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Strategies for Gene Therapy in the Liver
Gene therapy involves the transfer of normal, functional genetic material into cells to correct an abnormality due to a defective or deficient gene product.' Advances in molecular biology have led to the discovery of genetic defects in a variety of metabolic diseases, and the ability to clone the corresponding functionally normal genes.'Gene replacement or substitution involves removal of the entire gene or part of the mutant sequence and introduction of a normal gene or sequence necessary to correct the mutation.' This would be an ideal method for correcting single gene defects, especially those dominantly transmitted. Several laboratories have shown that gene substitution is possible but thus far on an inefficient Currently, most work is centered on gene augm e n t a t i ~ n .This ~ approach involves the introduction of DNA to express an absent gene product or modify the expression of a defective gene. In order to study the expression and regulation of foreign gene sequences, several techniques were developed-to introduce DNA into cells. These techniques include calcium phosphate coprecipitation,'-"' diethylaminoethyl (DEAE) dextran," electroporation,".13 scrape loading,I4 sonication,I4 particle bombardment,'5.'h microinjection, liposomes,"-'had ligandbased DNA carrier^.""^ Viral vectors have also been shown to be an effective mode of introducing DNA into The introduction of DNA by cell surcells in vitro.'.'"' face uptake is also referred to, in general terms, as transfe~tion.~ Another issue in gene therapy is the use of somatic versus germ cells. Correction of deficits at the germ cell level would intuitively seem ideal. This approach, however, has several problems. For example, there may be some degree of insertional mutagenesis with undesirable and unpredictable complication^.'^ Also, genetic manipulation of germ cells is currently the focus of ethical debate in that changes would be passed on to future generations and could alter normal human genetic variability. Because of these issues, much work has focused on the use of somatic cell lines for gene therapy.
From the Division of Gn.stroenterolojiy cirld Hepcrtolo~y.Utriversity c?fCor~rrc~ticut Hetilth Center. F~irtningtorr,Cotlt~ecticut. Reprint requests: Dr. G.Y. Wu, Division of Gastroenterology and Hepatology, AM044, University of Connecticut Health Center, Farmington, CT 06030.
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Fibroblasts, myocytes, renal cells, hematopoietic cells, and hepatocytes have all been successfully trans. ~limitation in many of these cell lines fected in ~ i t r o A is that secondary processing of the gene product is often necessary for activity and not all transfected cells may contain the cellular machinery to affect these alterations. Hepatocytes have received a great deal of attention because a wide variety of proteins are manufactured in the liver and therefore have the necessary cellular features to process these proteins. In addition, many genetic diseases result from an absence or deficiency of a hepatocyte-derived gene product. For these reasons, the liver is an attractive target organ for directed gene therapy. This article will detail current work in the transfer of DNA to hepatocytes in vitro, the advances made in the area of in vivo gene transfer, and the applicability of in vivo gene transfer to gene therapy in humans.
METHODS FOR IN VlTRO TRANSFECTION OF HEPATOCYTES Calcium Phosphate Coprecipitation Calcium phosphate coprecipitation with DNA was initially described in 1973.9This procedure involves precipitation of DNA with calcium phosphate to produce insoluble calcium phosphate DNA complexes that are internalized by endocytosis when they contact the cell surface. This results in delivery of many copies of the DNA of interest. The advantage of this system is that it is relatively easy to perform and is not cell-specific, although some cell lines are easier to transfect than others. The method can be used on any appropriate cell line and has been widely applied to hepatoma cell lines and in a few instances to primary hepatocytes. The disadvantage of this method is that although many copies of DNA are introduced into the cell, expression is, for the most part, transient. Stable transfection of the cell is inefficient, generally less than 1 %, and tends to be limited to continuous cell lines. Recently, stable transfection frequencies approaching 50% were reported when precipitates were formed slowly over a period of time by gradually increasing the pH in the incubation media.x A modification of this technique was used to obtain stable transfection of primary rat hepatocytes at frequencies up to 20%.'"
Copyright 0 1992 by Thieme Medical Publishers, Inc.. 381 Park Avenue South. New York. NY 10016. All rights reserved.
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While primarily limited to in vitro systems, there are reports of in vivo applications of the techniq~e.'~," In one report,'" a polyoma virus plasmid was precipitated with calcium phosphate. The precipitate was then injected directly into a mouse liver resulting in transient expression of the gene. In another study," investigators formed calcium phosphate precipitates with the genes for chloramphenicol acteyltransferase (CAT), a bacterial gene product, hepatitis B surface antigen, and human insulin. These were injected intraperitoneal in newborn mice with resulting transient expression of the gene products in the liver and spleen.
Diethylaminoethyl Dextran DEAE dextran and other compounds like it are large polycations that bind DNA electrostatically and are taken up by cells in much the same manner as calcium phosphate precipitates. This technique is basically a variation on the calcium phosphate method. It has been useful in improving the efficiency of transfection and in transfecting some cell lines less responsive to the calcium phosphate method. ' I
Electroporation Electroporation is a method whereby an electric pulse is transmitted to cells in culture that are incubated with DNA. The electrical pulse causes disruptions in the cell membranes allowing- entrance of the D N A . This generally results in transient expression of DNA at low frequencies. In primary hepatocyte cultures, cell viability is generally better than that reported for calcium phosphate or DEAE dextran."
Cell Sonication Cell sonication employs the use of high-frequency sound waves applied to cells in suspension with the DNA of interest. Sound waves result in transient disruption of the cell membranes allowing DNA to enter the cell. High rates of DNA transfer into cells are achieved with this method but expression is only transient. l 4
Scrape Loading Scrape loading is a technique whereby cells at confluence on a plate are mechanically scraped off in the presence of DNA in solution. As with cell sonication, this technique causes transient disruption of the cell membranes, allowing a high rate of DNA transfer into the cells. l 4
Viral-Mediated Transfection of Cells Viral agents have been demonstrated to be highly efficient vectors for the transfection of somatic cells. Retroviruses in particular have received a great deal of attention because they not only enter cells efficiently, but also provide a mechanism for stable integration into the host genome through the p r o v i r u ~ . ' ~Retroviral -~~ vectors have been used to introduce the P-galactosidase gene
into adult hepatocytes with expression of the marker gene approaching 25%.2"n another example, the gene for the LDL receptor was transferred into a primary culture of hepatocytes obtained from Watanabe hyperlipidemic rabbits with expression of the normal receptor.'* Retroviral vectors appear to be promising agents for gene transfer and expression. However, there have only been a small number of studies in v i ~ o . " , ' ~ Actively dividing cells are necessary for provirus integration. Also, obtaining sufficient titer of virus and the lack of targetability have limited in vivo application. Currently, investigators are attempting to address this last problem by use of organ-specific promoters and by chemically modifying the cell recognition sites on the viral envelope." In vivo application to hepatocytes has currently involved ex vivo transfection and subsequent reimplantation of the modified h e p a t ~ c y t e s . ' ~ .Details ~' of this technique are discussed elsewhere. Other viral vectors, such as vaccinia, adeno-associated, herpes, and papilloma viruses are also being studied. '.'X-40 The adeno-associated viruses (AAV) are particularly intriguing because of their dual life cycle. Alone, these viruses are nonpathogenic and exist in humans as an integrated provirus. However, in the presence of coinfection-with a helper virus, such as adenovirus or herpes virus, the AAV can become lytic and release high titers of viral progeny. Portions of the AAV genome can be deleted and DNA of interest inserted. The virus can then be introduced into a cell culture in the form of a provirus. High titers of the genetically modified provirus can then be produced by coinfection with a helper virus. The provirus produced can then be used to transfect cells of interest.?" With the recent discovery of defective forms of hepatitis B virus (HBV), there has been interest in the use of hepadnaviruses as hepatotropic vectors for the delivery of foreign genes. In vitro studies have shown that large portions of the hepadna viral genome are dispensable for helper-dependent packaging of foreign genes.4' This technique has been used to introduce the CAT gene, with stable expression of the gene product, into primary cultures of duck hepatocytes by use of a recombinant ' transfection, however, duck hepatitis v i r u ~ . ~Successful required coinfection with wild-type helper virus. Attempts at coinfection with a mutant helper virus was not successful in supporting expression of the CAT plasmid.
METHODS FOR GENE THERAPY FOR HEPATIC GENETIC DISEASE IN VlVO Particle Bombardment Particle bombardment is a unique modality for the delivery of DNA to both in vitro and in vivo systems. This method was first used successfully for the delivery of DNA to plant^.^' The technique involves the use of microspheres of an inert material such as gold or tungsten on which the plasmid DNA of interest is precipitated. These particles are then suspended on a Mylar film or similar arrangement and placed adjacent to two electrodes across which
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a high voltage discharge can be produced. The resulting electric arc provides the energy to accelerate the particles to high velocity into the desired tissue. By varying the voltage across the electrodes, and the size and density of the beads, the distribution of the particles in the target tissue can be c o n t r ~ l l e d . ~Utilizing ~.'~ this method, in vivo transfection of rat hepatocytes has been accomp l i ~ h e d . ~ ~Plasmid ." DNA was prepared using the bacterial gene for CAT along with a mammalian promoter. This enzyme is not found in mammalian cells and, thus, expression of the gene product would be a sensitive marker for a successful transfection. In two different experiments, CAT plasmids were coated onto microspheres. Rats then underwent laparotomy to expose a lobe of the liver. The plasmid-coated spheres were then "shot" into the exposed lobe, as already described. Animals were sacrificed at 24 hours in one experimentihand 48 hours in another.Is Liver samples were then taken from the area of bombardment and assayed for the presence of CAT gene expression. This was determined by demonstrating the appearance of the acetylated products of chloramphenicol. In both experiments, CAT gene expression was demonstrated. From these two studies, however, the duration of gene expression beyond 48 hours could not be determined because the animals were only sampled at one point in time. Stable transfection was obtained with this method in nonhepatocyte cell cultures at frequency of to l s This is similar to other methods such as calcium phosphate and electroporation. One of the problems with obtaining stable transfections is the need for actively dividing cells. When cells are dividing, there is a greater chance that the gene will be integrated into the host cell genome and the likelihood of persistent expression enhanced.44For this reason, stable transfections have been mainly reported in cell culture. With organs such as the liver, cell populations are generally stable and are not undergoing active replication. The liver, however, has a tremendous capacity for regeneration when there is loss of liver tissue or damage. Mechanical damage to liver tissue during particle bombardment might be beneficial to the transfection process because hepatocytes would be expected to undergo replication to repair the damage.
Liposomes Phospholipids placed in an aqueous solution can assume three possible forms: (1) micelles, which are small spherical structures with the hydrophilic heads of the molecules on the surface and the hydrophobic tails packed into the center; (2) lipid bilayers, which are sheets of phospholipids two molecules thick with the hydrophilic tails packed in the center; or (3) liposomes that are essentially spherical lipid bilayers. The latter are of interest because they form spontaneously in an aqueous medium and have been demonstrated to trap aqueous phase material. The size and shape of the liposomes can be varied by changing the mixture of phospholipids, degree of saturation of the fatty acid side chains, and the conditions under which the liposomes are formed.4s Liposomes are taken up by cells by endocytosis and this
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characteristic has been used to package, for example, drugs and enzymes for delivery to cell^.^'^^^ Liposomes have also been used to package DNA for delivery to cells in v i t r ~ . ~This ~ . ~ is' of interest because plasmids of large size could potentially be packaged and maintained in a soluble form that would allow direct application to in vivo systems by a simple intravenous injection. It has also been shown that these liposomes can be targeted by the selection of phospholipids present in the liposomal membrane or the addition of exposed sugar moieties on the surface of the liposome. Normally, liposomes injected into animals are preferentially taken up by reticuloendothelial cells. It has been shown, however, that small liposomes with a significant proportion of eggphosphatidyl choline in the membrane are preferentially taken up by hepatocytes when injected into rats.s0 The addition of side chains containing terminal galactose residues to the liposome has also been examined as a way of targeting liposomes to hepatocytes. This process can be inhibited by pretreatment of the animals with asialofetuin, a natural asialoglycoprotein, and is assumed to be mediated by surface receptors unique to h e p a t o c y t e ~ . ~ ' These asialoglycoprotein surface receptors recognize ligands with exposed terminal galactose residues and deliver the liposomes by endocytosis into clathrin-coated vesicles prior to fusion with lysosomes. DNA found in these coated vesicles has been demonstrated to be intact and can be transcribed when subsequently transfected ~ ' use of asialoglycoprotein reinto Escherichia c ~ l i . The ceptors for gene delivery is discussed in greater detail subsequently. Several investigators have taken advantage of the ability to package DNA into liposomes and selectively target them to the liver in vivo. 17-I' A plasmid containing the bacterial gene for neomycin resistance driven by the Rous sarcoma virus promoter was packaged into liposomes. These were then injected intravenously into rats. The neomycin resistance gene was chosen because this is a bacterial enzyme that is not made by mammalian cells and can be used to select cells possessing the gene. The presence of neomycin phosphotransferase activity in the cells could then be followed as an expression marker of the transferred gene. The animals were sacrificed at various time intervals and the liver homogenates assayed for neomycin phosphorylation activity. Activity was present at 4 hours and could still be detected 4 days after injection. Southern blot analysis of hepatocyte fractions showed no evidence of integration." In another example, the gene for rat preproinsulin was packaged into liposomes and again injected intravenously into rats. This is of interest in that the liver possesses the enzymes necessary to cleave precursor propeptides to the their active form. Serum from the animals was then analyzed for glucose levels, and livers and spleens were removed at various times. Serum glucose was demonstrated to fall in the treated animals during the first 10 hours, but then returned to levels above baseline by 18 hours. Elevated insulin levels appeared transiently in the liver and spleen, but by 18 hours were the same as control levels.s3 In a similar example, the gene for human insulin was introduced in vivo by intravenous injection of the lipo-
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some-encapsulated DNA along with a high molecular weight nuclear protein that binds to DNA and increases the amount of DNA reaching the nucleus of the cell. Expression of the gene was determined by assaying serum for circulating human insulin. Peak serum levels of human insulin were found 7 days after injection and persisted until about 10 days. Corresponding levels of RNA and DNA were also detected in the liver during this time period.'' A similar technique was also used to introduce the gene for hepatitis B surface antigen into rat livers in vivo. Expression of surface antigen peaked in the serum 2 days after injection and was detectable for 9 days. l 9 Of note, these investigators have also demonstrated a typical viral histologic inflammatory response in the livers of adult rats transfected with the gene for hepatitis B surface antigen.s4
Cell Surface Receptors
proteins6 to clathrin-coated vesicles that fuse with lysosomes where the ligand is degraded. The asialoglycoprotein receptor is then recycled back to the cell ~urface.'~ We have previously demonstrated that a variety of agents can be delivered specifically to hepatocytes by coupling them to asialoglycopr~teins.~~-~~ In these studies, agents were coupled to the asialoglycoprotein carrier by covalent bonds. 6' However, this method of coupling to nucleic acids could potentially alter them. To solve this problem, a complex was created that consisted of a polycation, poly L-lysine, covalently bound to asialoorosomucoid, a naturally occurring asialoglycoprotein with a high affinity for the receptor. The polycation could then bind the negatively charged DNA in an electrostatic, nondamaging manner, as illustrated in Figure 1.
Evidence for In Vitro Expression in Hepatocytes
Targeting of DNA to somatic cells by way of cell surface receptors is another potential method of gene therapy. Hepatocytes are uniquely suited to this form of receptor-mediated transfection. As opposed to some of the other methods of transfecting somatic cells, DNA delivery via cell surface receptors is applicable to both in vitro and in vivo systems. Hepatocytes are attractive because they possess cell surface receptors that are able to recognize and bind circulating galactose-terminal glycoproteins (asialoglycopr~teins).~~ Binding of the glycoprotein to the asialoglycoprotein receptor results in endocytosis of the receptor ligand complex and delivery of the glyco-
To determine whether the asialoorosomucoid-polylysine-DNA (AsOR-PL-DNA) complex could be delivered to hepatocytes, two continuous cell lines, HepG2 and SK-Hep 1, were used. HepG2 cells possess substantial asialoglycoprotein receptor activity on their surfaces and SK-Hep 1 cells do not. Each cell line was incubated with AsOR-PL-DNA complex or controls consisting of the various components of the complex. Cells were then assayed for activity of the CAT gene p r o d ~ c tNo . ~ CAT activity was detected with either the AsOR-PL-DNA or any of the controls in receptor-negative SK-Hep I cells. With receptor-positive HepG2 cells, CAT activity was
COMPONENTS OF A TARGETABLE DNA COMPLEX
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6 Polycation Covalent Bond Ligand
Receptors
FIG. 1. Soluble DNA carrier complex. Poly L-lysine is covalently bound to the carrier asialoglycoprotein that is the part of the complex that provides recognition for binding and internalization by the hepatocyte. DNA to be delivered is then electrostatically bound to the carrier.
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found in the cells incubated with the AsOR-PL-DNA complex, but not in the controls. Furthermore, the addition of excess AsOR to the media of HepG2 cells incubated with AsOR-PL-DNA inhibited the development of CAT activity in the cells. These data prove that DNA can be introduced into hepatocytes via the asialoglycoprotein receptor with resultant expression of the foreign gene. 2'.24 Similarly, we have also demonstrated that an ecotropic viral vector can be targeted to liver cells by chemically modifying the protein coat of the virus with lactose residue^.'^ Attachment of lactose residues to the viral coat results in exposed galactose molecules, in effect, creating a synthetic ASG. In this study, the P-galactosidase gene was introduced into a replication defective Moloney murine leukemia virus, an ecotropic viral vector. As would be expected, murine cell lines were infected by the unmodified virus, but not human SK-Hepl (asialoglycoprotein receptor-negative) or human HepG2 (asialoglycoprotein receptor-positive) cells. When modified virus was used, receptor-positive human HepG2 cells were successfully transfected with resultant gene expression. Attempts to transfect receptor-negative SKHepl cells with the modified virus were unsuccessful. As additional proof that the entry of the modified virus was via the asialoglycoprotein receptor, transfection of HepG2 cells could be competitively inhibited by addition of an excess of AsOR. This study demonstrated that viral vectors could be chemically modified to target them potentially to a specific organ. Furthermore, modification of the viral vector did not affect subsequent expression of the foreign gene.
Evidence for In Vivo Targeting and Expression in Hepatocytes To demonstrate that DNA could be targeted in vivo by a soluble DNA carrier system, rats were injected intravenously with a plasmid consisting of the bacterial CAT gene with a viral promoter, complexed to the asialoglycoprotein carrier system. Animals were sacrificed 24 hours later and liver homogenates assayed for CAT activity. Only those animals injected with the intact complex exhibited CAT activity. No evidence for CAT gene expression was seen in any of the animals injected with the various controls. This expression was also shown to be liver-specific, as determined by assaying other organs for CAT gene activity.'" A targeted foreign gene driven by mammalian regulatory elements can also result in persistent gene expression. In rats injected with the DNA complex without partial hepatectomy, the presence of detectable gene expression peaked between 24 and 48 hours but fell to undetectable levels over approximately 4 days. However, in rats that had two thirds hepatectomy, persistent CAT gene expression was achieved through 1 1 weeks." These results indicated that foreign genes could be targeted in vivo with persistent expression of the gene. A more detailed review of these studies has been previously published. h2
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Application of Targeted Gene Delivery to Gene Deficiency Models A major goal of targeted delivery of foreign DNA in vivo is to cure diseases resulting from deficiency of a gene product by introducing the normal gene into the host genome. To test the feasibility of this strategy, Nagase analbuminemic rats were employed. These animals have an mRNA splicing defect that results in virtually undetectable levels of serum albumin. A plasmid was constructed with the gene for human albumin driven by mouse enhancer-rat albumin promoter sequences. This plasmid was complexed to the asialoglycoprotein carrier and injected intravenously. The animals were then subjected to two thirds partial hepatectomy" and serum was assayed for albumin by Western Blot using a specific antibody to human albumin. By 48 hours, human albumin could be detected in the serum at a level of 0.05 pglml. By 2 weeks, this had increased to a maximum of 34 pglml and remained stable for 4 weeks.2' Results are shown in Figures 2 and 3. Analysis of cell lysates 2 weeks post-transfection showed that a majority of the plasmid DNA existed as an episomal, nonintegrated form. However, infrequent integration could not be excluded. In another example, the Watanabe heritable hyperlipidemic rabbit was used. This animal is hypercholesterolemic secondary to genetically defective LDL receptors and serves as an animal model for human familial hypercholesterolemia. A plasmid was constructed containing the gene for the human LDL receptor driven by a mouse albumin promoter and enhancer. This plasmid and a control plasmid construct using the CAT gene were then complexed to the soluble DNA carrier and injected into separate groups of rabbits in a two treatment crossover experiment. Serum was assayed for cholesterol levels at various time points (Fig. 4). At the end of 12 days, the same animals were again injected, this time with the other plasmid complex. Analysis of liver homogenates 10 minutes postinjection revealed about 1000 copies of the plasmid per cell. This rapidly decreased to less than 0.1 copies per cell after 48 hours. Expression of low density lipoprotein (LDL) receptor mRNA reached a maximum expression at approximately 24 hours after transfection and was estimated to be about 2 to 4% of normal. This expression was functionally correlated to a 25 to 30% reduction in serum cholesterol that was specific for the LDL receptor plasmid. The reduction, however, was transient and by 5 days had returned to baseline levels.h3 While complete normalization of serum cholesterol was not obtained in this experiment, the 30% decrease that was obtained would be clinically significant.
FUTURE OF GENE THERAPY The ultimate objective in gene therapy is its application to human disease states. At the present time, several studies are already underway and others have received Food and Drug Administration appro~al.~~.~%All these trials are utilizing retroviruses and ex vivo trans-
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FIG. 2. Western blot of serum samples obtained 2 weeks postinjection and partial hepatectomy. Lane 1 shows the electrophoretic m~grationof-standard human albumin identified bv immunoblottina with s~ecificantibodv. Lane 2 shows an identical amount of standard rat serum albuminto demonstrate a lack of cross-reactivity with the human albumin antibody. Lane 3 shows standard human albumin mixed with untreated analbuminemic rat serum to demonstrate identical migration and detection with the antibody. Lanes 4 and 5 show untreated Sprague-Dawley rat serum and analbuminemic rat serum, respectively, to demonstrate the absence of detectable protein with the human albumin antibody. Lane 6 shows human albumin in serum from an analbuminemic rat treated with the DNA carrier complex; lane 7 shows the absence of serum albumin when the vehicle (saline) alone was injected; and lane 8 shows the absence of human serum albumin when an enhancer-less plasmid complex was injected.
FIG. 3. Presence of serum albumin in a Nagase analbuminemic rat after transfection in vivo with the gene for human albumin using a soluble DNA-carrier system. Lanes 1 to 3 contain human albumin standards of increasing concentration. At 24 hours, albumin was not detectable, as shown in lane 4. Lane 5 demonstrates detectable human albumin in the serum of analbuminemic rats at a level of 0.05 wliml 48 hours post-transfection and partial hepatectomy. The concentration of albumin increased to 34 pgiml by 2 weeks and persisted at this level through 4 weeks (lanes 5 to 11).
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FIG. 4. Effect of hepatocyte targeted low density lipoprotein (LDL) receptor or chloramphenicol acetyltransferase (CAT) genes on serum cholesterol in Watanabe heritable hyperlipidemic rabbits. Animals shown in A to C received LDL receptor plasmid on d a y zero a n d CAT plasrnid on day 12. Animals D to F received CAT plasmid on day zero a n d LDL receptor plasrnid on day 12. A d e c r e a s e in serum cholesterol w a s s e e n only in t h e animals injected with t h e LDL receptor DNA complex. T h e maximum d e c r e a s e w a s seen after about 2 d a y s a n d by 5 d a y s had returned t o baseline.
fection. Although there are still many questions to be answered and problems to overcome, the future of gene therapy appears bright. This aspect of molecular medicine, which would have been considered science fiction a decade ago, will likely become part of the clinician's therapeutic armamentarium in the not too distant future. Acknowledgments. We gratefully acknowledge the collaboration and advice of Dr. David A. Shafritz, Dr. James M. Wilson, and Mariann Grossman on work presented in this manuscript. This work was supported in part by the National Institutes of Health grants CA468O 1 and DK42 182, Research Career Development Award CAOl110, and March of Dimes Grant 1-90-421 to G.Y.W., and a grant from TargeTech, Inc., to C.H. W.
REFERENCES 1.
Friedmann T: Progress toward human gene therapy. Science 244:1275-1281, 1989.
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Behart J , Caskey C: Developments leading to human gene therapy. In Kucherlapati R (Ed): Gene Transfer. New York, Plenum Press, 1986, pp. 41 1-435. Danks D, Cotton R: Future developments in phenylketonuria. Enzyme 38:296-301, 1987. Wilson J: Prospects for gene therapy of familial hypercholesterolemia. Mol Biol Med 7: 223-232, 1990. Frohman M, Martin G: Cut, paste and save: New approaches to altering specific genes in mice. Cell 56: 145-147, 1989. Doetschman T, Gregg R, Maeda N, et al: Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330576-78, 1987. Ledley F: Clinical application of somatic gene therapy in inborn errors of metabolism. J Inher Metab Dis 13:597-616, 1990. Chen C , Okayama H: High-efficiency transfection of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745-2752, 1987. Graham F, Van Der Eb A: A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456567, 1973. Rippe R, Brenner D, Leffert H: DNA-mediated gene transfer into adult rat hepatocytes in primary culture. Mol Cell Biol 10:689-695, 1990. Gopal T: Gene transfer method for transient gene expression, stable transfection, and co-transfection of suspension cell cultures. Mol Cell Biol 5: 1 188-1 190, 1985. Tur-Kaspa R, Teicher L, Levine B, et al: Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes. Mol Cell Biol 6:716-718, 1986. Potter H, Weir L, Leder P: Enhancer-dependent expression of human k immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci USA 81:7161-7165, 1984. Fechheimer M, Boylan J , Parker S , et al: Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci USA 84:8463-8467, 1987. Yang N, Burkholder J , Robert B, et al: In vivo and in virro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci USA 87:9568-9572, 1990. Zelenin A, Alimov A. Titomirov A, et al: High-velocity mechanical DNA transfer of the chloramphenicol acetyltransferase gene into rodent liver, kidney and mammary gland cells in organ explants and in vivo. FEBS Lett 280:94-96, 1991. Leibiger B, Leibiger I, Sarrach D, Zuhlke H: Expression of exogenous DNA in rat liver cells after liposome-mediated transfection in vivo. Biochem Biophys Res Commun 174: 1223-1231, 1991. Kaneda Y, lwai K , Uchida T: Introduction and expression of the human insulin gene in adult rat liver. J Biol Chem 264: l2I26-12l29, 1989. Kato K, Nakanishi M, Kaneda Y, et al: Expression of adult hepatitis B virus surface antigen in adult rat liver. J Biol Chem 266:3361-3364, 1991. Wu G, Wu C: Receptor-mediated gene delivery and expression in vivo. J Biol Chem 263: 14621-14624, 1988. Wu C, Wilson J , Wu G: Targeting genes: Delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J Biol Chem 264:16985-16987, 1989. Wu G, Wilson J , Shalaby F, et al: Receptor-mediated gene delivery in vivo. J Biol Chem 266: 14338-14342, 1991. Wu G, Wu C: Evidence for targeted gene delivery to HepG2 hepatoma cells in vitro. Biochemistry 27:887-892, 1988. Wu G, Wu C: Receptor-mediated in virro gene transfections by a soluble DNA carrier system. J Biol Chem 262:4429-4432, 1987. Wilson J , Jefferson D, Chowdhury J , et al: Retrovirus-me-
Downloaded by: University of British Columbia. Copyrighted material.
SEMINARS IN LIVER DISEASE-VOLUME
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diated transduction of adult hepatocytes. Proc Natl Acad Sci USA 85:3014-3018, 1988. Ledley F, Darlington G, Hahn T, Woo S: Retroviral gene transfer into primary hepatocytes: Implications for genetic therapy of liver-specific functions. Proc Natl Acad Sci USA 8453355339, 1987. Wolff J, Yee J, Skelly H, et al: Expression of retrovirally transduced gene in primary cultures of adult rat hepatocytes. Proc Natl Acad Sci USA 84:3344-3348, 1987. Armentano D, Thompson A, Darlington G, Woo S: Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene therapy of hemophilia B. Proc Natl Acad Sci USA 87:6141-145, 1990. Palmiter R, Brinster R: Transgenic mice. Cell 41:343-345, 1985. Dubensky T, Campbell B, Villarreal C: Direct transfection of viral and plasmid DNA into the liver or spleen of mice. Proc Natl Acad Sci USA 8 l:7529-7533, 1984. Benvenisty N, Neshif L: Direct introduction of genes into rats and expression of the genes. Proc Natl Acad Sci USA 83:955 1-9555, 1986. Wilson J, Johnston D, Jefferson D, Mulligan R: Correction of the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci USA 85:44214425, 1988. Hatzoglou M, Lamers W, Fatima B, et al: Hepatic gene transfer in animals using retroviruses containing the promoter from the gene phosphoenolpyruvate carboxykinase. J Biol Chem 265: 17285-17293. 1990. Ferry N, Duplessis 0,Houssin D, et al: Retroviral-mediated gene transfer into hepatocytes in vivo. Proc Natl Acad Sci USA 88:8377-8381, 1991. Neda H, Wu C, Wu G: Chemical modification of an ecotropic murine leukemia virus results in redirection of its target cell specificity. J Biol Chem 266: 14143-14146, 1991. Wilson J, Chowdhury N, Grossman M, et al: Temporary amelioration of hyperlipidemia in low density lipoprotein receptordeficient rabbits transplanted with genetically modified hepatocytes. Proc Natl Acad Sci USA 87:8437-8441. 1990. Fuller B: Transplantation of isolated hepatocytes: J Hepatol 7:368-376, 1988. Coupar B, Andrew M, Boyle D: A general method for the construction of recombinant vaccinia virus expressing multiple foreign genes. Gene 68:l-10, 1988. Hermonat P, Muzyczka N: Use of adeno-associated virus as a mammalian DNA cloning vector: Transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci USA 81:6466-6470, 1984. Rasmussen C, Simmen R, MacDougall E, Means A: Methods for analyzing bovine papilloma virus based calmodulin expression vectors. Methods Enzymol 139:642-654, 1987. Horwich A, Furtak K, Pugh J, Summers J: Synthesis of hepadnavirus particles that contain replication-defective duck hepatitis B virus genomes in cultured HuH7 cells. J Virol 64:642-650, 1990. Chang C, Ganem D, Lavine J: Foreign gene delivery and expression in hepatocytes using a hepatitis B virus vector. Hepatology 14: l24A, 199 1 . Klein T, Wolf E, Wu R, Sanford J: High velocity microprojectiles for deliveries nucleic acids into living cells. Nature 327:70-73, 1987. Varmus H, Padgette T, Heasley S , et al: Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA. Cell 11:307-319, 1977. Szoka F, Papahadjopoulos D: Procedure for preparations of li-
posomes with large internal aqueous space and high capture by reserve phase evaporation. Proc Natl Acad Sci USA 75:41944198, 1978. Gregoriadis G , Leathwood P, Ryman B: Enzyme entrapment in liposomes. FEBS Lett 14:95-99, 1971. Gregoriadis G, Ryman B: Fate of protein containing liposomes injected into rats: An approach to the treatment of storage diseases. Eur J Biochem 2 4 : 4 8 5 4 9 l , 1972. Wong T, Nicolau C, Hofschneider P: Appearance of p-lactamase activity in animal cells upon liposome mediated gene transfer. Gene 10:87-94, 1980. Nicolau C, Sene C: Liposome mediated DNA transfer in eukaryotic cells. Biochim Biophys Acta 721: 185-90, 1982. Spanjer H, VanGalen M, Roerdink F, et al: Intrahepatic distributor of small unilamellar liposomes as a function of liposomal lipid composition. Biochim Biophys Acta 863:224-230, 1986. Ghosh P. Bachhawat B: Targeting of liposomes to hepatocytes. In: Wu G, Wu C (Eds): Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. New York, Marcel Dekker, 199 1 . Nandi P, Legrand A, Nicolau C: Biologically active recombinant DNA in clathrin-coated vesicles isolated from rat livers after in vivo injection of liposome encapsulated DNA. J Biol Chem 261: 16722-16726, 1986. Nicolau C , Legrand A, Grosse E: Liposomes as carriers for in vivo gene transfer and expression. Methods Enzymol 149: 157176, 1987. Kato K , Kaneda Y, Sakurai M, et al: Direct injection of hepatitis B virus into liver induced hepatitis in adult rats. J Biol Chem 266:22O7 1-22074, 1991. Ashwell G, Morell A: Rule of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymo141:99-128, 1974. Wall D, Wilson G, Hubbard A: The galactose specific recognition system of mammalian liver: The route of ligand internalization in rat hepatocytes. Cell 21:79-83, 1980. Schwartz A: Trafficking of asialoglycoproteins and the asialoglycoprotein receptor. In: Wu G, Wu C (Eds): Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. New York, Marcel Dekker, 1991. Wu G, Wu C, Rubin M: Acetaminophen hepatotoxicity and targeted rescue: A model for specific chemotherapy of hepatocellular carcinoma. Hepatology 5:709-713, 1985. Wu G, Wu C, Stockert R: Model for specific rescue of normal hepatocytes during methotrexate treatment of hepatic malignancy. Prod Natl Acad Sci USA 80:3078-3080, 1983. Wu G, Keegan-Rogers V, Franklin S, et al: Targeted antagonism of galactosamine toxicity in normal rat hepatocytes in vitro. J Biol Chem 263:4719-4723, 1988. Jung G, Kohnlein W, Luders G: Biological activity of the antitumor protein neocarzinostatin coupled to a monoclonal antibody by N-succinimydyl 3-(2-pyridy1thio)-propionate. Biochem Biophys Res Commun 101:599-606, 1981. Wu G, Wu C: Delivery systems for gene therapy: Biotherapy 3:87-95, 199 1. Wilson J, Grossman M, Wu C, et al: Hepatocyte directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in LDL receptor-deficient rabbits. J Biol Chem (In press.) Marwick C: Gene replacement therapy enters second year. JAMA 266:2193, 1991. Marwick C: Pace of gene therapy picking up. JAMA 266:2668. 199 1.
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Strategies for Gene Therapy in the Liver
Gene therapy involves the transfer of normal, functional genetic material into cells to correct an abnormality due to a defective or deficient gene product.' Advances in molecular biology have led to the discovery of genetic defects in a variety of metabolic diseases, and the ability to clone the corresponding functionally normal genes.'Gene replacement or substitution involves removal of the entire gene or part of the mutant sequence and introduction of a normal gene or sequence necessary to correct the mutation.' This would be an ideal method for correcting single gene defects, especially those dominantly transmitted. Several laboratories have shown that gene substitution is possible but thus far on an inefficient Currently, most work is centered on gene augm e n t a t i ~ n .This ~ approach involves the introduction of DNA to express an absent gene product or modify the expression of a defective gene. In order to study the expression and regulation of foreign gene sequences, several techniques were developed-to introduce DNA into cells. These techniques include calcium phosphate coprecipitation,'-"' diethylaminoethyl (DEAE) dextran," electroporation,".13 scrape loading,I4 sonication,I4 particle bombardment,'5.'h microinjection, liposomes,"-'had ligandbased DNA carrier^.""^ Viral vectors have also been shown to be an effective mode of introducing DNA into The introduction of DNA by cell surcells in vitro.'.'"' face uptake is also referred to, in general terms, as transfe~tion.~ Another issue in gene therapy is the use of somatic versus germ cells. Correction of deficits at the germ cell level would intuitively seem ideal. This approach, however, has several problems. For example, there may be some degree of insertional mutagenesis with undesirable and unpredictable complication^.'^ Also, genetic manipulation of germ cells is currently the focus of ethical debate in that changes would be passed on to future generations and could alter normal human genetic variability. Because of these issues, much work has focused on the use of somatic cell lines for gene therapy.
From the Division of Gn.stroenterolojiy cirld Hepcrtolo~y.Utriversity c?fCor~rrc~ticut Hetilth Center. F~irtningtorr,Cotlt~ecticut. Reprint requests: Dr. G.Y. Wu, Division of Gastroenterology and Hepatology, AM044, University of Connecticut Health Center, Farmington, CT 06030.
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Fibroblasts, myocytes, renal cells, hematopoietic cells, and hepatocytes have all been successfully trans. ~limitation in many of these cell lines fected in ~ i t r o A is that secondary processing of the gene product is often necessary for activity and not all transfected cells may contain the cellular machinery to affect these alterations. Hepatocytes have received a great deal of attention because a wide variety of proteins are manufactured in the liver and therefore have the necessary cellular features to process these proteins. In addition, many genetic diseases result from an absence or deficiency of a hepatocyte-derived gene product. For these reasons, the liver is an attractive target organ for directed gene therapy. This article will detail current work in the transfer of DNA to hepatocytes in vitro, the advances made in the area of in vivo gene transfer, and the applicability of in vivo gene transfer to gene therapy in humans.
METHODS FOR IN VlTRO TRANSFECTION OF HEPATOCYTES Calcium Phosphate Coprecipitation Calcium phosphate coprecipitation with DNA was initially described in 1973.9This procedure involves precipitation of DNA with calcium phosphate to produce insoluble calcium phosphate DNA complexes that are internalized by endocytosis when they contact the cell surface. This results in delivery of many copies of the DNA of interest. The advantage of this system is that it is relatively easy to perform and is not cell-specific, although some cell lines are easier to transfect than others. The method can be used on any appropriate cell line and has been widely applied to hepatoma cell lines and in a few instances to primary hepatocytes. The disadvantage of this method is that although many copies of DNA are introduced into the cell, expression is, for the most part, transient. Stable transfection of the cell is inefficient, generally less than 1 %, and tends to be limited to continuous cell lines. Recently, stable transfection frequencies approaching 50% were reported when precipitates were formed slowly over a period of time by gradually increasing the pH in the incubation media.x A modification of this technique was used to obtain stable transfection of primary rat hepatocytes at frequencies up to 20%.'"
Copyright 0 1992 by Thieme Medical Publishers, Inc.. 381 Park Avenue South. New York. NY 10016. All rights reserved.
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While primarily limited to in vitro systems, there are reports of in vivo applications of the techniq~e.'~," In one report,'" a polyoma virus plasmid was precipitated with calcium phosphate. The precipitate was then injected directly into a mouse liver resulting in transient expression of the gene. In another study," investigators formed calcium phosphate precipitates with the genes for chloramphenicol acteyltransferase (CAT), a bacterial gene product, hepatitis B surface antigen, and human insulin. These were injected intraperitoneal in newborn mice with resulting transient expression of the gene products in the liver and spleen.
Diethylaminoethyl Dextran DEAE dextran and other compounds like it are large polycations that bind DNA electrostatically and are taken up by cells in much the same manner as calcium phosphate precipitates. This technique is basically a variation on the calcium phosphate method. It has been useful in improving the efficiency of transfection and in transfecting some cell lines less responsive to the calcium phosphate method. ' I
Electroporation Electroporation is a method whereby an electric pulse is transmitted to cells in culture that are incubated with DNA. The electrical pulse causes disruptions in the cell membranes allowing- entrance of the D N A . This generally results in transient expression of DNA at low frequencies. In primary hepatocyte cultures, cell viability is generally better than that reported for calcium phosphate or DEAE dextran."
Cell Sonication Cell sonication employs the use of high-frequency sound waves applied to cells in suspension with the DNA of interest. Sound waves result in transient disruption of the cell membranes allowing DNA to enter the cell. High rates of DNA transfer into cells are achieved with this method but expression is only transient. l 4
Scrape Loading Scrape loading is a technique whereby cells at confluence on a plate are mechanically scraped off in the presence of DNA in solution. As with cell sonication, this technique causes transient disruption of the cell membranes, allowing a high rate of DNA transfer into the cells. l 4
Viral-Mediated Transfection of Cells Viral agents have been demonstrated to be highly efficient vectors for the transfection of somatic cells. Retroviruses in particular have received a great deal of attention because they not only enter cells efficiently, but also provide a mechanism for stable integration into the host genome through the p r o v i r u ~ . ' ~Retroviral -~~ vectors have been used to introduce the P-galactosidase gene
into adult hepatocytes with expression of the marker gene approaching 25%.2"n another example, the gene for the LDL receptor was transferred into a primary culture of hepatocytes obtained from Watanabe hyperlipidemic rabbits with expression of the normal receptor.'* Retroviral vectors appear to be promising agents for gene transfer and expression. However, there have only been a small number of studies in v i ~ o . " , ' ~ Actively dividing cells are necessary for provirus integration. Also, obtaining sufficient titer of virus and the lack of targetability have limited in vivo application. Currently, investigators are attempting to address this last problem by use of organ-specific promoters and by chemically modifying the cell recognition sites on the viral envelope." In vivo application to hepatocytes has currently involved ex vivo transfection and subsequent reimplantation of the modified h e p a t ~ c y t e s . ' ~ .Details ~' of this technique are discussed elsewhere. Other viral vectors, such as vaccinia, adeno-associated, herpes, and papilloma viruses are also being studied. '.'X-40 The adeno-associated viruses (AAV) are particularly intriguing because of their dual life cycle. Alone, these viruses are nonpathogenic and exist in humans as an integrated provirus. However, in the presence of coinfection-with a helper virus, such as adenovirus or herpes virus, the AAV can become lytic and release high titers of viral progeny. Portions of the AAV genome can be deleted and DNA of interest inserted. The virus can then be introduced into a cell culture in the form of a provirus. High titers of the genetically modified provirus can then be produced by coinfection with a helper virus. The provirus produced can then be used to transfect cells of interest.?" With the recent discovery of defective forms of hepatitis B virus (HBV), there has been interest in the use of hepadnaviruses as hepatotropic vectors for the delivery of foreign genes. In vitro studies have shown that large portions of the hepadna viral genome are dispensable for helper-dependent packaging of foreign genes.4' This technique has been used to introduce the CAT gene, with stable expression of the gene product, into primary cultures of duck hepatocytes by use of a recombinant ' transfection, however, duck hepatitis v i r u ~ . ~Successful required coinfection with wild-type helper virus. Attempts at coinfection with a mutant helper virus was not successful in supporting expression of the CAT plasmid.
METHODS FOR GENE THERAPY FOR HEPATIC GENETIC DISEASE IN VlVO Particle Bombardment Particle bombardment is a unique modality for the delivery of DNA to both in vitro and in vivo systems. This method was first used successfully for the delivery of DNA to plant^.^' The technique involves the use of microspheres of an inert material such as gold or tungsten on which the plasmid DNA of interest is precipitated. These particles are then suspended on a Mylar film or similar arrangement and placed adjacent to two electrodes across which
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a high voltage discharge can be produced. The resulting electric arc provides the energy to accelerate the particles to high velocity into the desired tissue. By varying the voltage across the electrodes, and the size and density of the beads, the distribution of the particles in the target tissue can be c o n t r ~ l l e d . ~Utilizing ~.'~ this method, in vivo transfection of rat hepatocytes has been accomp l i ~ h e d . ~ ~Plasmid ." DNA was prepared using the bacterial gene for CAT along with a mammalian promoter. This enzyme is not found in mammalian cells and, thus, expression of the gene product would be a sensitive marker for a successful transfection. In two different experiments, CAT plasmids were coated onto microspheres. Rats then underwent laparotomy to expose a lobe of the liver. The plasmid-coated spheres were then "shot" into the exposed lobe, as already described. Animals were sacrificed at 24 hours in one experimentihand 48 hours in another.Is Liver samples were then taken from the area of bombardment and assayed for the presence of CAT gene expression. This was determined by demonstrating the appearance of the acetylated products of chloramphenicol. In both experiments, CAT gene expression was demonstrated. From these two studies, however, the duration of gene expression beyond 48 hours could not be determined because the animals were only sampled at one point in time. Stable transfection was obtained with this method in nonhepatocyte cell cultures at frequency of to l s This is similar to other methods such as calcium phosphate and electroporation. One of the problems with obtaining stable transfections is the need for actively dividing cells. When cells are dividing, there is a greater chance that the gene will be integrated into the host cell genome and the likelihood of persistent expression enhanced.44For this reason, stable transfections have been mainly reported in cell culture. With organs such as the liver, cell populations are generally stable and are not undergoing active replication. The liver, however, has a tremendous capacity for regeneration when there is loss of liver tissue or damage. Mechanical damage to liver tissue during particle bombardment might be beneficial to the transfection process because hepatocytes would be expected to undergo replication to repair the damage.
Liposomes Phospholipids placed in an aqueous solution can assume three possible forms: (1) micelles, which are small spherical structures with the hydrophilic heads of the molecules on the surface and the hydrophobic tails packed into the center; (2) lipid bilayers, which are sheets of phospholipids two molecules thick with the hydrophilic tails packed in the center; or (3) liposomes that are essentially spherical lipid bilayers. The latter are of interest because they form spontaneously in an aqueous medium and have been demonstrated to trap aqueous phase material. The size and shape of the liposomes can be varied by changing the mixture of phospholipids, degree of saturation of the fatty acid side chains, and the conditions under which the liposomes are formed.4s Liposomes are taken up by cells by endocytosis and this
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characteristic has been used to package, for example, drugs and enzymes for delivery to cell^.^'^^^ Liposomes have also been used to package DNA for delivery to cells in v i t r ~ . ~This ~ . ~ is' of interest because plasmids of large size could potentially be packaged and maintained in a soluble form that would allow direct application to in vivo systems by a simple intravenous injection. It has also been shown that these liposomes can be targeted by the selection of phospholipids present in the liposomal membrane or the addition of exposed sugar moieties on the surface of the liposome. Normally, liposomes injected into animals are preferentially taken up by reticuloendothelial cells. It has been shown, however, that small liposomes with a significant proportion of eggphosphatidyl choline in the membrane are preferentially taken up by hepatocytes when injected into rats.s0 The addition of side chains containing terminal galactose residues to the liposome has also been examined as a way of targeting liposomes to hepatocytes. This process can be inhibited by pretreatment of the animals with asialofetuin, a natural asialoglycoprotein, and is assumed to be mediated by surface receptors unique to h e p a t o c y t e ~ . ~ ' These asialoglycoprotein surface receptors recognize ligands with exposed terminal galactose residues and deliver the liposomes by endocytosis into clathrin-coated vesicles prior to fusion with lysosomes. DNA found in these coated vesicles has been demonstrated to be intact and can be transcribed when subsequently transfected ~ ' use of asialoglycoprotein reinto Escherichia c ~ l i . The ceptors for gene delivery is discussed in greater detail subsequently. Several investigators have taken advantage of the ability to package DNA into liposomes and selectively target them to the liver in vivo. 17-I' A plasmid containing the bacterial gene for neomycin resistance driven by the Rous sarcoma virus promoter was packaged into liposomes. These were then injected intravenously into rats. The neomycin resistance gene was chosen because this is a bacterial enzyme that is not made by mammalian cells and can be used to select cells possessing the gene. The presence of neomycin phosphotransferase activity in the cells could then be followed as an expression marker of the transferred gene. The animals were sacrificed at various time intervals and the liver homogenates assayed for neomycin phosphorylation activity. Activity was present at 4 hours and could still be detected 4 days after injection. Southern blot analysis of hepatocyte fractions showed no evidence of integration." In another example, the gene for rat preproinsulin was packaged into liposomes and again injected intravenously into rats. This is of interest in that the liver possesses the enzymes necessary to cleave precursor propeptides to the their active form. Serum from the animals was then analyzed for glucose levels, and livers and spleens were removed at various times. Serum glucose was demonstrated to fall in the treated animals during the first 10 hours, but then returned to levels above baseline by 18 hours. Elevated insulin levels appeared transiently in the liver and spleen, but by 18 hours were the same as control levels.s3 In a similar example, the gene for human insulin was introduced in vivo by intravenous injection of the lipo-
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some-encapsulated DNA along with a high molecular weight nuclear protein that binds to DNA and increases the amount of DNA reaching the nucleus of the cell. Expression of the gene was determined by assaying serum for circulating human insulin. Peak serum levels of human insulin were found 7 days after injection and persisted until about 10 days. Corresponding levels of RNA and DNA were also detected in the liver during this time period.'' A similar technique was also used to introduce the gene for hepatitis B surface antigen into rat livers in vivo. Expression of surface antigen peaked in the serum 2 days after injection and was detectable for 9 days. l 9 Of note, these investigators have also demonstrated a typical viral histologic inflammatory response in the livers of adult rats transfected with the gene for hepatitis B surface antigen.s4
Cell Surface Receptors
proteins6 to clathrin-coated vesicles that fuse with lysosomes where the ligand is degraded. The asialoglycoprotein receptor is then recycled back to the cell ~urface.'~ We have previously demonstrated that a variety of agents can be delivered specifically to hepatocytes by coupling them to asialoglycopr~teins.~~-~~ In these studies, agents were coupled to the asialoglycoprotein carrier by covalent bonds. 6' However, this method of coupling to nucleic acids could potentially alter them. To solve this problem, a complex was created that consisted of a polycation, poly L-lysine, covalently bound to asialoorosomucoid, a naturally occurring asialoglycoprotein with a high affinity for the receptor. The polycation could then bind the negatively charged DNA in an electrostatic, nondamaging manner, as illustrated in Figure 1.
Evidence for In Vitro Expression in Hepatocytes
Targeting of DNA to somatic cells by way of cell surface receptors is another potential method of gene therapy. Hepatocytes are uniquely suited to this form of receptor-mediated transfection. As opposed to some of the other methods of transfecting somatic cells, DNA delivery via cell surface receptors is applicable to both in vitro and in vivo systems. Hepatocytes are attractive because they possess cell surface receptors that are able to recognize and bind circulating galactose-terminal glycoproteins (asialoglycopr~teins).~~ Binding of the glycoprotein to the asialoglycoprotein receptor results in endocytosis of the receptor ligand complex and delivery of the glyco-
To determine whether the asialoorosomucoid-polylysine-DNA (AsOR-PL-DNA) complex could be delivered to hepatocytes, two continuous cell lines, HepG2 and SK-Hep 1, were used. HepG2 cells possess substantial asialoglycoprotein receptor activity on their surfaces and SK-Hep 1 cells do not. Each cell line was incubated with AsOR-PL-DNA complex or controls consisting of the various components of the complex. Cells were then assayed for activity of the CAT gene p r o d ~ c tNo . ~ CAT activity was detected with either the AsOR-PL-DNA or any of the controls in receptor-negative SK-Hep I cells. With receptor-positive HepG2 cells, CAT activity was
COMPONENTS OF A TARGETABLE DNA COMPLEX
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6 Polycation Covalent Bond Ligand
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FIG. 1. Soluble DNA carrier complex. Poly L-lysine is covalently bound to the carrier asialoglycoprotein that is the part of the complex that provides recognition for binding and internalization by the hepatocyte. DNA to be delivered is then electrostatically bound to the carrier.
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found in the cells incubated with the AsOR-PL-DNA complex, but not in the controls. Furthermore, the addition of excess AsOR to the media of HepG2 cells incubated with AsOR-PL-DNA inhibited the development of CAT activity in the cells. These data prove that DNA can be introduced into hepatocytes via the asialoglycoprotein receptor with resultant expression of the foreign gene. 2'.24 Similarly, we have also demonstrated that an ecotropic viral vector can be targeted to liver cells by chemically modifying the protein coat of the virus with lactose residue^.'^ Attachment of lactose residues to the viral coat results in exposed galactose molecules, in effect, creating a synthetic ASG. In this study, the P-galactosidase gene was introduced into a replication defective Moloney murine leukemia virus, an ecotropic viral vector. As would be expected, murine cell lines were infected by the unmodified virus, but not human SK-Hepl (asialoglycoprotein receptor-negative) or human HepG2 (asialoglycoprotein receptor-positive) cells. When modified virus was used, receptor-positive human HepG2 cells were successfully transfected with resultant gene expression. Attempts to transfect receptor-negative SKHepl cells with the modified virus were unsuccessful. As additional proof that the entry of the modified virus was via the asialoglycoprotein receptor, transfection of HepG2 cells could be competitively inhibited by addition of an excess of AsOR. This study demonstrated that viral vectors could be chemically modified to target them potentially to a specific organ. Furthermore, modification of the viral vector did not affect subsequent expression of the foreign gene.
Evidence for In Vivo Targeting and Expression in Hepatocytes To demonstrate that DNA could be targeted in vivo by a soluble DNA carrier system, rats were injected intravenously with a plasmid consisting of the bacterial CAT gene with a viral promoter, complexed to the asialoglycoprotein carrier system. Animals were sacrificed 24 hours later and liver homogenates assayed for CAT activity. Only those animals injected with the intact complex exhibited CAT activity. No evidence for CAT gene expression was seen in any of the animals injected with the various controls. This expression was also shown to be liver-specific, as determined by assaying other organs for CAT gene activity.'" A targeted foreign gene driven by mammalian regulatory elements can also result in persistent gene expression. In rats injected with the DNA complex without partial hepatectomy, the presence of detectable gene expression peaked between 24 and 48 hours but fell to undetectable levels over approximately 4 days. However, in rats that had two thirds hepatectomy, persistent CAT gene expression was achieved through 1 1 weeks." These results indicated that foreign genes could be targeted in vivo with persistent expression of the gene. A more detailed review of these studies has been previously published. h2
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Application of Targeted Gene Delivery to Gene Deficiency Models A major goal of targeted delivery of foreign DNA in vivo is to cure diseases resulting from deficiency of a gene product by introducing the normal gene into the host genome. To test the feasibility of this strategy, Nagase analbuminemic rats were employed. These animals have an mRNA splicing defect that results in virtually undetectable levels of serum albumin. A plasmid was constructed with the gene for human albumin driven by mouse enhancer-rat albumin promoter sequences. This plasmid was complexed to the asialoglycoprotein carrier and injected intravenously. The animals were then subjected to two thirds partial hepatectomy" and serum was assayed for albumin by Western Blot using a specific antibody to human albumin. By 48 hours, human albumin could be detected in the serum at a level of 0.05 pglml. By 2 weeks, this had increased to a maximum of 34 pglml and remained stable for 4 weeks.2' Results are shown in Figures 2 and 3. Analysis of cell lysates 2 weeks post-transfection showed that a majority of the plasmid DNA existed as an episomal, nonintegrated form. However, infrequent integration could not be excluded. In another example, the Watanabe heritable hyperlipidemic rabbit was used. This animal is hypercholesterolemic secondary to genetically defective LDL receptors and serves as an animal model for human familial hypercholesterolemia. A plasmid was constructed containing the gene for the human LDL receptor driven by a mouse albumin promoter and enhancer. This plasmid and a control plasmid construct using the CAT gene were then complexed to the soluble DNA carrier and injected into separate groups of rabbits in a two treatment crossover experiment. Serum was assayed for cholesterol levels at various time points (Fig. 4). At the end of 12 days, the same animals were again injected, this time with the other plasmid complex. Analysis of liver homogenates 10 minutes postinjection revealed about 1000 copies of the plasmid per cell. This rapidly decreased to less than 0.1 copies per cell after 48 hours. Expression of low density lipoprotein (LDL) receptor mRNA reached a maximum expression at approximately 24 hours after transfection and was estimated to be about 2 to 4% of normal. This expression was functionally correlated to a 25 to 30% reduction in serum cholesterol that was specific for the LDL receptor plasmid. The reduction, however, was transient and by 5 days had returned to baseline levels.h3 While complete normalization of serum cholesterol was not obtained in this experiment, the 30% decrease that was obtained would be clinically significant.
FUTURE OF GENE THERAPY The ultimate objective in gene therapy is its application to human disease states. At the present time, several studies are already underway and others have received Food and Drug Administration appro~al.~~.~%All these trials are utilizing retroviruses and ex vivo trans-
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FIG. 2. Western blot of serum samples obtained 2 weeks postinjection and partial hepatectomy. Lane 1 shows the electrophoretic m~grationof-standard human albumin identified bv immunoblottina with s~ecificantibodv. Lane 2 shows an identical amount of standard rat serum albuminto demonstrate a lack of cross-reactivity with the human albumin antibody. Lane 3 shows standard human albumin mixed with untreated analbuminemic rat serum to demonstrate identical migration and detection with the antibody. Lanes 4 and 5 show untreated Sprague-Dawley rat serum and analbuminemic rat serum, respectively, to demonstrate the absence of detectable protein with the human albumin antibody. Lane 6 shows human albumin in serum from an analbuminemic rat treated with the DNA carrier complex; lane 7 shows the absence of serum albumin when the vehicle (saline) alone was injected; and lane 8 shows the absence of human serum albumin when an enhancer-less plasmid complex was injected.
FIG. 3. Presence of serum albumin in a Nagase analbuminemic rat after transfection in vivo with the gene for human albumin using a soluble DNA-carrier system. Lanes 1 to 3 contain human albumin standards of increasing concentration. At 24 hours, albumin was not detectable, as shown in lane 4. Lane 5 demonstrates detectable human albumin in the serum of analbuminemic rats at a level of 0.05 wliml 48 hours post-transfection and partial hepatectomy. The concentration of albumin increased to 34 pgiml by 2 weeks and persisted at this level through 4 weeks (lanes 5 to 11).
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FIG. 4. Effect of hepatocyte targeted low density lipoprotein (LDL) receptor or chloramphenicol acetyltransferase (CAT) genes on serum cholesterol in Watanabe heritable hyperlipidemic rabbits. Animals shown in A to C received LDL receptor plasmid on d a y zero a n d CAT plasrnid on day 12. Animals D to F received CAT plasmid on day zero a n d LDL receptor plasrnid on day 12. A d e c r e a s e in serum cholesterol w a s s e e n only in t h e animals injected with t h e LDL receptor DNA complex. T h e maximum d e c r e a s e w a s seen after about 2 d a y s a n d by 5 d a y s had returned t o baseline.
fection. Although there are still many questions to be answered and problems to overcome, the future of gene therapy appears bright. This aspect of molecular medicine, which would have been considered science fiction a decade ago, will likely become part of the clinician's therapeutic armamentarium in the not too distant future. Acknowledgments. We gratefully acknowledge the collaboration and advice of Dr. David A. Shafritz, Dr. James M. Wilson, and Mariann Grossman on work presented in this manuscript. This work was supported in part by the National Institutes of Health grants CA468O 1 and DK42 182, Research Career Development Award CAOl110, and March of Dimes Grant 1-90-421 to G.Y.W., and a grant from TargeTech, Inc., to C.H. W.
REFERENCES 1.
Friedmann T: Progress toward human gene therapy. Science 244:1275-1281, 1989.
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Behart J , Caskey C: Developments leading to human gene therapy. In Kucherlapati R (Ed): Gene Transfer. New York, Plenum Press, 1986, pp. 41 1-435. Danks D, Cotton R: Future developments in phenylketonuria. Enzyme 38:296-301, 1987. Wilson J: Prospects for gene therapy of familial hypercholesterolemia. Mol Biol Med 7: 223-232, 1990. Frohman M, Martin G: Cut, paste and save: New approaches to altering specific genes in mice. Cell 56: 145-147, 1989. Doetschman T, Gregg R, Maeda N, et al: Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330576-78, 1987. Ledley F: Clinical application of somatic gene therapy in inborn errors of metabolism. J Inher Metab Dis 13:597-616, 1990. Chen C , Okayama H: High-efficiency transfection of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745-2752, 1987. Graham F, Van Der Eb A: A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456567, 1973. Rippe R, Brenner D, Leffert H: DNA-mediated gene transfer into adult rat hepatocytes in primary culture. Mol Cell Biol 10:689-695, 1990. Gopal T: Gene transfer method for transient gene expression, stable transfection, and co-transfection of suspension cell cultures. Mol Cell Biol 5: 1 188-1 190, 1985. Tur-Kaspa R, Teicher L, Levine B, et al: Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes. Mol Cell Biol 6:716-718, 1986. Potter H, Weir L, Leder P: Enhancer-dependent expression of human k immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc Natl Acad Sci USA 81:7161-7165, 1984. Fechheimer M, Boylan J , Parker S , et al: Transfection of mammalian cells with plasmid DNA by scrape loading and sonication loading. Proc Natl Acad Sci USA 84:8463-8467, 1987. Yang N, Burkholder J , Robert B, et al: In vivo and in virro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci USA 87:9568-9572, 1990. Zelenin A, Alimov A. Titomirov A, et al: High-velocity mechanical DNA transfer of the chloramphenicol acetyltransferase gene into rodent liver, kidney and mammary gland cells in organ explants and in vivo. FEBS Lett 280:94-96, 1991. Leibiger B, Leibiger I, Sarrach D, Zuhlke H: Expression of exogenous DNA in rat liver cells after liposome-mediated transfection in vivo. Biochem Biophys Res Commun 174: 1223-1231, 1991. Kaneda Y, lwai K , Uchida T: Introduction and expression of the human insulin gene in adult rat liver. J Biol Chem 264: l2I26-12l29, 1989. Kato K, Nakanishi M, Kaneda Y, et al: Expression of adult hepatitis B virus surface antigen in adult rat liver. J Biol Chem 266:3361-3364, 1991. Wu G, Wu C: Receptor-mediated gene delivery and expression in vivo. J Biol Chem 263: 14621-14624, 1988. Wu C, Wilson J , Wu G: Targeting genes: Delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J Biol Chem 264:16985-16987, 1989. Wu G, Wilson J , Shalaby F, et al: Receptor-mediated gene delivery in vivo. J Biol Chem 266: 14338-14342, 1991. Wu G, Wu C: Evidence for targeted gene delivery to HepG2 hepatoma cells in vitro. Biochemistry 27:887-892, 1988. Wu G, Wu C: Receptor-mediated in virro gene transfections by a soluble DNA carrier system. J Biol Chem 262:4429-4432, 1987. Wilson J , Jefferson D, Chowdhury J , et al: Retrovirus-me-
Downloaded by: University of British Columbia. Copyrighted material.
SEMINARS IN LIVER DISEASE-VOLUME
WU, WU
diated transduction of adult hepatocytes. Proc Natl Acad Sci USA 85:3014-3018, 1988. Ledley F, Darlington G, Hahn T, Woo S: Retroviral gene transfer into primary hepatocytes: Implications for genetic therapy of liver-specific functions. Proc Natl Acad Sci USA 8453355339, 1987. Wolff J, Yee J, Skelly H, et al: Expression of retrovirally transduced gene in primary cultures of adult rat hepatocytes. Proc Natl Acad Sci USA 84:3344-3348, 1987. Armentano D, Thompson A, Darlington G, Woo S: Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene therapy of hemophilia B. Proc Natl Acad Sci USA 87:6141-145, 1990. Palmiter R, Brinster R: Transgenic mice. Cell 41:343-345, 1985. Dubensky T, Campbell B, Villarreal C: Direct transfection of viral and plasmid DNA into the liver or spleen of mice. Proc Natl Acad Sci USA 8 l:7529-7533, 1984. Benvenisty N, Neshif L: Direct introduction of genes into rats and expression of the genes. Proc Natl Acad Sci USA 83:955 1-9555, 1986. Wilson J, Johnston D, Jefferson D, Mulligan R: Correction of the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci USA 85:44214425, 1988. Hatzoglou M, Lamers W, Fatima B, et al: Hepatic gene transfer in animals using retroviruses containing the promoter from the gene phosphoenolpyruvate carboxykinase. J Biol Chem 265: 17285-17293. 1990. Ferry N, Duplessis 0,Houssin D, et al: Retroviral-mediated gene transfer into hepatocytes in vivo. Proc Natl Acad Sci USA 88:8377-8381, 1991. Neda H, Wu C, Wu G: Chemical modification of an ecotropic murine leukemia virus results in redirection of its target cell specificity. J Biol Chem 266: 14143-14146, 1991. Wilson J, Chowdhury N, Grossman M, et al: Temporary amelioration of hyperlipidemia in low density lipoprotein receptordeficient rabbits transplanted with genetically modified hepatocytes. Proc Natl Acad Sci USA 87:8437-8441. 1990. Fuller B: Transplantation of isolated hepatocytes: J Hepatol 7:368-376, 1988. Coupar B, Andrew M, Boyle D: A general method for the construction of recombinant vaccinia virus expressing multiple foreign genes. Gene 68:l-10, 1988. Hermonat P, Muzyczka N: Use of adeno-associated virus as a mammalian DNA cloning vector: Transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci USA 81:6466-6470, 1984. Rasmussen C, Simmen R, MacDougall E, Means A: Methods for analyzing bovine papilloma virus based calmodulin expression vectors. Methods Enzymol 139:642-654, 1987. Horwich A, Furtak K, Pugh J, Summers J: Synthesis of hepadnavirus particles that contain replication-defective duck hepatitis B virus genomes in cultured HuH7 cells. J Virol 64:642-650, 1990. Chang C, Ganem D, Lavine J: Foreign gene delivery and expression in hepatocytes using a hepatitis B virus vector. Hepatology 14: l24A, 199 1 . Klein T, Wolf E, Wu R, Sanford J: High velocity microprojectiles for deliveries nucleic acids into living cells. Nature 327:70-73, 1987. Varmus H, Padgette T, Heasley S , et al: Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA. Cell 11:307-319, 1977. Szoka F, Papahadjopoulos D: Procedure for preparations of li-
posomes with large internal aqueous space and high capture by reserve phase evaporation. Proc Natl Acad Sci USA 75:41944198, 1978. Gregoriadis G , Leathwood P, Ryman B: Enzyme entrapment in liposomes. FEBS Lett 14:95-99, 1971. Gregoriadis G, Ryman B: Fate of protein containing liposomes injected into rats: An approach to the treatment of storage diseases. Eur J Biochem 2 4 : 4 8 5 4 9 l , 1972. Wong T, Nicolau C, Hofschneider P: Appearance of p-lactamase activity in animal cells upon liposome mediated gene transfer. Gene 10:87-94, 1980. Nicolau C, Sene C: Liposome mediated DNA transfer in eukaryotic cells. Biochim Biophys Acta 721: 185-90, 1982. Spanjer H, VanGalen M, Roerdink F, et al: Intrahepatic distributor of small unilamellar liposomes as a function of liposomal lipid composition. Biochim Biophys Acta 863:224-230, 1986. Ghosh P. Bachhawat B: Targeting of liposomes to hepatocytes. In: Wu G, Wu C (Eds): Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. New York, Marcel Dekker, 199 1 . Nandi P, Legrand A, Nicolau C: Biologically active recombinant DNA in clathrin-coated vesicles isolated from rat livers after in vivo injection of liposome encapsulated DNA. J Biol Chem 261: 16722-16726, 1986. Nicolau C , Legrand A, Grosse E: Liposomes as carriers for in vivo gene transfer and expression. Methods Enzymol 149: 157176, 1987. Kato K , Kaneda Y, Sakurai M, et al: Direct injection of hepatitis B virus into liver induced hepatitis in adult rats. J Biol Chem 266:22O7 1-22074, 1991. Ashwell G, Morell A: Rule of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymo141:99-128, 1974. Wall D, Wilson G, Hubbard A: The galactose specific recognition system of mammalian liver: The route of ligand internalization in rat hepatocytes. Cell 21:79-83, 1980. Schwartz A: Trafficking of asialoglycoproteins and the asialoglycoprotein receptor. In: Wu G, Wu C (Eds): Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. New York, Marcel Dekker, 1991. Wu G, Wu C, Rubin M: Acetaminophen hepatotoxicity and targeted rescue: A model for specific chemotherapy of hepatocellular carcinoma. Hepatology 5:709-713, 1985. Wu G, Wu C, Stockert R: Model for specific rescue of normal hepatocytes during methotrexate treatment of hepatic malignancy. Prod Natl Acad Sci USA 80:3078-3080, 1983. Wu G, Keegan-Rogers V, Franklin S, et al: Targeted antagonism of galactosamine toxicity in normal rat hepatocytes in vitro. J Biol Chem 263:4719-4723, 1988. Jung G, Kohnlein W, Luders G: Biological activity of the antitumor protein neocarzinostatin coupled to a monoclonal antibody by N-succinimydyl 3-(2-pyridy1thio)-propionate. Biochem Biophys Res Commun 101:599-606, 1981. Wu G, Wu C: Delivery systems for gene therapy: Biotherapy 3:87-95, 199 1. Wilson J, Grossman M, Wu C, et al: Hepatocyte directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in LDL receptor-deficient rabbits. J Biol Chem (In press.) Marwick C: Gene replacement therapy enters second year. JAMA 266:2193, 1991. Marwick C: Pace of gene therapy picking up. JAMA 266:2668. 199 1.
Downloaded by: University of British Columbia. Copyrighted material.
STRATEGIES FOR GENE THERAPY-VERSLAND,
