic and diagnostic potentials. For example, trifluorothymidine [3] and adenine 9-fl-D-arabinofuranoside [4] have been coupled chemically to asialoglycoproteins and demonstrated a selected inhibition of viral DNA synthesis in mouse hepatocytes infected with Ectromelia virus. Similarly, the antimalarial drug, primaquine, was linked to a asialoglycoprotein for the treatment of the hepatic stage of malaria. Conjugates given to mice infected with P. berghei resulted in improvement in the numbers of long-term survivors [5]. Potential diagnostic agents have also been developed. For example, a synthetic asialoglycoprotein, galactosyl-albumin, was radiolabeled and found to be capable of being bound by hepatocytes in vitro [6] and in vivo [7]. Because tumors and other space-occupying lesions do not contain asialoglycoprotein receptors, this type of agent was expected to be of value for radio-imaging purposes. In a somewhat different approach, the lysosomotropic drug, primaquine, was coupled to an asialoglycoprotein. This class of compounds has been shown to raise the pH of endosomal vesicles. This is of interest because iron is normally taken up by cells, from transferrin, and released to the cell during acidification of endosomal vesicles. Elevation of the endosomal pH would be expected to decrease the iron release, and uptake by cells. When primaquine coupled to an asialoglycoprotein was administered to cells, uptake of iron mediated by transferrin was dramatically decreased but only in those cells that possessed asialoglycoprotein receptor activity. By using radiolabeled iron, the results raised the possibility that this strategy could be used to form images of space-occupying lesions in liver by excluding uptake of radionuclides in normal liver cells. The concept of targeting has been extended therapeutically in a technique called 'targeted rescue'. In this case, advantage was taken of the fact that most hepatocellular carcinomas have markedly decreased asialoglycoprotein activity compared to normal hepatocytes [8,9]. Instead of targeting toxins to tumors, which is inherently difficult due to the lack of distinguishable markers on most transformed cells, asialoglycoproteins have been used as a carrier to target protective agents to normal cells in the presence of corresponding cytotoxins. In initial studies, folinic acid, a methotrexate antagonist, was chemically coupled to an asialoglycoprotein. In the presence of methotrexate, the asialoglycoprotein-folinic
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acid conjugate protected only cells that possessed asialoglycoprotein receptors while those that did not possess these receptors were destroyed [10] in vitro. This work was subsequently extended using the selective hepatotoxin toxin, acetaminophen, in order to prevent toxicity to non-hepatic tissues [11].
III. Targeted delivery and expression of genes in hepatocytes In all of the above-described previous studies, agents were chemically linked to asialoglycoproteins for targeting. This was convenient for small molecules which could survive chemical modification and still remain functional. However, it seemed possible that, under appropriate conditions, macromolecules such as DNA could also survive a receptor-mediated endocytotic event. However, it appeared to be likely that an alternative linkage strategy would be required because covalent linkage to an asialoglycoprotein would alter the nucleic acid bases likely resulting in undesirable transcriptional events. To circumvent this potential problem, a soluble DNA carrier system [12,13] was developed consisting of an asialoglycoprotein carrier chemically coupled to poly-L-lysine. The latter was introduced to allow binding of DNA in a strong, non-damaging electrostatic interaction [14] to form a soluble complex. If it retained its targetability, the system could result in concentrated delivery of genetic material to a specific (organ) cell type. The solubility property could make possible delivery of foreign genes to hepatocytes in vivo and raise the possibility that foreign genes could be targeted to hepatocytes in vivo by intravenous injection. Parenchymal liver cells have a low turnover rate, are very active metabolically, and possess an excellent blood supply in vivo. Because of these properties, hepatocytes are attractive as recipients for delivery and expression of genes that could have therapeutic function [15]. III.1. A targetable DNA carrier system
To test this system, plasmid DNA was used which contained an SV-40 viral promoter driving the marker gene for the bacterial enzyme, chloramphenicol acetyltransferase (CAT), which catalyzes the acetylation of chloramphenicol. The gene was selected because mammalian cells lack the CAT gene. Therefore, the appearance of acetylated products of chloramphenicol could provide a simple marker for detection of foreign gene expression in target cells. The asialoglycoprotein carrier, a human serum protein derivative, asialoorosomucoid (AsOR), was prepared by desialylation of orosomucoid [16]. This protein was conjugated to L-lysine using disulfide bonds [17]. DNA was then mixed with the conjugate in order to form a soluble complex. Optimal conjugate to DNA ratios were determined by a gel retardation assay system [18]. That DNA was actually retarded by the presence of conjugate was confirmed using DNA labeled with 32p [19]. 111.2. Receptor-mediated gene delivery and expression in hepatocytes in vitro
In order to test DNA delivery and expression of foreign genes, an in vitro system was used consisting of two cell lines: HepG2, asialoglycoprotein receptor ( + ) cells
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and SK-Hep 1 ( - ) cells. Incubation of the receptor ( - ) cells with either targetable DNA complex or controls did not produce detectable CAT activity under any conditions [19]. However, HepG2, receptor ( + ) cells incubated with the targetable DNA complex did produce CAT enzyme activity as demonstrated by the formation of acetylated chloramphenicol derivatives. Control experiments in which HepG2 under identical conditions were incubated with DNA alone, DNA plus poly-Llysine, or DNA plus AsOR, all present in the same concentrations as provided by the complete complex did not result in foreign gene expression [19]. Addition of an excess of the asialoglycoprotein itself to the complex to compete for asialoglycoprotein receptor uptake blocked expression of the CAT gene in HepG2 cells, supporting the idea that the delivery of the D N A was directed by the asialoglycoprotein component of the complex [19]. There was no evidence of toxicity in cells treated with the DNA complex as they proliferated at the same rate as untreated cells [12]. These data taken together indicated that a DNA carrier system based on internalization of asialoglycoproteins by hepatocytes could result in targeting of a foreign gene in a soluble form specifically to asialoglycoprotein receptor-bearing cells in a non-toxic fashion, and that this gene could be expressed by those cells in vitro. 111.3. Receptor-mediated gene delivery and expression in vivo
There are many techniques available for introduction of foreign genes into cells in vitro. The main advantage to the receptor-mediated asialoglycoprotein-based system lay in the fact that the DNA complex was soluble and targetable to a specific cell type. This raised the possibility that foreign genes could be targeted in vivo by simple intravenous injection. To investigate this possibility, radiolabeled plasmid DNA alone, or in the form of a targetable complex, was injected intravenously into adult rats. Ten minutes after injection of the labeled DNA only, radioactivity in various organs [20] demonstrated that only 17% of the injected counts were taken up by the liver. However, labeled DNA injected in the form of a targetable complex resulted in liver uptake of 85% of the injected counts. The organ distribution of the complex was similar to that for radiolabeled asialoglycoprotein only, indicating that the complex had retained its ability to be recognized by asialoglycoprotein receptors in vivo [20]. To determine whether CAT gene sequences could be detected in intact form beyond the immediate post-injection period, rats where injected intravenously with unlabeled, complexed plasmid containing the CAT gene. Twenty-four hours later, liver DNA was extracted, and CAT sequences detected by dot blot using a labeled cDNA-CAT probe [20]. CAT gene sequences in transfected liver were easily detected by dot blot. Equal quantities of control liver DNA failed to hybridize to the cDNA probe, indicating that the hybridization found in liver transfected with complex was due to targeted DNA and not due to cross-hybridization to host DNA sequences
[20]. To determine whether the targeted DNA was actually functional, rats were injected with complexed DNA in saline or controls: saline only, DNA only, poly-Llysine plus DNA or a mixture of components of the complex all present in the same concentrations as provided by the complete complex. After 24 hours, CAT enzyme
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activity was assayed [21]. Targeted CAT gene expression was found in liver in vivo, but not in controls, under identical conditions. In order to determine whether the foreign gene expression was localized to the liver, other organs were assayed for CAT activity after administration of complexed DNA. Kidney, spleen, lungs, and liver were assayed, but only liver was found to have detectable quantities of CAT activity [20]. To determine whether natural mammalian regulatory elements could be targeted and used to drive a foreign gene in vivo, a construct containing the CAT gene driven by mouse albumin enhancer-rat promoter elements was prepared by Dr. James M. Wilson, University of Michigan. Complexed plasmid DNA, prepared as described previously, was injected intravenously into groups of rats. After 24 hours, it was determined that livers from rats that received the D N A in the form of a soluble complex produced CAT gene expression. However, livers from control rats that received saline only, D N A only, a mixture of components of the complex: AsOR/ poly-L-lysine/DNA did not produce detectable CAT activity. As seen with the previous plasmid construct, CAT gene expression was localized to liver and absent in heart, lung, spleen or kidney. The time course of targeted gene expression was determined in vivo. Injection of complexed D N A into groups of rats which were sacrificed at regular time intervals thereafter, and hepatic CAT activity assayed showed that targeted gene expression was transient with a maximum occurring between 24 and 48 hours after injection. Thereafter, CAT activity slowly declined, until by day 4 it was no longer detectable [17].
111.4. Strategies for obtaining persistence of receptor-mediated gene transfection In considering strategies to obtain persistent targeted gene expression, previous studies were noted in which transfection of cultured cells that were rapidly dividing had a higher likelihood of developing persistent expression. This had been attributed to a higher probability of integration of the foreign gene into host D N A during cell replication [22]. Because normal adult hepatocytes, under appropriate conditions, can be stimulated to replicate, this raised the possibility that stimulation of hepatocyte replication might enhance the chances for persistent targeted gene expression in adult liver. To examine the effect of hepatocyte replication on targeted gene expression, two-thirds of livers were removed after intravenous injection of targetable D N A complex into groups of rats. The two-thirds partial hepatectomy is a well-established model for stimulation of hepatic regeneration, producing a predictable response in D N A synthesis beginning by 12 hours after surgery and resulting in at least one round of cell division in almost all remaining hepatocytes within 1-2 weeks [15]. CAT gene expression was not detectable 24 hours after injection; but by 48 hours, CAT enzyme activity had become detectable and reached a maximum level by the 8th week. Activity remained high through the 1 lth week post-surgery [22]. In a model of a genetic metabolic disorder, the Watanabe heritable hyperlipidemic rabbit was employed [23]. This mutant contains a defect in receptors for low-density lipoprotein (LDL). As a result, these animals develop severe cardiovascular complications of hypercholesterolemia due to elevated levels of LDL. To study
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targeted gene delivery in this model, a plasmid construct was prepared to contain the gene for human LDL receptor driven by mouse albumin promoter and enhancer elements. Targetable complexes were made and injected intravenously. Quantitation of targeted D N A using a labeled vector-specific probe demonstrated that approximately 1000 copies of plasmid/cell were present 10 minutes after injection. Use of a complex in which the carrier was radiolabeled showed that more than 90% of the radioactivity detected by autoradiography was present in liver parenchymal cells. However, the abundance of the targeted D N A declined rapidly with time such that the copy number at 24 hours had decreased to a value of 1 copy/cell and to 0.1 copy/cell by 48 hours. Liver tissues were analyzed for the presence of human LDL receptor m R N A by RNase protection assays. This demonstrated that an appropriately-sized fragment was first detected 4 hours after injection and maximal levels of 2 4 % of endogenous levels were obtained by 24 hours after injection. Functionality of the foreign LDL receptor was demonstrated by a maximal drop of 25-30% in cholesterol levels 48 hours after injection. This decrease was highly significant (P < 0.001) compared to control that received CAT plasmid complex. The effect was transient with cholesterol levels rising to pre-injection levels by the 6th day. Cross-over experiments confirmed these results. No antibody against the carrier was detected circulating in rabbit serum 2 weeks after injection [24]. The targetable D N A delivery system was used to introduce another normal gene to correct an inherited metabolic disorder. In this second model, the Nagase analbuminemic rat was selected. This mutant rat strain possesses a defect in splicing of m R N A for serum albumin, resulting in virtually undetectable levels of circulating serum albumin [25]. A plasmid was constructed containing the structural gene for human serum albumin driven by mouse albumin enhancer-rat albumin promoter elements [26]. The human albumin gene was selected to avoid possible confusion between potential reversion of the mutation with expression of the endogenous rat albumin gene as opposed to actual foreign gene expression. A control construct was prepared identical to the first but without the albumin enhancer elements. Because the enhancer regions are required for high levels of expression by the albumin promoter [27], the control plasmid served to assay the contribution of potential nonspecific effects of plasmid D N A or the human albumin structural gene. Groups of mutant rats were injected with complexed D N A followed by two-thirds partial hepatectomies [27]. D N A was extracted from livers 2 weeks after injection. Blot hybridization analysis of total cellular D N A restricted with an enzyme which excised the human albumin gene insert from an enhancer-containing plasmid revealed a copy number of 1000/diploid genome. No bands larger than the albumin insert were detected, indicating that significant re-arrangements involving the albumin structural gene had not occurred. When total cellular D N A was digested with another restriction enzyme that cuts at a single site in the plasmid, hybridization with the albumin c D N A probe showed that livers from rats treated with complete plasmid produced a band that corresponded in size to linearized plasmid. Hybridization analysis of total cellular D N A from livers from the rat treated with complexes of the complete and the control plasmid were digested with an enzyme which does not cut the plasmid. Samples were probed with a fragment of the plasmid spanning a portion of the bacterial region of the plasmid but lacking any albumin sequences. These analyses showed two predominant bands corresponding
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to nicked circular and supercoiled forms of the plasmid. A small band was also seen corresponding in position to that of the linearized plasmid. These data indicate that the overwhelmingly predominant portion of the retained DNA in liver, in these experiments, existed as unintegrated circular plasmid DNA [27]. However, the possibility of random, non-clonal integration of some plasmid DNA into the host genome could not be excluded. Rats treated with the enhancerless plasmid showed similar patterns. To determine whether the targeted DNA had been transcribed, anaibuminemic rat livers were assayed by dot blots for the presence of human serum albumin mRNA 2 weeks after injection and partial hepatectomy. Total RNA from livers of rats that received the complete plasmid did produce a strong hybridization signal. RNA from liver of normal untreated rats did not hybridize with the probe, indicating that the signal detected previously was not due to hybridization to endogenous rat messenger sequences. The presence of human mRNA was confirmed by RNase protection analysis using a vector-specific RNA probe that is complementary to vector-derived sequences in the 3'-untranslated region of the recombinant-derived transcript. Analysis of RNA from liver harvested 2 weeks after transfection of analbuminemic rats with the complete plasmid in the form of a complex followed by partial hepatectomy resulted in a protected band of the expected size. However, liver from analbuminemic rats harvested 2 weeks after transfection and partial hepatectomy using identical molar quantities of complexed enhancerless plasmid did not generate any protected sequences under identical conditions. Formation of targeted gene product was assessed by Western blot using standard albumins and rat serum samples taken 2 weeks after treatment of analbuminemic rats with targeted complete plasmid DNA. These animals were shown to have circulating human serum albumin quantitated at 30 ~g/ml by 2 weeks after injection. Control animals that received saline alone, or the enhancerless plasmid in the form of a complex, did not produce detectable human albumin under identical conditions [27]. A time course of the appearance of human albumin in the circulation was determined by measuring serum samples by Western blot as a function of time. Analbuminemic rats treated with complete plasmid DNA complex did not have detectable circulating albumin after 24 hours. However, human albumin was detectable in serum in rats treated with the complete plasmid in the form of a complex when measured 24 hours after injection. The level of human albumin rose from a value of 0.05 #g/ml at 48 hours reaching a plateau of 34 #g/ml by the 2nd week and remained at this level without significant change through the 4th week post-injection [27]. The mechanism of persistent of foreign gene expression was further elucidated by a detailed examination of the state of the transfected DNA following partial hepatectomy. Southern blots of DNA extracted from livers at various time intervals ranging from 10 minutes to 11 weeks after injection and partial hepatectomy revealed that the vast majority of the transfected DNA was in the form of intact bacterial plasmid. The bacterial portion of the plasmid remained functional, as demonstrated by re-transfection of the extracted DNA from rats, which resulted in successful transfection of bacteria. Plasmids recovered from these bacteria produced restriction patterns identical to those of the original plasmid.
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Virtually all the plasmid DNA extracted from the liver was sensitive to a bacterial enzyme that cleaves only double-stranded methylated DNA of bacterial origin. It was concluded that the transfected DNA remained as stabilized episomal forms without evidence of clonal integration or replication within the limits of detection
[28]. Acknowledgements The assistance of Brenda Kawecki and Rosemary Pavlick is gratefully acknowledged. This work was supported in part by grants by the U.S. Public Health Service (NIH, DK42182) (G.Y.W.), a grant from the March of Dimes (91-0485) (G.Y.W.), and a grant from TargeTech, Inc. (C.H.W.).
References 1 Ashwell, G. and Morell, AIG. (1974) The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv. Enzymol. 41, 99 128. 2 Wall, D.A., Wilson, G, and Hubbard, A.L. (1980) The galactose-specific recognition system of mammalian liver: the route of ljgand internalization in rat hepatocytes. Cell 21, 79 83. 3 Fiume, k., Mattioli, A.; Balboni, P.G., Tognon, M., Barbanti-Brodano, G., De Vries, J., et al. (1979) Enhanced inhibition of virus DNA synthesis in hepatocytes by trifluorothymidine coupled to asialofetuin. FEBS Lett. 103,~47-51. 4 Fiume, L., Mattioli, A., Busi, C., Balboni, P.G., Barbanti-Brodano, G., De Vries, J., et al. (1980) Selective inhibition of Ectromelia virus DNA synthesis in hepatocytes by adenine-9-/3-Darabinofuranoside (ara-A) and adenine-9-/3-D-arabinofuranoside monophosphate (ara-AMP) conjugated to asialofetuin. FEBS Lett. 116, 185 188. 5 Trouet, A., Baurain, R., Deprez De Campeneere, D., Masquelier, M. and Pirson, P. (I 981) Targeting of antitumor and antiprotozoal drugs by covalent linkage to protein carriers. In: G. Gregoriadis, J. Senior, and A. Trouet, (Eds.), Targeting of Drugs, Plenum, New York, p. 28. 6 Vera, D.R., Krohn, K.A., Stadalnik, R.C. and Scheibe, P.O. (1984) Tc-99m-galactosyl-neoglycoalbumin: In vivo characterization of receptor-mediated binding to hepatocytes. Radiology 151, 191 196. 7 Vera, D.R., Stadalnik, R.C. and Krohn, K.A. (1985) Technetium 99m galactosyl-neoglycoalbumin: Preparation and preclinical studies. J. Nucl. Med. 26, 1157 1167. 8 Stockert, R.J. and Becker, F.F. (1980) Diminished hepatic binding protein for desialylated glycoproteins during chemical hepatocarcinogenesis. Cancer Res. 40, 3632 3634. 9 Hickman, J. and Ashwell, G. (1974) Enzyme therapy in lysosomal storage diseases. In: J.M. Tager, G.J.M. Hooghwinkel and W.T. Daems (Eds.), Proceedings, Workshop on Cell Biological and Enzymological Aspects of the Therapy of Lysosomal Storage Diseases, North-Holland, Publ. Co., Amsterdam, p. 168. 10 Wu, G.Y., Wu, C.H. and Stockert, R.J. (1983) A model for the specific rescue of normal hepatocytes during methotrexate treatment of hepatic malignancy. Proc. Natl. Acad. Sci. USA 80, 3878 3880. I1 Wu, G.Y., Wu, C.H. and Rubin, M.I. (1985) Acetaminophen hepatotoxicity and targeted rescue: A model for specific chemotherapy for hepatocellular carcinoma. Hepatology 5, 709 713. 12 Wu, G.Y. and Wu, C.H. (1991) Targeted delivery and expression of foreign genes in hepatocytes. In: G.Y. Wu and C.H. Wu, (Eds.), Liver Diseases: Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Marcel Dekker, Inc., New York, pp. 127 148. 13 Wu, G.Y. and Wu, C.H. (1991) Delivery systems for gene therapy. Biotherapy 3, 87 95. 14 Chang, C., Weiskopf, M. and Li, H.J. (1973) Conformational studies of nucleoprotein. Circular dichroism of deoxyribonucleic acid base pairs bound by polylysine. Biochemistry 12, 3028 3032. 15 Leffert, H.L., Koch, K.S., Moran, T. and Rubalcava, B. (1979) Hormonal control of rat liver regeneration. Gastroenterology 76, 1470 1482.
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16 Oka, J.A. and Weigel, P.H. (1983) Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. J. Biol. Chem. 258, 10253 10262. 17 Wu, G.Y., Wilson, J.M. and Wu, C.H. (1989) Targeting genes: Delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J. Biol. Chem. 264, 16985 16987. 18 Wu, G.Y. and Wu, C.H. (1987) Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. 262, 4429-4432. 19 Wu, G.Y. and Wu, C.H. (1988) Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro. Biochemistry 27, 887 892. 20 Wu, G.Y. and Wu, C.H. (1988) Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263, 14621 14624. 21 Gorman, C.M., Moffat, L.F. and Howard, B.H. (1982) Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell Biol. 2, 1044-1051. 22 Varmus, H.E., Padgett, T., Heasley, S., Simon, G. and Bishop, J.M. (1977) Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA. Cell l l, 307 319. 23 Watanabe, Y. (1980) The effect of selective breeding on the development of coronary atherosclerosis in W H H L rabbits. A model for familial hypercholesterolemia. Atherosclerosis 36, 261 268. 24 Wilson, J.M., Grossman, M., Wu, C.H., Roy Chowdhury, N., Wu, G.Y. and Roy Chowdhury, J. (1992) Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in LDL receptor-deficient rabbits. J. Biol. Chem. 267, 963 967. 25 Nagase, S., Shimamune, S. and Shumiya, S. (1979) Albumin-deficient rat mutant. Science 205, 590 591. 26 Shalaby, F. and Shafritz, D.A. (1990) Exon-skipping during splicing of albumin mRNA precursors on Nagase analbuminemic rats. Proc. Natl, Acad. Sci. USA 87, 26752 26756. 27 Wu, G.Y., Wilson, J.M., Grossman, M. and Wu, C.H. (1991) Receptor-mediated gene delivery in vivo: Partial correction of genetic analbuminemia in Nagase rats. J. Biol. Chem. 266, 14338 14342. 28 Wilson, J.M., Grossman, M., Cabrera, J.A., Wu, C.H. and Wu, G.Y. (1992) A novel mechanism for achieving transgene persistence in vivo following somatic gene transfer into hepatocytes; J. Biol. Chem. 267, 11483 11489.
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© 1993 Elsevier Science Publishers B.V. All rights reserved. / 0169-409X/93/$24.00 ADR 00133
Liver-directed gene delivery G e o r g e Y. W u a n d C a t h e r i n e H. W u Department of Medicine, Division of Gastroenterology-Hepatology, University of Connecticut School of Medicine, Farmington, CT, USA (Received April 27, 1992) (Accepted May 20, 1992)
Key words: Genes; Targeting; Asialoglycoproteins; Receptors
Contents Summary .................................................................................................................
159
I. Introduction ....................................................................................................
160
II. Targeted delivery of small molecules to hepatocytes .................................................
160
III. Targeted delivery and expression of genes in hepatocytes .......................................... 1. A targetable D N A carrier system .................................................................... 2. Receptor-mediated gene delivery and expression in hepatocytes in vitro ................... 3. Receptor-mediated gene delivery and expression in vivo ....................................... 4. Strategies for obtaining persistence of receptor-mediated gene transfection ...............
161 161 161 162 163
Acknowledgements .....................................................................................................
166
References ................................................................................................................
166
Summary Cell surface receptors can provide a convenient natural mechanism for delivery and internalization of substances to cells. We have utilized asialoglycoprotein receptors on hepatocytes to target DNA specifically to these cells. Studies initially began with marker genes in cell culture studies. More recently, normal genes in the form of targetable protein-DNA complexes have been introduced into animals Correspondence to: George Y. Wu, M.D., Ph.D., Department of Medicine, Division of GastroenterologyHepatology, University of Connecticut School of Medicine, 263 Farmington Avenue, AM-044, #1845, Farmington, CT 06030, USA.
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possessing inherited metabolic disorders. Partial, transient corrections of these diseases have been achieved by intravenous injection of the complexed genes. I. Introduction Normal hepatocytes possess cell-surface receptors that can recognize and internalize glycoproteins that possess clustered galactose residues [1]. Exposure of this class of proteins, known as 'asialoglycoproteins', to their receptor results in a complex series of cell biological events culminating in internalization of the ligand within membrane-limited endosomal vesicles [2]. The virtually unique presence of asialoglycoprotein receptors on normal hepatocytes has been to used to target substances specifically to these cells. II. Targeted delivery of small molecules to hepatocytes This concept has been studied for both therapeutic and diagnostic potentials. For example, trifluorothymidine [3] and adenine 9-fl-D-arabinofuranoside [4] have been coupled chemically to asialoglycoproteins and demonstrated a selected inhibition of viral DNA synthesis in mouse hepatocytes infected with Ectromelia virus. Similarly, the antimalarial drug, primaquine, was linked to a asialoglycoprotein for the treatment of the hepatic stage of malaria. Conjugates given to mice infected with P. berghei resulted in improvement in the numbers of long-term survivors [5]. Potential diagnostic agents have also been developed. For example, a synthetic asialoglycoprotein, galactosyl-albumin, was radiolabeled and found to be capable of being bound by hepatocytes in vitro [6] and in vivo [7]. Because tumors and other space-occupying lesions do not contain asialoglycoprotein receptors, this type of agent was expected to be of value for radio-imaging purposes. In a somewhat different approach, the lysosomotropic drug, primaquine, was coupled to an asialoglycoprotein. This class of compounds has been shown to raise the pH of endosomal vesicles. This is of interest because iron is normally taken up by cells, from transferrin, and released to the cell during acidification of endosomal vesicles. Elevation of the endosomal pH would be expected to decrease the iron release, and uptake by cells. When primaquine coupled to an asialoglycoprotein was administered to cells, uptake of iron mediated by transferrin was dramatically decreased but only in those cells that possessed asialoglycoprotein receptor activity. By using radiolabeled iron, the results raised the possibility that this strategy could be used to form images of space-occupying lesions in liver by excluding uptake of radionuclides in normal liver cells. The concept of targeting has been extended therapeutically in a technique called 'targeted rescue'. In this case, advantage was taken of the fact that most hepatocellular carcinomas have markedly decreased asialoglycoprotein activity compared to normal hepatocytes [8,9]. Instead of targeting toxins to tumors, which is inherently difficult due to the lack of distinguishable markers on most transformed cells, asialoglycoproteins have been used as a carrier to target protective agents to normal cells in the presence of corresponding cytotoxins. In initial studies, folinic acid, a methotrexate antagonist, was chemically coupled to an asialoglycoprotein. In the presence of methotrexate, the asialoglycoprotein-folinic
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161
acid conjugate protected only cells that possessed asialoglycoprotein receptors while those that did not possess these receptors were destroyed [10] in vitro. This work was subsequently extended using the selective hepatotoxin toxin, acetaminophen, in order to prevent toxicity to non-hepatic tissues [11].
III. Targeted delivery and expression of genes in hepatocytes In all of the above-described previous studies, agents were chemically linked to asialoglycoproteins for targeting. This was convenient for small molecules which could survive chemical modification and still remain functional. However, it seemed possible that, under appropriate conditions, macromolecules such as DNA could also survive a receptor-mediated endocytotic event. However, it appeared to be likely that an alternative linkage strategy would be required because covalent linkage to an asialoglycoprotein would alter the nucleic acid bases likely resulting in undesirable transcriptional events. To circumvent this potential problem, a soluble DNA carrier system [12,13] was developed consisting of an asialoglycoprotein carrier chemically coupled to poly-L-lysine. The latter was introduced to allow binding of DNA in a strong, non-damaging electrostatic interaction [14] to form a soluble complex. If it retained its targetability, the system could result in concentrated delivery of genetic material to a specific (organ) cell type. The solubility property could make possible delivery of foreign genes to hepatocytes in vivo and raise the possibility that foreign genes could be targeted to hepatocytes in vivo by intravenous injection. Parenchymal liver cells have a low turnover rate, are very active metabolically, and possess an excellent blood supply in vivo. Because of these properties, hepatocytes are attractive as recipients for delivery and expression of genes that could have therapeutic function [15]. III.1. A targetable DNA carrier system
To test this system, plasmid DNA was used which contained an SV-40 viral promoter driving the marker gene for the bacterial enzyme, chloramphenicol acetyltransferase (CAT), which catalyzes the acetylation of chloramphenicol. The gene was selected because mammalian cells lack the CAT gene. Therefore, the appearance of acetylated products of chloramphenicol could provide a simple marker for detection of foreign gene expression in target cells. The asialoglycoprotein carrier, a human serum protein derivative, asialoorosomucoid (AsOR), was prepared by desialylation of orosomucoid [16]. This protein was conjugated to L-lysine using disulfide bonds [17]. DNA was then mixed with the conjugate in order to form a soluble complex. Optimal conjugate to DNA ratios were determined by a gel retardation assay system [18]. That DNA was actually retarded by the presence of conjugate was confirmed using DNA labeled with 32p [19]. 111.2. Receptor-mediated gene delivery and expression in hepatocytes in vitro
In order to test DNA delivery and expression of foreign genes, an in vitro system was used consisting of two cell lines: HepG2, asialoglycoprotein receptor ( + ) cells
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and SK-Hep 1 ( - ) cells. Incubation of the receptor ( - ) cells with either targetable DNA complex or controls did not produce detectable CAT activity under any conditions [19]. However, HepG2, receptor ( + ) cells incubated with the targetable DNA complex did produce CAT enzyme activity as demonstrated by the formation of acetylated chloramphenicol derivatives. Control experiments in which HepG2 under identical conditions were incubated with DNA alone, DNA plus poly-Llysine, or DNA plus AsOR, all present in the same concentrations as provided by the complete complex did not result in foreign gene expression [19]. Addition of an excess of the asialoglycoprotein itself to the complex to compete for asialoglycoprotein receptor uptake blocked expression of the CAT gene in HepG2 cells, supporting the idea that the delivery of the D N A was directed by the asialoglycoprotein component of the complex [19]. There was no evidence of toxicity in cells treated with the DNA complex as they proliferated at the same rate as untreated cells [12]. These data taken together indicated that a DNA carrier system based on internalization of asialoglycoproteins by hepatocytes could result in targeting of a foreign gene in a soluble form specifically to asialoglycoprotein receptor-bearing cells in a non-toxic fashion, and that this gene could be expressed by those cells in vitro. 111.3. Receptor-mediated gene delivery and expression in vivo
There are many techniques available for introduction of foreign genes into cells in vitro. The main advantage to the receptor-mediated asialoglycoprotein-based system lay in the fact that the DNA complex was soluble and targetable to a specific cell type. This raised the possibility that foreign genes could be targeted in vivo by simple intravenous injection. To investigate this possibility, radiolabeled plasmid DNA alone, or in the form of a targetable complex, was injected intravenously into adult rats. Ten minutes after injection of the labeled DNA only, radioactivity in various organs [20] demonstrated that only 17% of the injected counts were taken up by the liver. However, labeled DNA injected in the form of a targetable complex resulted in liver uptake of 85% of the injected counts. The organ distribution of the complex was similar to that for radiolabeled asialoglycoprotein only, indicating that the complex had retained its ability to be recognized by asialoglycoprotein receptors in vivo [20]. To determine whether CAT gene sequences could be detected in intact form beyond the immediate post-injection period, rats where injected intravenously with unlabeled, complexed plasmid containing the CAT gene. Twenty-four hours later, liver DNA was extracted, and CAT sequences detected by dot blot using a labeled cDNA-CAT probe [20]. CAT gene sequences in transfected liver were easily detected by dot blot. Equal quantities of control liver DNA failed to hybridize to the cDNA probe, indicating that the hybridization found in liver transfected with complex was due to targeted DNA and not due to cross-hybridization to host DNA sequences
[20]. To determine whether the targeted DNA was actually functional, rats were injected with complexed DNA in saline or controls: saline only, DNA only, poly-Llysine plus DNA or a mixture of components of the complex all present in the same concentrations as provided by the complete complex. After 24 hours, CAT enzyme
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activity was assayed [21]. Targeted CAT gene expression was found in liver in vivo, but not in controls, under identical conditions. In order to determine whether the foreign gene expression was localized to the liver, other organs were assayed for CAT activity after administration of complexed DNA. Kidney, spleen, lungs, and liver were assayed, but only liver was found to have detectable quantities of CAT activity [20]. To determine whether natural mammalian regulatory elements could be targeted and used to drive a foreign gene in vivo, a construct containing the CAT gene driven by mouse albumin enhancer-rat promoter elements was prepared by Dr. James M. Wilson, University of Michigan. Complexed plasmid DNA, prepared as described previously, was injected intravenously into groups of rats. After 24 hours, it was determined that livers from rats that received the D N A in the form of a soluble complex produced CAT gene expression. However, livers from control rats that received saline only, D N A only, a mixture of components of the complex: AsOR/ poly-L-lysine/DNA did not produce detectable CAT activity. As seen with the previous plasmid construct, CAT gene expression was localized to liver and absent in heart, lung, spleen or kidney. The time course of targeted gene expression was determined in vivo. Injection of complexed D N A into groups of rats which were sacrificed at regular time intervals thereafter, and hepatic CAT activity assayed showed that targeted gene expression was transient with a maximum occurring between 24 and 48 hours after injection. Thereafter, CAT activity slowly declined, until by day 4 it was no longer detectable [17].
111.4. Strategies for obtaining persistence of receptor-mediated gene transfection In considering strategies to obtain persistent targeted gene expression, previous studies were noted in which transfection of cultured cells that were rapidly dividing had a higher likelihood of developing persistent expression. This had been attributed to a higher probability of integration of the foreign gene into host D N A during cell replication [22]. Because normal adult hepatocytes, under appropriate conditions, can be stimulated to replicate, this raised the possibility that stimulation of hepatocyte replication might enhance the chances for persistent targeted gene expression in adult liver. To examine the effect of hepatocyte replication on targeted gene expression, two-thirds of livers were removed after intravenous injection of targetable D N A complex into groups of rats. The two-thirds partial hepatectomy is a well-established model for stimulation of hepatic regeneration, producing a predictable response in D N A synthesis beginning by 12 hours after surgery and resulting in at least one round of cell division in almost all remaining hepatocytes within 1-2 weeks [15]. CAT gene expression was not detectable 24 hours after injection; but by 48 hours, CAT enzyme activity had become detectable and reached a maximum level by the 8th week. Activity remained high through the 1 lth week post-surgery [22]. In a model of a genetic metabolic disorder, the Watanabe heritable hyperlipidemic rabbit was employed [23]. This mutant contains a defect in receptors for low-density lipoprotein (LDL). As a result, these animals develop severe cardiovascular complications of hypercholesterolemia due to elevated levels of LDL. To study
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targeted gene delivery in this model, a plasmid construct was prepared to contain the gene for human LDL receptor driven by mouse albumin promoter and enhancer elements. Targetable complexes were made and injected intravenously. Quantitation of targeted D N A using a labeled vector-specific probe demonstrated that approximately 1000 copies of plasmid/cell were present 10 minutes after injection. Use of a complex in which the carrier was radiolabeled showed that more than 90% of the radioactivity detected by autoradiography was present in liver parenchymal cells. However, the abundance of the targeted D N A declined rapidly with time such that the copy number at 24 hours had decreased to a value of 1 copy/cell and to 0.1 copy/cell by 48 hours. Liver tissues were analyzed for the presence of human LDL receptor m R N A by RNase protection assays. This demonstrated that an appropriately-sized fragment was first detected 4 hours after injection and maximal levels of 2 4 % of endogenous levels were obtained by 24 hours after injection. Functionality of the foreign LDL receptor was demonstrated by a maximal drop of 25-30% in cholesterol levels 48 hours after injection. This decrease was highly significant (P < 0.001) compared to control that received CAT plasmid complex. The effect was transient with cholesterol levels rising to pre-injection levels by the 6th day. Cross-over experiments confirmed these results. No antibody against the carrier was detected circulating in rabbit serum 2 weeks after injection [24]. The targetable D N A delivery system was used to introduce another normal gene to correct an inherited metabolic disorder. In this second model, the Nagase analbuminemic rat was selected. This mutant rat strain possesses a defect in splicing of m R N A for serum albumin, resulting in virtually undetectable levels of circulating serum albumin [25]. A plasmid was constructed containing the structural gene for human serum albumin driven by mouse albumin enhancer-rat albumin promoter elements [26]. The human albumin gene was selected to avoid possible confusion between potential reversion of the mutation with expression of the endogenous rat albumin gene as opposed to actual foreign gene expression. A control construct was prepared identical to the first but without the albumin enhancer elements. Because the enhancer regions are required for high levels of expression by the albumin promoter [27], the control plasmid served to assay the contribution of potential nonspecific effects of plasmid D N A or the human albumin structural gene. Groups of mutant rats were injected with complexed D N A followed by two-thirds partial hepatectomies [27]. D N A was extracted from livers 2 weeks after injection. Blot hybridization analysis of total cellular D N A restricted with an enzyme which excised the human albumin gene insert from an enhancer-containing plasmid revealed a copy number of 1000/diploid genome. No bands larger than the albumin insert were detected, indicating that significant re-arrangements involving the albumin structural gene had not occurred. When total cellular D N A was digested with another restriction enzyme that cuts at a single site in the plasmid, hybridization with the albumin c D N A probe showed that livers from rats treated with complete plasmid produced a band that corresponded in size to linearized plasmid. Hybridization analysis of total cellular D N A from livers from the rat treated with complexes of the complete and the control plasmid were digested with an enzyme which does not cut the plasmid. Samples were probed with a fragment of the plasmid spanning a portion of the bacterial region of the plasmid but lacking any albumin sequences. These analyses showed two predominant bands corresponding
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to nicked circular and supercoiled forms of the plasmid. A small band was also seen corresponding in position to that of the linearized plasmid. These data indicate that the overwhelmingly predominant portion of the retained DNA in liver, in these experiments, existed as unintegrated circular plasmid DNA [27]. However, the possibility of random, non-clonal integration of some plasmid DNA into the host genome could not be excluded. Rats treated with the enhancerless plasmid showed similar patterns. To determine whether the targeted DNA had been transcribed, anaibuminemic rat livers were assayed by dot blots for the presence of human serum albumin mRNA 2 weeks after injection and partial hepatectomy. Total RNA from livers of rats that received the complete plasmid did produce a strong hybridization signal. RNA from liver of normal untreated rats did not hybridize with the probe, indicating that the signal detected previously was not due to hybridization to endogenous rat messenger sequences. The presence of human mRNA was confirmed by RNase protection analysis using a vector-specific RNA probe that is complementary to vector-derived sequences in the 3'-untranslated region of the recombinant-derived transcript. Analysis of RNA from liver harvested 2 weeks after transfection of analbuminemic rats with the complete plasmid in the form of a complex followed by partial hepatectomy resulted in a protected band of the expected size. However, liver from analbuminemic rats harvested 2 weeks after transfection and partial hepatectomy using identical molar quantities of complexed enhancerless plasmid did not generate any protected sequences under identical conditions. Formation of targeted gene product was assessed by Western blot using standard albumins and rat serum samples taken 2 weeks after treatment of analbuminemic rats with targeted complete plasmid DNA. These animals were shown to have circulating human serum albumin quantitated at 30 ~g/ml by 2 weeks after injection. Control animals that received saline alone, or the enhancerless plasmid in the form of a complex, did not produce detectable human albumin under identical conditions [27]. A time course of the appearance of human albumin in the circulation was determined by measuring serum samples by Western blot as a function of time. Analbuminemic rats treated with complete plasmid DNA complex did not have detectable circulating albumin after 24 hours. However, human albumin was detectable in serum in rats treated with the complete plasmid in the form of a complex when measured 24 hours after injection. The level of human albumin rose from a value of 0.05 #g/ml at 48 hours reaching a plateau of 34 #g/ml by the 2nd week and remained at this level without significant change through the 4th week post-injection [27]. The mechanism of persistent of foreign gene expression was further elucidated by a detailed examination of the state of the transfected DNA following partial hepatectomy. Southern blots of DNA extracted from livers at various time intervals ranging from 10 minutes to 11 weeks after injection and partial hepatectomy revealed that the vast majority of the transfected DNA was in the form of intact bacterial plasmid. The bacterial portion of the plasmid remained functional, as demonstrated by re-transfection of the extracted DNA from rats, which resulted in successful transfection of bacteria. Plasmids recovered from these bacteria produced restriction patterns identical to those of the original plasmid.
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Virtually all the plasmid DNA extracted from the liver was sensitive to a bacterial enzyme that cleaves only double-stranded methylated DNA of bacterial origin. It was concluded that the transfected DNA remained as stabilized episomal forms without evidence of clonal integration or replication within the limits of detection
[28]. Acknowledgements The assistance of Brenda Kawecki and Rosemary Pavlick is gratefully acknowledged. This work was supported in part by grants by the U.S. Public Health Service (NIH, DK42182) (G.Y.W.), a grant from the March of Dimes (91-0485) (G.Y.W.), and a grant from TargeTech, Inc. (C.H.W.).
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