Gene therapy as an alternative to liver ... - Wiley Online Library

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Gene Therapy as an Alternative to Liver Transplantation Betsy T. Kren,* Namita Roy Chowdhury,‡ Jayanta Roy Chowdhury,‡ and Clifford J. Steer*† Liver transplantation has become a well-recognized therapy for hepatic failure resulting from acute or chronic liver disease. It also plays a role in the treatment of certain inborn errors of metabolism that do not directly injure the liver. In fact, the liver maintains a central role in many inherited and acquired genetic disorders. There has been a considerable effort to develop new and more effective gene therapy approaches, in part, to overcome the need for transplantation as well as the shortage of donor livers. Traditional gene therapy involves the delivery of a piece of DNA to replace the faulty gene. More recently, there has been a growing interest in the use of gene repair to correct certain genetic defects. In fact, targeted gene repair has many advantages over conventional replacement strategies. In this review, we will describe a variety of viral and nonviral strategies that are now available to the liver. The ever-growing list includes viral vectors, antisense and ribozyme technology, and the Sleeping Beauty transposon system. In addition, targeted gene repair with RNA/DNA oligonucleotides, small-fragment homologous replacement, and triplex-forming and single-stranded oligonucleotides is a long-awaited and potentially exciting approach. Although each method uses different mechanisms for gene repair and therapy, they all share a basic requirement for the efficient delivery of DNA. (Liver Transpl 2002;8:1089-1108.)

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iver transplantation has become a successful method in treating end-stage liver disease and has led to a dramatic increase in the number of prospective patients. Thousands of liver transplants are performed worldwide, and about half of those are performed in the United States. The 1- and 5-year survival rates in the United States are 85% to 90%, and 70% to 75%, respectively. Although the number of cadaver donors is relatively constant, increased demand has led to an overall shortage of organs, and more patients are dying while awaiting transplantation. To overcome this problem, innovative surgical approaches have been developed to address the organ shortage, including split livers and living donor transplants. In addition to acute and chronic liver failure, hepatic transplantation can correct a variety of inborn errors of metabolism, such as familial amyloidosis, hereditary oxalosis, ␣1-antitrypsin deficiency, Wilson’s disease, tyrosinemia, type I and IV glycogen storage diseases, Niemann-Pick disease, Crigler-Najjar syndrome type I,

urea cycle enzyme deficiencies, C protein deficiency, and hemophilias A and B. In many of these cases, the liver architecture remains normal; therefore, the prospect of circumventing the need for liver transplantation by replacing or correcting the errant gene is an attractive prospect. The last decade has witnessed great advances in our fundamental understanding of the genetic basis for many of these diseases. The new era of gene therapy undoubtedly will impact the role of liver transplantation in treating inborn errors of metabolism and a variety of other genetic disorders. The potential targets for genomic modification have increased considerably and include chronic viral diseases as well as diverse disorders involving the cell cycle, apoptotic cascade, and the immune system. A major barrier to all types of gene therapy is the lack of a safe and effective delivery system. Viral vectors such as adenovirus, adeno-associated virus, and retroviruses are commonly used for a variety of applications.1-3 However, their successful clinical application is hampered by the difficulty and expense of production. Additional potential problems include intrinsic toxicity of the viral proteins, host immune response against the vectors or transgene products, or both. The genomic size constraints imposed by the vectors also can influence significantly the choice of expression system and its regulatory elements. These problems have led investigators to explore nonviral nucleic acid transfer systems. From the Departments of *Medicine and †Genetics, Cell Biology, and Development, University of Minnesota School of Medicine, Minneapolis, MN; and the ‡Departments of Medicine and Molecular Genetics, and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY. Supported in part by grants P01-HD32652 (B.T.K.), R01DK39137 (N.R.C.), R01-DK46057 (J.R.C.), and P01-HL655578 and P01-HL55552 (C.J.S.) from the National Institutes of Health. Address reprint requests to Clifford J. Steer, MD, Department of Medicine, Mayo Mail Code 36, Mayo Building, Room A536, 420 Delaware Street SE, Minneapolis, MN 55455. Telephone: 612-6246648; FAX: 612-625-5620; E-mail: [email protected] Copyright © 2002 by the American Association for the Study of Liver Diseases 1527-6465/02/0812-0001$35.00/0 doi:10.1053/jlts.2002.36844

Liver Transplantation, Vol 8, No 12 (December), 2002: pp 1089-1108

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The main objectives of gene therapy to the liver are 1. gene augmentation; 2. expression of pharmacologically active products, such as suicide genes for adjuvant cancer therapy; 3. interference or extinction of gene expression; 4. expression of gene products not normally produced in the liver, such as hormones and immunomodulatory factors, and 5. targeted in situ gene repair under endogenous regulation. In addition to the myriad hepatic disorders, the liver has many other attributes that make it a unique target for gene therapy. The capillary fenestrae are prominent in the liver sinusoids and allow passage of complexes less than 100 nm in size into the space of Disse. The high volume of venous outflow ensures adequate biodistribution of a transgene product. The asialoglycoprotein receptor is unique to hepatocytes and expressed in high copy number at the plasma membrane,4 providing a mechanism for hepatocyte-specific delivery. The liver may be targeted in situ or hepatocytes may be isolated, treated ex vivo and then returned to the host. In fact, the later approach initially was used to correct animal models of hypercholesterolemia.5 Gene modification may even find a therapeutic role in liver transplantation. For example, short-term expression of superoxide dismutase6 may prevent acute ischemia, whereas longer-term expression of immunomodulatory molecules may reduce organ rejection and improve tolerance.7,8 Gene replacement involves delivery and sustained expression of a transgene in the target cell. In comparison, gene repair is directed precisely at the genetic mutation, thereby correcting the defect in situ and allowing the corrected gene to be regulated endogenously. Gene targeting strategies are associated with a number of advantages: 1. Site-specific repair can address both recessive and dominant disorders; 2. gene repair uses small, synthetic molecules that are less expensive and easier to produce than transgene complexes; 3. correction implies that the target gene remains under endogenous control; 4. gene augmentation may lead to random genomic integration and the potential for insertional mutation; 5. targeted repair strategies do not typically induce an immunologic response or alter endogenous promoter function; and 6. a repair process may require only a single treatment. It has recently been determined that viral-mediated gene integration can result in significant changes in genome-wide methylation,9 which could result in epigenetic gene silencing and tumorigenesis.10,11 Despite the obvious advantages of gene repair, only relatively low levels of gene correction have been achieved. Successful targeting approaches require pre-

cise knowledge of the target loci and, thus, would have to be tailored to an individual’s specific mutation (i.e., designer gene repair). This article will review the current status of both viral and nonviral gene therapy and gene repair in the treatment of liver diseases. We will discuss strategies from conventional viral-mediated gene transfer to the awakening of Sleeping Beauty to site-specific repair of genetic sequences.

Viral Vectors for Gene Transfer Into Hepatocytes In Vitro and In Vivo Mammalian viruses have specific mechanisms to enter the nucleus of the host cell, express viral genes, and replicate. Some viruses integrate their DNA genome, or the reverse-transcribed DNA product of their RNA genome, into the host chromosomes. For some viruses, integration into the host genome is required for viral gene expression. Others may express their genes with or without integration, whereas some viruses have evolved to remain episomal throughout their life cycle. These characteristics are exploited for transgene delivery by packaging the target DNA into a viral genome. The wild-type virus is made replication defective by deleting most of their genes or a critical transactivating gene. An ideal recombinant virus for gene transfer into the liver or cultured hepatocytes should be (1) able to efficiently infect quiescent cells, (2) integrate into the host genome, (3) possible to generate at high titer, (4) have a large “gene stuffing” space, (5) non-toxic, and (6) capable of being administered repeatedly. However, none of the recombinant viruses that are currently available for liver-directed gene therapy fulfills all these requirements. The viral vectors commonly used for gene transfer into cultured hepatocytes and intact liver is a relatively small group. Recombinant Retroviruses Retroviruses contain RNA genomes that are reversetranscribed into complementary DNA (cDNA) after entering the cell. The double-stranded cDNA provirus integrates into the host genome. Integration is required for the expression of retroviral genes. Recombinant retroviruses can be grown at relatively high titers, can accept large DNA inserts, and do not elicit significant host immune response. Parts of the long terminal repeats (LTRs) of the virus that contain the viral promoters and enhancers can be deleted, so that the viral LTR is self-inactivated (SIN) during replication. The target gene is expressed from a separate internal promoter. Although this strategy had been developed to

Gene Therapy for Liver Diseases

reduce the risk of insertional activation of host oncogenes, removing the enhancer may not be sufficient to achieve this objective. However, because the major portion of the mammalian genome does not contain functional genes, the risk of tumorigenesis by chance activation of oncogenes is, in fact, very small. Conventionally, retroviral vectors are engineered from Moloney’s murine leukemia virus (MoMuLV). In a recombinant plasmid, the viral genes are replaced by the target gene, but the packaging signal (␺) is kept intact. The recombinant plasmid is transfected into packaging cells, which contain stable integrations of the viral genes and provide the necessary viral proteins in trans. A prototypical method of generating recombinant MoMuLV is summarized in Figure 1. Recombinant MoMuLV has been used both for ex vivo gene transfer into isolated hepatocytes5 and for in vivo gene transfer to the liver by ex sanguineous perfusion of the liver with this preparation.12 However, the efficiency of gene transfer into hepatocytes by MoMuLV is low, because this type of retrovirus requires cell division for integration and hepatocytes do not divide frequently. Thus, to achieve high transduction efficiency, the host needs to be manipulated to induce mitosis. For these reasons, another group of retroviruses, termed lentiviruses have been engineered for gene transfer (Fig. 2). After entering the cell and reverse transcription of the viral RNA genome, the cDNA version of the lentiviral genome becomes incorporated in a

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preintegration complex (PIC), which is transported into the nucleus without the need for resolution of the nuclear envelope. Thus, recombinant lentiviruses can transduce cells that are not undergoing cell division. However, although gene transfer into the intact liver by lentiviral vectors has been shown, experimental results indicate that efficient lentiviral transduction of the hepatocytes requires cell cycling in vivo.13,14 For example, serum levels of human coagulation factor IX were 4- to 6-fold higher after lentiviral gene transfer in partially hepatectomized mice than in nonhepatectomized recipients.15 To expand the host cell range of the vectors and to stabilize the recombinant virus, the retroviral envelope has been replaced by the VSV-G protein. This permits concentration of the virus by centrifugation.13 Adenoviral Vectors Recombinant adenoviruses are highly efficient in delivering genes to the liver. On systemic administration, they localize predominantly to the liver16,17 and transduce both dividing and nondividing cells. In sufficient doses, these agents can transduce over 90% of all hepatocytes of the liver and so have been used extensively for in vivo hepatic gene transfer. Recombinant adenoviruses are usually generated from human adenovirus types 5 and 2. The target transgene disrupts the E1 region that encodes transactivating factors required for viral gene expression. The recombinant viruses are generated at high titers in cell lines that

Figure 1. Generation of a recombinant MoMuLV. The packaging cell is transfected stably with multiple DNA segments expressing different viral proteins (gag, pol, and env). All viral genes are replaced by a target transgene in a plasmid containing the cDNA of the viral genome, keeping the packaging signal (␺) intact. The recombinant plasmid is transfected into a packaging cell that provides the viral proteins in trans. The integrated recombinant viral DNA gives rise to the recombinant viral RNA genome that can be assembled with the viral proteins because of the presence of the packaging signal. The assembled recombinant virus, which is secreted into the culture medium, can infect other cells but cannot replicate because it lacks the viral genes.

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Figure 2. Generation of a VSV-pseudotyped lentivirus. A typical first generation lentivirus is generated by cotransfecting three plasmids into packaging 293T cells. A helper plasmid expresses gag, pol, and other accessory proteins required for generating the virus, excepting env. The helper construct lacks the viral LTR and the packaging signal ␺, whereas a CMV promoter drives expression. A second plasmid expresses the VSV-G protein, which serves as the envelope of the pseudotyped recombinant lentivirus, also from a CMV promoter. A third plasmid is the transducing vector, which contains cis-acting sequences of HIV-1 that are necessary for reverse transcription and integration (3’ and 5’ LTR’s and primer-binding site), the packaging sequence (␺), splice signals, Rev response elements (RRE) for nuclear export, and the transgene sequence. After transient transfection, the expressed recombinant mRNA containing the transgene is packaged with the viral proteins expressed from the other two plasmids and the assembled recombinant virus buds out of the packaging cell into the media.

provide E1 encoded proteins in trans. Recombinant adenoviruses can accept large transgene inserts, and deleting several or all of the viral genes increases the space further. Despite the above characteristics, some major shortcomings limit the utility of these agents. Adenoviruses are episomal and, thus, the transgene expression is limited in duration. In addition, the adenoviral protein evokes strong humoral and cell-mediated immune responses in the host. This leads to a rapid elimination of the vector and cytotoxic lymphocyte-mediated liver damage, precluding repeated administration. A large segment of the general population becomes naturally exposed to adenoviral infections and, therefore, has pre-existing immunity against adenoviral proteins. Injection of recombinant adenoviruses could result in a rapid and strong booster response, which can be potentially life-threatening. The danger posed by acquired or innate immune response to recipients of adenoviral vectors was exemplified by the recent tragic death of a young man with ornithine transcarbamylase deficiency. After receiving a large dose of a recombinant adenovirus through the hepatic artery, he developed acute liver injury and died from multiorgan failure. Strategies for abrogation of the immune response by

modification of the host or the vector have been explored. Administration of recombinant adenovirus into newborn rats results in life-long tolerance to the viral antigens, permitting repeated administration.17 Tolerance has been induced also by injecting adenoviral proteins into the thymus of young adult rats.18 More interestingly, oral feeding of adenoviral proteins in low doses results in tolerance to the viral proteins, permitting repeated administration both in naive hosts and those that are preimmunized against adenoviral antigens.19 Alternatively, coadministration of a strong immunosuppressive agent, such as tacrolimus, for 3 days around the time of administration of adenoviruses prevents both cellular and humoral immune responses.20 However, this procedure produces immune ignorance, rather than tolerance. Thus, to maintain this ignorance, the immunosuppressing agent must be administered with each dose of the adenoviral vector. Modification of the vector also has been used to reduce its immunogenecity. Viral structural genes have been deleted to prevent any viral gene expression in vivo. The genome of these recombinant adenoviral vectors contain only the necessary cis-acting elements, including the two origins of DNA replication at the

Gene Therapy for Liver Diseases

physical ends of the viral genome and the packaging sequences within the first 500 base pairs of the left-hand end.21 Deletion of large segments of the viral DNA also provides increased target gene stuffing space in these so called gutless vectors. These fully viral gene-deleted vectors require helper adenoviral infection for provision of the viral proteins. Subsequently, the helper virus is removed by gradient centrifugation, although some contamination by replication-competent adenovirus cannot be avoided. However, despite deletion of all viral genes, these vectors retain a degree of immunogenicity because of the antigenic viral proteins provided in trans in the producer cells.22 In most adenoviral vectors, viral proteins are not expressed because of the disruption of the E1 region, an actual deletion of the viral genes, or both. However, the adenoviral E3 gene encodes proteins that can downregulate immune attack against adenoviral-infected cells. Recombinant viruses that express the immunomodulatory E3 proteins through a separate promoter have been constructed. These vectors express transgenes for a longer duration and can be administered at least twice.23 Another strategy involves construction of adenoviral vectors that coexpress the immunomodulatory protein CTLA4Ig along with the target gene. CTLA4Ig inhibits the costimulatory interaction between the antigen presenting cells and the cytotoxic T-lymphocytes. These modified vectors permit high levels of transgene expression for at least 6 months after a single dose and can be readministered multiple times.24 Although long-term gene transfer is now possible by abrogating the immune response against adenoviral vectors, other significant issues have arisen. In particular, the adenoviral receptor on the mammalian cell surface (CAR) is a transmembrane protein of the tight junction complex25,26 that is highly expressed in murine and rodent hepatocytes, but to a much lower extent in human hepatocytes. In fact, a recent clinical trial has shown very low levels of expression of adenovirus-mediated transgenes in human liver.27 This coupled with safety concerns about tolerizing human hosts to a potentially pathogenic virus has provided the impetus to develop other viral agents, such as those based on SV40 and adeno-associated viruses. Recombinant Simian Virus 40 (SV40) SV40 is a DNA virus of the papova family. The recombinant SV40 viral vector is constructed by replacing the T-antigen coding sequences from its genome with the gene of interest. The viruses are packaged in COS cells that provide the required T-antigen in trans. They can be grown and purified at high titers. Removal of the

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T-antigen renders the recombinant SV40 vector both nonimmunogenic and replication-deficient. SV40-based vectors integrate into the host genome and the gene product is expressed permanently.28 Although the integration is not site-specific, no noticeable adverse effects of SV40 vector integration have been observed in vivo. In fact, a follow-up of more than 40 years for thousands of patients who got inadvertently infected with SV40 viruses contaminating their polio vaccines showed no untoward effects associated with the infection by the wild-type virus. However, SV40 viruses do not localize specifically into the liver and, thus, transgene expression occurs in many organs. Recombinant Adeno-Associated Virus Adeno-associated virus-2 (AAV-2) is a papova virus, containing a single-stranded DNA genome. The wildtype virus integrates preferentially on the q13.4-ter arm of human chromosome 19 and may remain dormant for extended periods.29 On infection with a helper virus, such as adeno- or herpes simplex virus, AAV causes productive lytic infection. AAV replication also can be induced by genotoxic stimuli, such as heat shock, hydoxyurea, ultraviolet (UV) light or irradiation.30 AAV type 2 has been used most extensively for vector development. AAV is internalized by binding to a cell surface heparin sulfate proteoglycan. The AAV genes are flanked by 145 base pair inverted terminal repeats (ITRs) in hairpin conformation that are needed for integration into the host genome. Target genes can replace most of the viral genome. However, the viral Rep protein also directs the site specificity of AAV integration into chromosome 19, and recombinant vectors lacking this gene lose site-specificity of integration. To generate AAV vectors, three plasmids, one containing the transgene flanked by AAV ITRs; one encoding AAV rep, cap; and another encoding adenoviral E4 proteins are cotransfected into 293 cells, which provides adenoviral E1A in trans (Fig. 3). Within the cells, AAV vectors may persist as episomes or may integrate into the host genome.31 Moreover, AAV vectors can infect nondividing cells. Infusion of recombinant AAV into the portal vein of factor IX-deficient dogs resulted in the appearance of factor IX activity in plasma at 5% of the normal level.32 Unfortunately, recombinant AAV causes a humoral immune response, which may be a problem if the vector needs to be readministered. Recombinant Herpes Simplex Virus-1 and Baculovirus Recombinant herpes simplex virus-1 (HSV-1), a DNA virus has a broad host range and can infect nondividing

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Figure 3. Generation of recombinant adeno-associated viruses. To generate AAV vectors, three plasmids, one containing the transgene flanked by the ITRs, another encoding AAV rep and cap and a third one encoding adenoviral E4 proteins are cotransfected into 293 cells.

cells. However, long-term gene expression in the liver has not been achieved with the current HSV vectors.33 The Autographa californica nuclear polyhedrosis virus (AcNPV), which is used commonly for generating recombinant proteins in insect cells,34 can infect mammalian hepatocytes. Chimeric vectors consisting of segments of the baculovirus and other viruses are being evaluated for in vivo application.

Comparative Efficiency of Recombinant Viral Vectors Recombinant adenoviruses can be generated in high enough titers to express the transgene in about 90% of hepatocytes in rodents after a single injection; however, current evidence suggests that such high efficiency may not occur in the human liver. In a series of patients with ornithine transcarbamylase deficiency, administration of relatively high doses of a recombinant adenovirus resulted in a very modest level of transgene expression.27 Thus, the development of new generations of recombinant adenoviruses for liver gene therapy must be directed not only at abrogating the immune response, but also delivering the vector through alternative cellentry pathways.35 The gene transfer efficiency of recombinant adenoassociated viruses has increased progressively, so that therapeutically useful plasma levels of some transgene products, such as the coagulation factor IX may be generated.36 However, despite intraportal infusion, it has been possible to transduce only about 5% of hepa-

tocytes in the intact liver. As described above, the leukemia type retroviruses are inefficient in transducing nondividing cells. However, using vectors at very high titers, usually in combination with surgical or pharmacologic maneuvers to induce mitosis, can transduce a significant percentage of hepatocytes. Lentiviral vectors are difficult to generate at a scale that is sufficient for transducing a high percentage of hepatocytes in large experimental animals. However, recent methodologic improvement has permitted the generation of the recombinant vector at higher titer, increasing the proportion of transduced hepatocytes. Among the vectors that lead to integration in the host genome, recombinant SV40 vectors can be generated at the highest infectious titer (1012 to 1013 per mL), and can be used to transduce a majority of hepatocytes by repeated administration.37 The myriad challenges and drawbacks associated with viral vectors have accelerated our efforts to develop other methods of gene therapy that are independent of viruses. Transposons, naked DNA, ribozymes, and gene repair are examples of just some of the nonviral gene methods that are now available at the bench and the bedside. These strategies are the subject of the remainder of this review.

Nonviral Methods of Liver-Directed Gene Therapy Sleeping Beauty Transposon System Transposable elements may provide a novel tool for vertebrate genetic engineering by their ability to move exogenous fragments of DNA into the genome.38,39 Two major classes of the mobile genetic elements are distinguished by their different modes of transposition. Class I elements are retrotransposons that use reverse transcription to transpose through an RNA intermediate. Class II elements transpose directly from DNA to DNA and include the Tc1/mariner superfamily. Members of the Tc1/mariner family of DNA transposons do not require host-specific factors for activity in vitro. Transposons represent unique nonviral vectors for gene therapy that provide stable integration and potentially long-term expression of transgenes. Although efficient nuclear delivery remains key, a single integrant per cell genome may provide a therapeutic effect. Despite their wide spread presence in nature, not a single active transposable element has been identified in vertebrates, caused predominately by mutations in their transposase genes. An active transposase called Sleeping Beauty (SB) was reconstructed recently from an ances-

Gene Therapy for Liver Diseases

tral Tc1-like fish element.39 SB mediates transposition through a cut-and-paste mechanism. The nonviral plasmid vector contains the transgene (transposon), flanked by inverted repeat/direct repeat (IR/DR) sequences that bind the active transposase. The bound SB catalyses the excision of the flanked transgene and mediates its integration into the target host genome. The catalytically active transposase can be expressed on a separate (trans) or same (cis) plasmid system. The mechanism of insertion (Fig. 4) is assumed to be similar to that of Tc3 in Caenorhabditis elegans.40 The activity and thus the potential for the SB-mediated transposition has been shown in fish, mouse, and human cells.41 The SB transposon system was originally shown to work in mouse embryonic stem cells.42 The first mammalian in vivo application of SB was shown recently in normal mice and a transgenic mouse model of hemophilia B.43 With a hydrodynamic delivery system, the IR/DR-flanked transgene was transposed into the genome of mouse liver with an efficiency of 5% to 6%. Chromosomal transposition resulted in long-term expression of human blood coagulation factor IX for longer than 5 months at therapeutic levels. Repeated dosing in the transgenic mouse model led to increased transgene expression with no apparent toxicity and significantly reduced bleeding times. SB recently has been inserted into the mouse germ line and may prove to be a powerful tool to induce mutagenesis.44 Somatic integration and long-term transgene expression with the SB transposon system provides a number of advantages over traditional viral-mediated gene therapy. It allows delivery of large genetic coding sequences containing regulatory elements, although there may be size restrictions for efficient transposition. Additionally, genomic transposition occurs in quiescent and nonreplicating cells. Combined with an efficient delivery system, significant transposition could ensure a therapeutic response with a single administration. This would, of course, reduce the risk of adverse immunologic reactions from both the carrier system, as well as the plasmid itself, especially by immunostimulatory CpG motifs.45 As with all gene therapy strategies, the SB system is associated with some potential problems. In particular, the observed random genomic integration could result in insertional mutagenesis as well as inactivation of an essential gene(s). It remains to be determined whether integration by SB also is associated with changes in the methylation state observed with certain viral vectors. Such chromosomal changes have been associated with carcinoma, gene silencing, and genetic imprinting.

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Figure 4. The SB transposon system. Transposons, or transposable elements, have evolved as DNA elements that can mediate horizontal gene translocation by hopping between regions within the cell’s genome. The fish-derived SB transposon system is a unique functioning transposon system for vertebrates. In nature, the transposase (SB) is found between the inverted repeat/direct repeat sequences (IR/DRs) of the transposon. When reconstructed, the transposase was relocated and replaced by a designated gene. The dual component system requires a transposon carrying the target gene and a source of active transposase. The catalytically active transposase enzyme binds to and cleaves the DNA at either end of the IR/DRs, producing a circularized transposon/transpoase element. Insertion of this protein/DNA element occurs at a TA nucleotide site in the genomic DNA. Each end of the IR/DRs is now flanked by the distinctive TA border sequence duplicated during the insertion event.

Ribozymes, Antisense, and DNA Ribonucleases Ribozymes are RNA enzymes that bind to specific substrate sequences and catalyze endoribonucleolytic cleavage.46-48 RNA cleavage is a naturally occurring process involved in the removal of specific introns to form

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mature RNA. Ribozymes hybridize to complementary RNA sequences to mediate the directed cleavage of the target RNA. The ribozymal domains that base pair to substrate RNAs are functionally separate from the moieties that effect cleavage. As a result, the substrate specificity of ribozymes can be altered within certain constraints to allow catalytic, trans-cleavage of specified sites within the target RNA. Two major classes of ribozymes have been characterized as 1. hairpin ribozymes that require only a guanosine (G) residue immediately 3’ to the cleavage site49 and 2. the hammerhead ribozyme, requiring only a UN dinucleotide for cleavage. The resulting RNA fragments are rapidly degraded, rendering the molecule nonfunctional. The minimal sequence design coupled with their trans-acting specific cleavage allows ribozymes to target viral RNAs in a variety of genetic disorders. Ribozymes can either be synthesized and packaged for cellular uptake or expressed in cells. Moreover, they remain catalytically active for weeks in liver after somatic gene transfer. In one study, recombinant adenoviral vectors containing human growth hormone ribozyme expression cassettes were used to ablate growth hormone in mouse liver.50 Both hammerhead and hairpin ribozymes have been used successfully to inhibit hepatitis B and C viral production in cells.51,52 Hammerhead ribozymes, individually or in combination, either reduced or eliminated the hepatitis C virus RNAs in cultured cells and primary human hepatocytes from chronic hepatitis C infected patients.53 Also, it is possible to target a variety of highly conserved hepatitis C viral RNA sequences simultaneously with multiple ribozyme genes expressed from a single vector.54 This therapeutic approach could, in fact, result in a constant and continuous supply of multiple intracellular ribozymes, thereby decreasing the potential development of drug-resistant viral variants. Hepatitis B virus, a partially double-stranded DNA virus, replicates through a pregenomic RNA intermediate, which has been successfully targeted by hairpin ribozymes.55 Hammerhead ribozymes also have been used to inhibit hepatitis B replication. The messenger RNA (mRNA) for hepatitis X protein, which is implicated in viral genomic integration and development of hepatocellular carcinoma, was successfully targeted and reduced in cultured cells.56 A hammerhead ribozyme targeted to the poly(A) signal sequence of hepatitis B in HepG2 cells also reduced viral replication. Interestingly, the mutated ribozyme that exhibited no target mRNA cleavage in vitro slightly reduced viral replication, suggesting some antisense effect or cleavage activity in vivo.57

These RNA enzymes are being used also to treat inherited genetic disorders as well as cancer. Ribozymes against mRNA for human apolipoprotein B reduced both apolipoprotein B protein and serum lipid levels in a transgenic mouse model of hyperlipidemia.58 Interestingly, a hammerhead ribozyme directed against vascular endothelial growth factor receptors mRNAs induced substantial growth inhibition of a human colonic carcinoma liver metastasis in a xenograft model.59 DNA Ribonucleases DNA ribonucleases are catalytic molecules consisting of synthetic single-stranded DNA that also specifically cleave targeted RNA substrates. DNA ribonucleases are designed with two substrate-recognition domains that bind and target RNA through Watson-Crick base pairing and flank a catalytic domain consisting of 15 nucleotides. These molecules have several significant advantages over ribozymes, including ease of preparation and delivery to cells, improved resistance to degradation, and increased catalytic activity.60 They have been shown to be effective against both the hepatitis B and C viral genomes where specific cleavage of the targeted RNA occurred inhibiting viral replication.61 Interestingly, like ribozymes, the mutated DNA ribonucleases exhibited baseline antisense and cleavage activity in vivo. Antisense In contrast to ribonucleases, antisense molecules inhibit gene expression by hybridization with complementary nucleic acid sequences, thus inhibiting transcription or translation of the target sequence, or both. Antisense oligonucleotides have been used in the treatment of hepatitis B. Antisense 21-mer phosphorothioate-linked DNA oligonucleotides complementary to the hepatitis B virus polyadenylation signal and 5’-upstream sequences were used to transfect HuH-7 cells before introduction of B virus DNA. Pre-exposure of the HuH-7 cells to antisense DNA substantially blocked viral gene expression and replication in the virus-infected cells, resulting in specific inhibition of viral protein synthesis and replication in vitro.62 More recently, antisense phosphodiester or phosphorothioate oligonucleotides were targeted against duck hepatitis B virus in vivo. Although both antisense oligonucleotides significantly reduced viral replication, the natural phosphodiester linked DNA was five times more effective than its phosphorothioate counterpart in inhibiting viral replication.63 The potential of antisense technology is not con-

Gene Therapy for Liver Diseases

fined to antiviral therapy. Recently, antisense oligonucleotides have been used for inhibition of hepatoma growth by reducing vascular endothelial growth factor levels,64 reduction of FAS levels in the mouse liver to protect against acute liver failure,65 inhibition of hepatic protein synthesis resulting in reversal of hyperglycemia in diabetic mice,66 and development of a tumor vaccine with molecules targeting transforming growth factor (TGF)-␤2 with a reduction in tumor burden.67 In addition, a variety of methods have been used for delivery of the antisense molecules including liposomal, receptor-targeted, nanoparticle, oral, and intradermal approaches.68-72 Naked DNA Naked DNA injected into the systemic circulation appears to be cleared rapidly by the liver. In fact, Kupffer cells initially were implicated as the major site of first-pass clearance after DNA binding to surface membrane scavenger receptors. Biodistribution of naked DNA is limited by efficient hepatic uptake and degradation by circulating nucleases.73,74 Moreover, methylated CpG motifs within the DNA molecules also may increase the likelihood of immune response and subsequent removal of complexes.45 Interestingly, it has been proposed recently that the liver parenchymal cells internalize naked DNA by receptor-mediated endocytosis,75 although specific receptor(s) have not been identified. Direct injection of naked plasmid DNA into the liver of cats and rats produced expression of different reporter genes, as well as human ␣1-antitrypsin.76 Although the study showed a dose response, expression was relatively low and short-lived. More recently, particle-mediated gene delivery77 with DNA-coated projectiles discharged directly into the liver produced effective protection against malarial parasites in a mouse model.78 Unfortunately, despite relatively high levels of gene expression, the technique is limited by the tissue thickness. Two reports79,80 have compared the expression of a luciferase reporter gene after various modes of delivery to mouse liver. Increased hydrostatic pressure within the liver sinusoids by vascular occlusion resulted in prolonged interaction between DNA and hepatocytes. DNA delivery in a hypertonic solution also increased expression but, again, it was transient.81,82 Rapid tail vein injection of a plasmid construct produced detectable levels of hepatocyte growth factor after 4 hours, with peak expression in the liver at 12 hours. Weekly administrations led to both liver enlargement and a rise in overall body mass of 31% and 16%, respectively.83 It

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has been shown also that systemic delivery of naked DNA can be used to study hepatic viral replication and antiviral agents.84

DNA Delivery Systems Three major nonviral gene delivery approaches to the liver have been developed over the last decade.85 Polyplex systems consist of complexes formed with nucleic acids and cationic polymers. These polycation delivery systems, such as poly-L-lysine (PLL),86 polyethylenimine (PEI),87 polyglucosamines,88,89 lipopolyamines,90 and cationic peptides91 form water soluble complexes that can provide simple and efficient delivery systems. The accessible free amino groups on these agents permit easy conjugation of a variety of targeting ligands. Lipid-based DNA delivery systems include both lipid-encapsulated92 and cationic lipid/nucleic acid complexes (lipoplex).85 Lipopolyplex systems are hybrid complexes containing both polycationic polymers and lipids.93-95 Compaction of the large molecular-weight DNA with polycations before lipid encapsulation produces a DNA core surrounded by a lipid shell. Two major advantages of the hybrid over the lipoplex systems are the reduction in particle size and increased protection of nucleic acid from nucleases. The virosome combines fusion proteins from the hamaggutinating virus of Japan (HVJ) with liposomes into a novel transfecting agent with reduced immunogenicity.96 It has been used to deliver plasmids to the liver by both the portal vein as well as the biliary system.97,98 The later report is intriguing when one considers the accessibility of the biliary system with standard endoscopic techniques. Over the past decade, there has been major progress in transferring nucleic acids across the plasma membrane. Cationic lipid transfecting agents such as lipofectamine; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); and the cationic polymers, including poly-L-lysine and PEI, have significantly improved nucleic acid delivery into the cytoplasm.99,100 The substantially enhanced transfection efficacy observed with these delivery vehicles in vitro is associated with small, stable uniform particle sizes; however, these nucleic acid/cationic complexes often exhibit some cellular toxicity as well as reduced transfection efficiency in serum. The cellular toxicity of these cationic complexes may, in part, result from their large size101 and the high positive zeta potential required for efficient uptake.87,102-104 Serum proteins can reduce transfection by neutralization of the positive charge.105,106 The binding of serum proteins reduces transfection effi-

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ciency by increasing particle size, thereby reducing passage through fanestrae as well as endocytosis by coated pits.107 The hydrophilic polyethylene glycol (PEG) prevents aggregation, stabilizes and maintains the small size required for endocytosis, and diminishes binding of serum proteins. However, shielding of the cationic charge by PEG has resulted in reduced transfection efficiency.106 To overcome these hurdles, some delivery systems have used specific ligands to mediate uptake by receptor-mediated endocytosis. For example, asialoorosomucoid,86,108 asialofetuin,109 and galactose90,110,111 have been conjugated to PLL, lipopolyamines, and PEI for uptake by the asialoglycoprotein receptor. Lipid based delivery systems have used galactocerebrosides94,111,112 for targeting this receptor. Each of these ligand-targeted systems increased gene delivery to hepatocytes both in vitro and in vivo.86,90,94,110-114 Additionally, ligand/receptor-mediated endocytosis does not require an overall net positive charge. In fact, negatively charged particles promote efficient ligand-receptor mediated nucleic acid delivery.93 PEI conjugation with small linear tetragalactose resulted in greater transfection efficiency than complexes formed with lactose-conjugated PEI because, in part, of an increased binding affinity.115 Finally, DNA entry into the cells through endosomal/lysosomal trafficking pathways appears to improve efficient nuclear translocation of foreign DNA.116 Hepatocyte targeting also may be increased by delivery of complexes through the portal vein or hepatic artery, and this may reduce potential systemic side effects. Selective hepatic arterial delivery may be a benefit also in tumor-specific targeting. For example, most primary liver tumors possess large and abnormally leaky blood supplies that may be more favorable for gene transfer.117 Direct injection of tumors with cationic lipid complexes has shown potential in the treatment of hepatocellular carcinoma and was shown to be less toxic than systemic or portal administration.118 Both PEI and other polycations have been used in compacting and protecting DNA from nuclease degradation.119 A variety of peptides increase endosomal release of DNA into the cytosol.120 For example, the hemaglutinin HA2 protein of the influenza virus is a potent membrane disruptor.121 The protein undergoes a conformational change on exposure to an acidic environment producing an amphipathic ␣-helix and facilitating escape by endosomal membrane fusion.122 PEI exhibits a novel characteristic in its ability to efficiently disrupt the endosome by acting as a proton sponge.123 Interestingly, it has recently been shown that PEI persists in the endosome/lysosome for days, thereby acting

to protect the plasmid from degradation and acting as a slow release mechanism.124,125 Once released from the endosome, a number of factors including particle size and delivery agent appear to influence the nuclear uptake of molecules greater than ⬃125 nucleotides.100,126 As a rule, small particle size is associated with increased nuclear localization and transgene expression. In addition, unlike the cationic lipids, polyplex delivery agents do not appear to inhibit nuclear expression of the transgene.126 Nuclear localization signals have provided the most dramatic increase in the delivery of transgenes from cytosol to nucleus.127,128 Thus, the translocation of the transgene into the nucleus exploits active transport pathways that can increase nuclear localization/expression 10- to 10,000fold. Nuclear localizing signal sequences derived from the SV40 virus have been incorporated into, and shown to improve delivery of, cationic liposomal systems.129 Finally, newly identified membrane binding sites and their ligands have improved the delivery of molecules to mitochondria.130 Mitochondrial DNA mutations may play a central role in many hepatic disorders characterized by extensive steatosis and gene therapy may become a therapeutic option. Taken together, there has been considerable progress in just the last few years in developing dramatically improved nonviral delivery systems and overcoming the known barriers for efficient nucleic acid delivery to the nuclei of quiescent cells.

Gene Repair Strategies Chimeric RNA/DNA Oligonucleotides The concept of a chimeric RNA/DNA molecule (chimeraplast) mediating genomic alteration originated from studies on homologous recombination.131,132 The initial chimeraplast design was a contiguous stretch of 68 nucleotides containing both RNA and DNA residues (Fig. 5A). One strand of the heteroduplex structure consisted of a central pentameric block of DNA bases flanked on both sides by ten RNase H resistant 2’-O-methylated RNA residues. The opposing strand consisted of all-DNA bases complimentary to the RNA/DNA strand.133 Two polythymidine hairpin caps and a 5-base pair 3’ GC clamp help provide secondary structure and increased nuclease resistance. The 3’ and 5’ ends were juxtaposed but not joined, thereby providing the molecule with greater flexibility for unwinding and interaction with recombinase proteins responsible for base pairing. The chimeric oligonucleotide aligns in perfect register with its genomic target except for an

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Figure 5. Methods of in situ gene repair. (A) A prototypic chimeraplast and its proposed mechanism of repair. The hybrid strand consists of 5 DNA nucleotides (black) flanked by two 10-base stretches of 2’-O-methylated RNA residues (green). The complementary all-DNA strand of the oligonucleotide is shown in black. The targeted DNA sequence is identical to the homology region of the duplex structure except for the single engineered G-C base pair mismatch. 1. Watson-Crick base pairing between the chimeraplast and genomic DNA. 2. The G-C mismatch activates endogenous DNA repair pathway(s) that converts the genomic A-T base pair to G-C. (B) Repair of point mutations by short SSOs. An SSO (red) is synthesized that is identical to its homologous genomic DNA target except for a single C mismatch. 1. A D-loop structure is formed between the SSO and its genomic target with the C-C mismatch. 2. This triggers endogenous DNA repair mechanisms, including strand transfer, to convert the genomic C to a G (red). 3. The internal genomic mismatch then activates a repair process that substitutes a C for the endogenous G at the target site. (C) A tethered domain-triplex forming oligonucleotide can also alter DNA sequence. 1. Target site recognition is facilitated by triplex formation between the triple-helix forming domain (red) and complementary purine-rich genomic sequence. The donor DNA fragment is tethered to the triplex-forming domain by a flexible linker indicated by black circles. Donor DNA is identical to the target genomic sequence except for the targeted G-C base pair. 2. The triple helix activates DNA repair/recombination pathways. (D) Modification of genomic DNA by small fragment homologous replacement uses single-stranded DNA fragments between 400 and 800 nucleotides. 1. The single-stranded DNA (red) is generated homologous to a genomic target except for the desired mismatch(s) (purple). 2. A D-loop precedes strand transfer. 3. The D-loop structure is resolved by endogenous DNA modification/repair processes.

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intended base mismatch that produces a structural distortion and subsequent repair of the targeted bases. Chimeraplasty begins with homologous targeting by the recombinase family of proteins (i.e., RAD 51/52 in humans and the RECA/2 system in yeast).134 The second phase involves mismatch recognition, and then base removal and exchange by the complex action of DNA polymerases and ligases.135 Chimeraplasty was originally performed on episomal targets,136 the sickle cell mutation in cultured lymphoblasts,137 cell-free extracts,138 and a variety of cultured cells,139,140 including HuH-7 cells for the endogenous alkaline phosphatase gene.141 To increase hepatocyte uptake, chimeric molecules were either complexed with lactosylated PEI or encapsulated with anionic, cationic, or neutral liposomes containing galactocerebroside for targeting to the asialoglycoprotein receptor. Delivery was assessed by confocal microscopy, and single base correction rates by polymerase chain reaction (PCR)/filter lift hybridizations. The improved delivery systems resulted in base conversion rates of 19-24% in isolated hepatocytes.111 In the first in vivo studies, chimeraplasts were complexed to lactosylated PEI and injected into the tail vein of normal rats. The RNA/DNA oligonucleotide was designed to induce a point mutation in the factor IX gene resulting in altered clotting activity.142 Conversion rates were dose-dependent, ranged from 15% to 40% and correlated with reduction in factor IX activity. These results remained unchanged for almost 2 years and also were reproduced in a group of rats that underwent 70% partial hepatectomy 3 weeks after treatment.143 The Gunn rat model of Crigler-Najar type 1 syndrome is characterized by a single base deletion in the UDP-glucuronyltransferase gene (UGT1A1), producing a frame shift and premature stop codon. The genomic defect results in an absence of enzymatic activity required for bilirubin glucuronidation and biliary excretion. The rat phenotype is characterized by hyperbilirubinemia, and the absence of mono- and di-bilirubin glucuronides in bile. In the study, the liver was targeted with chimeraplasts complexed with lactosylated PEI or encapsulated in the anionic liposomes. With chimeraplasty, the deleted guanosine base was replaced at frequencies greater than 20%. Serum bilirubin levels were reduced by 25% to 50% and coincided with the appearance of mono- and di-bilirubin glucuronyl salts in bile. No phenotypic or genomic changes were noted in the control groups. The genomic and phenotypic changes were stable over an 18-month study period.144

In a more recent study, the mutant isoform of apolipoprotein E (apoE2) was targeted and converted to the wild-type apoE3 in both isolated cells and in situ.145 Conversion rates in a clonally expanded stably transfected Chinese hamster ovary (CHO) cell line were 14% to 54% as assessed by PCR. The conversion event was confirmed at the phenotypic level by western blot detection of the apoE3 isoform. The human apoE2 gene also was targeted in a transgenic mouse model. Chimeraplasts were complexed with linear unmodified PEI, administered by intraperitoneal injection and induced a 25% conversion frequency in the liver after one week. Interestingly, these studies showed an ⬃50% improvement in conversion with chimeraplasts containing 15 modified RNA residues (i.e., an 88-mer) compared with the standard 68-mer molecule, reflecting either improved binding activity, an increased resistance to nucleases, or both. Chimeraplasty has been showed in bacteria,133 and yeast146 as well as in vitro and in vivo in a wide variety of target cells and tissues, including skin melanocytes,139,147 kidney,148,149 and the skeletal muscle in both dog150 and mouse151,152 models of Duchenne muscular dystrophy as well as plants.153-155 These numerous studies have established correction by showing phenotypic and non–PCRbased genotypic correction both in cultured cells and in vivo. Single-Stranded Oligonucleotides (SSOs) Cell-free extracts were initially used to optimize the chimeric RNA/DNA structure and elucidate the underlying mechanisms of chimeraplasty. Modified chimeric molecules were targeted to plasmids containing point mutations in antibiotic resistance genes and coincubated in cell-free extracts. After recovery of the plasmids, they were then electroporated into bacteria that were grown on antibiotic containing selection media. These studies suggested that the all-DNA strand of the chimeraplast mediated base conversion. It was subsequently shown that a short SSO containing a single base mismatch could itself activate the DNA repair systems and induce a base change (Fig. 5B). Although the precise mechanism is unknown and probably different from that of chimeraplasty, it appears to involve mismatch repair proteins and the recombinase family of proteins.156,157 To further characterize their potential for DNA repair, SSOs were capped with varying numbers of phosphorothioate nucleotides to increase nuclease resistance. These modified molecules were four times more active than their unmodified counterparts. In contrast, all-phosphorothioate molecules were devoid of activity,

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suggesting that unmodified nucleotides are required to stimulate recognition and repair of the mismatched bases. Mutant strains of bacteria were used to identify some of the essential proteins involved in the base conversion.157 The study showed that nucleotide conversion was independent of the mismatch repair proteins MSH2 and MSH3, in contrast to chimeraplasty. A similar broad potential application of SSOs for sitespecific nucleotide alteration was confirmed in diverse systems of both mammalian and plant cell–free extracts. In a recent study, SSOs were characterized for their ability to corrected a mutated ␤-galactosidase plasmid reporter construct in cell-free extracts.158 The optimal length was determined to be 25 to 61 nucleotides and equivalent base conversion was observed irrespective of the strand targeted. Interestingly, a chimeric 25-mer SSO consisting of a pentameric all-DNA portion flanked by ten 2’-O-methylated RNA residues showed significantly less activity than those modified with only four 2’-O-methylated RNA residues at either end. Episomal conversion using CHO cells transiently cotransfected with the mutated ␤-galactosidase plasmid and SSOs resulted in a significantly higher conversion frequency. Surprisingly, there was a 1,000-fold higher conversion when the nontranscribed strand was targeted. With genomic DNA, conversion rates were higher than those in the cell-free extract but an order of magnitude lower than the episomal studies. The optimal length of the molecule was 45 nucleotides and higher conversion rates were again observed when the nontranscribed strand was targeted. It remains to be determined if targeting the nontranscribed strand is more likely to promote genomic DNA repair, whereas targeting the transcribed strand may lead to repair of the oligonucleotide. These studies have generated great interest in the potential for in vivo application. The SSO system would provide advantages over conventional chimeraplasty, including lower cost of production and ease of synthesis and purification; however, both chimeraplasty and SSOs remain controversial technologies for gene repair. This is attributable, in part, to a lack of reproducible and efficient delivery systems, cell-to-cell variation in expression of the essential proteins required for conversion, and accessibility of target sequences. In addition, the processes may rely heavily on the simple laws of mass action. Therefore, to be successful, the oligonucleotides must be functional and delivered in high copy number.

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Triplex-Forming Oligonucleotides (TFOs) and Small-Fragment Homologous Replacement (SFHR) Targeted repair by chimeraplasty and SSOs is limited to several bases and thus would restrict their potential applications. Recently, an alternative gene repair strategy has been developed. TFOs derive from the observations that single-stranded DNA or RNA molecules can form stable and specific triple helical structures with homopurine-rich areas of the genome. The oligonucleotide binds to its target site in the major groove through hydrogen bonds with the target sequence.159 However, the technology is somewhat limited to purine-rich sequence tracts. Purine oligonucleotides have the disadvantage that their ability to form triplexes is considerably hindered by monovalent cations such as Na⫹ and K⫹, which promote formation of stable self-aggregates, thus preempting their function under normal physiological conditions. Potentially, TFOs could be used to target viral replication. One could design an SSO to bind a target strand by Watson-Crick base pairing and fold back on itself through a linker with the remaining portion of the molecule. This would result in a triplex within the major groove of the newly formed viral DNA/oligonucleotide duplex. These oligonucleotide clamps are very stable and would thus hinder the replicative cycle of the targeted virus. In addition, the antigene effect could block the binding of transcription factors to their promoter sites or, alternatively, inhibit transcription elongation. TFO activity has been significantly increased by coupling DNA damaging adducts, such as psoralen, to the oligonucleotides. The increased frequency of homologous recombination within the targeted area has been shown in both stem and somatic cells160-162 and appears to be dependent on nucleotide excision repair (NER) pathway(s). Novel hybrid or bifunctional TFO molecules have recently been developed (Fig. 5C). These molecules consist of a triple- helix forming sequence linked to a double-stranded DNA fragment homologous to an area close to the TFO binding site. They exploit the ability of the TFO to form stable complexes and to induce localized homologous recombination/NER. Combining this triplex-forming domain and the homologous DNA fragment permits formation of a heteroduplex at the target site. Then, the ability of the TFO to form a stable anchor and induce homologous recombination/ NER provides an improved design for triplex-mediated DNA repair. In a recent study, the hybrid TFO molecules targeted an episomal reporter system and medi-

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ated conversion frequencies of less than 1%. The genetic modifications were accomplished with both single- and double-stranded targeting DNA and seemed dependent on the presence of XPA protein and the NER system.161 In a separate study, bifunctional TFOs were used to correct point mutations in the endogenous adenosine deaminase (ADA) and p53 genes at frequencies of of 1-2% and 7.5%, respectively, in different cell lines.163 Homologous recombination has been used effectively, albeit as low frequencies, to disrupt genes for functional genomic studies and to create transgenic models of human disease. The low levels of success are attributable, in part, to the large, nonhomologous delivery vectors and selectable marker genes (e.g., neomycin resistance). This results in significantly reduced fidelity of a homologous pairing event and, thus, recombination. A modified approach recently has been described in which smaller fragments of DNA are targeted to the homologous regions of DNA. Known as small-fragment homologous replacement (SFHR), it may be entirely different than homologous recombination164 and typically uses SSOs several hundred bases in length (Fig. 5D). The precise mechanism of action is unknown and may involve previously uncharacterized pathways for DNA repair. The true potential of this technology will be realized as it is tested in a variety of animal models. SFHR was initially used to target the 3– base pair ⌬F508 deletion in exon 10 of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in transformed epithelial cells.164 A 500 nucleotide, single-stranded DNA fragment was used to correct the trinucleotide defect at a frequency of ⬃1% with a similar detection of functional CFTR protein. In a follow up study, the same group used a 488-mer oligonucleotide containing a silent mutation 100 nt from the CFTR deletion to target normal and transformed epithelial airway cells.165 The system was tested for the ability of the single-stranded DNA to correct or create the 3– base pair deletion, despite the presence of an upstream mismatch. The transfected oligonucleotides produced site-specific conversion in both transformed and normal cells. The conversion was not affected by the upstream mismatch and was confirmed by mRNA transcript expression. Subsequently, SFHR was used to correct a 4 – base pair deletion in the Zeocin antibiotic resistance gene166 at frequencies of up to 4%, as confirmed by Southern blot analysis. Recently, SFHR was tested in the mdx mouse model of Duchenne muscular dystrophy.167 Although repair in myoblast cultures was 15% to 20% by PCR analysis,

there was no detectable normal dystrophin protein. Repair of 0.1% or less was found in the animals that underwent direct injection of the muscle, but, again, there was no detectable functional gene expression at either the transcript or protein level. These results, of course, were in contrast to the positive results reported by chimeraplasty in similar animal models.150-152 Also, it was recently reported that SFHR introduced the CFTR ⌬F508 mutation in normal mouse lung.168 Interestingly, four different transfecting agents were used in the study with varying results, thus reaffirming the importance of delivery. SFHR has emerged as an important strategy for targeted gene repair. Despite the overlap in the various strategies, each clinical disorder probably will have to be individually and systematically assessed to define the best single approach or even combination of strategies. Recently, it has been reported that adeno-associated viral vectors were used to introduce single base substitutions at relatively high frequencies in normal human cells.169

Conclusions This overview has described some of the existing options for gene therapy of the liver. Although many obstacles exist for both viral and nonviral approaches, efficient delivery remains one of the most critical factors. In part, the ability to use gene therapy as a replacement for liver transplantation depends on the ability to deliver small and large pieces of DNA into the nucleus. A number of new methods are being developed to achieve this goal. Oligonucleotides as well as entire genes are being delivered to hepatocytes with molecules that bind surface membrane receptors, disrupt endosomal membranes, and translocate the DNA to the nucleus. Additional strategies have been developed to reduce enzymatic degradation and increase delivery of functional molecules to the nucleus. Both peptide and locked nucleic acids enhance nuclease resistance and could increase the number of oligonucleotides reaching their target site, thereby potentially improving repair frequencies. The rich blood supply to the liver makes it an optimal target for gene therapy. Both direct modification of the nucleic acid molecules and manipulation of the delivery vehicle have significantly improved the potential for success of gene therapy to the liver. Numerous clinical conditions could ultimately benefit from gene therapy, repair, or both.143 For example, the long list of genetic diseases resulting from singlenucleotide mutations are logical targets for the repair processes of chimeraplasty, SFHR, TFOs, and SSOs.

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Even small corrections could benefit such diseases as hemophilia, ornithine transcarbamylase deficiency, and Crigler-Najjar syndrome type 1. Higher correction frequencies probably would be necessary for dominant negative diseases such as ␣1-antitrypsin deficiency. As an alternate approach, RNA interference (RNAi) degradation of cytotoxic mRNA170 may someday replace the more traditional strategies with ribozymes, DNA enzymes, or both. In fact, a novel nonviral vector has recently been developed to express RNAi molecules in cultured cells, and may represent a potential strategy for in vivo use.171 A number of nonliver diseases also result from point mutations, including sickle cell and Gaucher diseases. The in utero deliver of these repair molecules could possibly even correct neurodegenerative disorders at a time when transfer across the blood-brain barrier is less restricted. The ability to manipulate single nucleotides within the genome also allows the precise introduction of mutations. Thus, the technology ultimately could permit us to introduce stop codons upstream of genes expressing certain dominant-negative proteins such as ␣1-antitrypsin and the huntingtin CAG repeat gene product of Huntington’s disease. With the explosive interest in single-nucleotide polymorphisms (SNPs) and their application to pharmacogenetics, we now have the ability to alter single nucleotides in an effort to engineer any number of ex vivo changes to mediate in vivo effects. For example, soon it may be possible to alter the genome of bystander cells to improve overall response to chemotherapy. The observed plasticity of hematopoietic stem cells suggests the possibility of correcting a defect ex vivo and then stimulating them to transform into different cell types in vivo such as hepatocytes. Overall, the gap between our expectations and achievements in gene therapy has narrowed considerably in the last several years. However, additional difficulties continue to develop while we transition from bench to bedside, and attempt to scale up from small animals to humans. With improved techniques in molecular design, gene targeting, nuclear delivery and site-specific repair, the reality of successful therapeutic application is at hand. The new millennium invites the dawn of a paradigm shift in medicine and the advent of the era of genomics. One answer to the growing shortage of donor livers lies in our ability to treat and even correct these myriad disorders the ultimate fate of which lies in gene therapy. The application of viral vectors, naked DNA, Sleeping Beauty, and the different functional oligonucleotides has provided us with the

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methods to potentially cure liver diseases without the need for liver transplantation.

Acknowledgments The authors thank the members of our laboratories for their support and encouragement during the course of this work. The authors acknowledge the important contributions by many investigators in the field of liver transplantation and gene therapy who were not cited because of limited space.

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