Introduction

The research on liver-directed gene therapy and hepatocyte transplantation has progressed in parallel. Hepatocytes, with or without genetic modification have been used to introduce normal genes into patients or animal models with inherited disorders, while gene transfer can be used to expand hepatocytes in culture or abrogate allograft rejection. In this article, we discuss the current state of the two areas of research, as well as the hurdles that remain to be crossed for widespread application of these advances in the treatment of liver diseases.

Hepatocyte Transplantation

Although liver transplantation has dramatically improved the prognosis for patients with acute or end-stage liver failure, and inherited metabolic disorders, because of the complexity and associated morbidity and mortality of this procedure, hepatocyte transplantation is being explored as a convenient and safer alternative. Transplanting isolated hepatocytes by percutaneous or transjugular infusion into the portal vein, or injecting into the splenic pulp or the peritoneal cavity, is a less invasive procedure compared with liver transplantation. As the host liver is not removed or resected, the loss of graft function should not worsen liver function. Furthermore, isolated hepatocytes could be, potentially, cryopreserved for ready access.¹ After extensive evaluation in experimental animals, clinical trials of hepatocyte transplantation have been initiated at several institutions for acute or chronic liver failure and inherited metabolic disorders. Hepatocytes transplantation has been explored as a vehicle for ex vivo gene therapy and is being considered for rescuing patients from radiation-induced liver damage resulting from radiotherapy for liver tumors.

Hepatocyte Transplantation for Liver-Based Metabolic Diseases

Hepatocyte transplantation has been tested on a number of animal models with liver-based metabolic disorders. The Gunn rat is an animal model of Crigler-Najjar syndrome type 1 (CN1). This mutant strain of Wistar rats lacks bilirubin-UDP-glucuronosyltransferase (UGT1A1) activity and consequently, accumulate toxic plasma levels of unconjugated bilirubin.² Hepatocyte transplantation has been tested most extensively in Gunn rats and Nagase genetically analbuminemic (NAR) rats. In both models, transplantation of normal donor hepatocytes ameliorated the metabolic deficit.³ When the liver is structurally normal, which is the case in many inherited liver based disorders, hepatocytes injected into the splenic pulp or infused into the portal vein migrate to the liver and become integrated into the hepatic cords within days. These cells become morphologically identical to the host cells and function throughout life, and can be recognized only by virtue of biochemical or genetic markers.^4,5 Hepatocyte transplantation has also resulted in partial correction of metabolic disorders in low density lipoprotein receptor-deficient⁶ Watanabe heritable hyperlipidemic rabbits (an animal model of familial hypercholesterolemia), and the Long-Evans Cinnamon rat (an animal model for Wilson's disease).⁷ Most functional liver proteins are present in great excess, therefore, the initial assumption was that the transplantation of a small fraction of the total hepatocyte mass (˜1–5%) should correct many metabolic disorders. However, most experimental and clinical studies have not borne out this expectation. Although repeated hepatocyte infusions could improve the response to transplantation to some extent,⁸ the most dramatic results have been obtained by massive preferential repopulation of the host liver by engrafted hepatocytes.

Massive Repopulation of the Liver

In inherited disorders which result in death of host hepatocytes, such as fumarylacetoacetate hydrolase (FAH) deficient mutant mice (a model of hereditary tyrosinemia type I) or transgenic mice overexpressing the plasminogen activator (uPA), transplanted normal hepatocytes can spontaneously repopulate the liver, eventually replacing almost all host hepatocytes.⁹ However, the transplant recipient mice remain susceptible to the development of hepatocellular carcinomas. Therefore, continuing cancer risk or recurrent disease should be considered in determining the clinical indications for hepatocyte transplantation for specific diseases.

In most metabolic diseases, however, the host hepatocytes do not have a rapid turnover. In these cases, the host hepatocytes respond to proliferative stimuli to the same extent, as do the engrafted cells, precluding preferential growth of transplanted cells. Therefore, for a more general application, strategies are being developed to inhibit host hepatocyte regeneration, while providing a proliferative stimulus to the transplanted cells. In experimental model, the proliferative pressure is exerted by performing partial hepatectomy, causing reperfusion injury, inducing Fas-mediated apoptosis of host hepatocytes or administering pharmacological doses of thyroid hormone.^10,11 For preventing the proliferation of host hepatocytes, a plant alkaloid, retrorsine, which blocks the hepatocyte cell cycle has been utilized.¹² However, as retrorsine is potentially carcinogenic, X-irradiation of the liver has been evaluated as a part of the preparative regimen.^13,14 In initial studies, the livers of recipient dipeptidylpeptidase IV-deficient rats were irradiated (50 Gy) and the rats were subjected to partial hepatectomy. Following transplantation of hepatocytes from congeneic normal rats (˜0.1% of the rat liver hepatocyte mass), the engrafted cells proliferated preferentially, forming small clusters in 3 weeks, and replacing nearly all host hepatocytes in 3 months (Fig. 1). Transplantation of normal hepatocytes from congeneic donor rats into Gunn rats that had undergone partial hepatectomy and preparative hepatic irradiation resulted in massive repopulation of the liver by the normal cells (Fig. 2), resulting in complete normalization of serum bilirubin levels.¹⁵ Interestingly, hepatocyte transplantation completely prevented radiation-induced morphological injury of the liver and the synthetic function of the liver was fully retained in the massively repopulated liver.¹⁴ Efforts to stimulate the proliferation of the transplanted hepatocytes by non-surgical means, such as controlled expression of FasL in the liver by gene transfer are underway.¹⁶

Figure 1

Massive repopulation of the liver by preparative irradiation. DPPIV-deficient F344 rats received preparative hepatic irradiation (50 Gy) and 66% hepatectomy, followed by transplantation of 2 × 10⁶ hepatocytes from congeneic DPPIV-positive F344 (more...)

Figure 2

Repolulation of the Gunn rat liver by engrafted normal hepatocytes. Gunn rats were subjected to preparative irradiation of the liver and partial hepatectomy as in Figure 1 and were transplanted 2 × 10⁶ hepatocytes from congeneic normal Wistar (more...)

Clinical Experience with Hepatocyte Transplantation for Inherited Metabolic Disorders

Patients with CN1, ornithine transcarbamylase (OTC) deficiency and α1-antitrypsin deficiency have been transplanted with allogeneic hepatocytes. Two children with OTC deficiency showed significant evidence of the enzyme activity. However, one of these patients died shortly from hyperammonemia. In the other, hyperammonemia was corrected for a 10-day period, but recurred after that time, and liver transplantation was required for correction of the metabolic abnormality (I.J. Fox, personal communication). Direct evidence of function of transplanted hepatocytes was obtained in a CN1 patient (UGT1A1 deficiency).^17,18 Allograft rejection was prevented with standard Tacrolimus and prednisone therapy. After transplantation, the serum bilirubin concentration declined to half of the pretransplantation level and UGT1A1 activity in a liver biopsy specimen increased to ˜5% of normal. Although the procedure did not cause portal hypertension or other significant complications, but the extent of replacement of hepatic UGT1A1 activity was not sufficient to eliminate the need for phototherapy.

Liver-Directed Gene Therapy

Potential Indications

Liver-directed gene therapy is being contemplated for both inherited disorders and acquired conditions, such as infectious and neoplastic diseases, cirrhosis of the liver and immune rejection of transplants. Missing gene products causing inherited diseases could be replaced by transferring genes expressing those proteins. In other cases, specific genes could be overexpressed for therapeutic purposes, such as the overexpression of metalloproteases for the treatment of cirrhosis. In some situations, such as CN1 or low-density lipoprotein receptor deficiency (familial hypercholesterolemia), the missing gene product must be expressed in the liver for appropriate metabolic effect. In other cases, the large size of liver and secretory characteristics of the liver could be exploited to generate “biodrugs” for export out of the hepatocytes. Such proteins include coagulation factors, hormones or vaccines. In some cases, proteins that are normally expressed at extrahepatic sites could be expressed in the liver for specific purposes. For example, the catalytic subunit of the apolipoprotein B mRNA editing enzyme (APOBEC-1), which is normally expressed in the intestinal epithelial cells, could be expressed ectopically in the liver to switch the hepatic apolipoprotein production from apo B100 to apo B48, thereby reducing the production of low density lipoproteins.⁴¹ Similarly, hepatic expression of PDX, a homeobox protein that is responsible for pancreatic differentiation, could result in the secretion of insulin from hepatocytes.⁴² In other cases, nucleic acids may be used to inhibit the expression of deleterious proteins, such as viral proteins or mutant a1-antitrypsin. This could be accomplished by transfecting synthetic antisense RNAs, ribozymes⁴³ or inhibitory double-stranded RNAi.^44,45 Genes expressing antisense RNAs, RNAi, ribozymes or dominant negative proteins⁴⁶ could be transferred into the liver. Finally, technologies are being developed to correct mutations within the endogenous genes in intact organisms. This could be accomplished by targeted replacement of the defective gene,⁴⁷ or by site-directed correction of a target genomic sequence.⁴⁸ These newer strategies permit the repaired gene to remain under the control of the endogenous promoter, whereby physiological regulation is retained. A partial list of inherited and acquired disorders targeted for liver-directed gene therapy is provided in Table 1. Gene therapies for cancer and infectious diseases of the liver pose some special problems, and have been discussed separately.

Table 1

Some liver-based disorders that are current targets for gene therapy.

Gene Therapy for Cancer

Nucleic acid transfer approaches are being designed to kill the tumor cells or inhibiting their growth, reduce tumor blood supply, evoke anti-tumor immune response, or to augment the effect of chemotherapy and radiotherapy. P53, a sentinel gene of the cell cycle, has been transferred to induce apoptosis of tumor cells.⁴⁹ The herpes simplex virus thymidine kinase (HSV-TK), which converts a prodrug, ganciclovir, to toxic ganciclovir phosphate,⁵⁰ or cytosine deaminase and purine nucleoside phosphorylase (which converts fludarabine to a diffusible toxic metabolite)⁵¹ have been expressed as a “suicide gene” in an attempt to ablate tumors. Ganciclovir phosphate may diffuse to neighboring cells, killing them by “by-stander effect”, extending number of tumor cells killed. To reduce the toxic effect on normal cells, the suicide genes have been targeted to tumor cells by tagging the DNA to monoclonal antibody directed at cell surface proteins that are preferentially expressed in tumor cells, such as AF-20, a 180-kDa tumor-specific cell surface glycoprotein, expressed in hepatoma cell lines.⁵² Another strategy to increase the therapeutic ration is to use tumor-specific promoters (e.g., alpha-fetoprotein or carcinoembryonic antigen) to drive the transgene expression. A different approach uses E1B-mutant adenoviruses that are replicate preferentially in P53-deficient tumor cells,⁵³ although P53 deficiency may not be always required for the replication of these mutant adenoviruses.

Since killing the tumor cells by topical expression of toxic gene products does not eliminate tumors at metastatic sites, efforts are being made to inhibit neovascularization, that is needed for both primary and metastatic tumor growth, by the expressing angiostatin or endostatin genes.^54,55

Host immune response against tumor antigens is important in eliminating tumor cells that are often present as micrometastasis. Expression of tumor-specific “neoantigens” may evoke this necessary immune response, provided they are presented properly to the immune system. Tumor antigens, such as those from melanoma cells, have been used for DNA-based tumor vaccination. Genetic manipulation of antigen-presenting cells may augment the host immune response against tumor cells. Since cytokines, such as TGFβ or IL10, secreted by large tumors, suppress immune response⁵⁶ “debulking” the tumor by surgery, radiotherapy, chemotherapy or gene therapy may enhance the immune response.

Currently, the greatest potential of gene therapy for cancer is an adjunct to chemotherapy or radiotherapy. Irradiation of tumors augments tumor cell transduction using recombinant viruses. Transgene expression could be driven by radiation inducible promoters.⁵⁷ On the other hand, inhibition of the expression in the tumor cells of radioprotective proteins, such as ATM (“mutated in ataxia telangiectasia”), could increase radiosensitivity of the tumors.⁵⁸

Gene Therapy for Infectious Diseases

Nucleic acid-based approaches are being explored both for prophylaxis and treatment of hepatic viral infections. DNA vaccination⁵⁹ can be safer and cheaper by eliminating the possibility of vaccine contamination infectious agents from tissue culture. The injected DNA itself can enhance the immunoreactivity of the expressed antigen. Expression of the antigenic peptides directly in antigen presenting cells could augment the immunoresponse by improving presentation. Expression of specific cytokines can be used to promote the proliferation of antigen presenting cells, as well as the maturation of T cells to helper T cells. Gene transfer is also being used to express single chain antibodies or antibody fragments in hepatocytes to make them resistant to viral infections.⁶⁰

Gene therapy can also be used to interfere with the viral life-cycle by introducing synthetic antisense RNAs, ribozymes, or DNA ribonucleases (see below). Ribozymes, antisense RNAs or dominant negative proteins⁶¹ can also be expressed within the target cells by gene transfer

Gene Therapy for Inherited Disorders

As many inherited disorders are caused by the abnormality of a single gene, the effect of gene therapy can be evaluated directly and precisely in these conditions. For this reason, rare single gene abnormalities continue to be important targets of liver-directed gene therapy. Table 1 contains a partial list of inherited disorders that are currently targets of gene therapy.

Methods of Gene Delivery to the Liver

Genes may be delivered to the liver by systemic administration, infusion into the portal vein, hepatic artery or bile duct, or direct injection into the liver. Alternatively, hepatocytes isolated from the liver, or immortalized liver cells can be transduced with a therapeutic gene and then transplanted into the liver as a part of an “ex vivo” gene therapy approach. Commonly used methods for delivering genes to the liver of intact organisms, such as those using replication-deficient recombinant viruses, or non-viral vehicles are briefly discussed below. Frequently used methods of gene transfer to the liver are listed in Table 2.

Vectors Based on Recombinant Viruses

The attainable infectious titer, ability of the virus to infect non-dividing cells, integration into the host genome, repeatability of administration and safety of the vector system are important considerations in selecting recombinant viruses for gene therapy for specific diseases.

Retrovirus-Based Vectors

Proviral DNA, complimentary for the RNA genome of retroviruses, is integrated into the host genome and are transmitted to the progeny of the transduced cells.⁶² Tumor retroviruses, such as, Moloney's murine leukemia virus (MoMuLV), have been used extensively for generating recombinant vectors. The viral structural genes are replaced by target transgenes, leaving packaging signal (ψ) intact.⁶³ The viral proteins are provided in trans by “packaging cell lines”, generating a replication-deficient recombinant virus. The envelope protein determines the range of species infectable by the recombinant retrovirus. The host range can be expanded by engineering proteins from other viruses, such as the G-protein of the vesicular stomatitis virus (VSV) into the retroviral envelope. After the recombinant virus enters the cell, the RNA genome is reverse-transcribed into double-stranded DNA provirus, which is transported to the nucleus and is integrated into the host genome. For the tumor viruses, this process requires resolution of the nuclear envelope that occurs during cell division. Therefore, the tumor retroviruses are inefficient in infecting primary hepatocytes, which undergo mitosis infrequently. Lentiviruses, which are retroviruses of the immunodeficiency group, form preintegration complexes that can be translocated into non-dividing nuclei. Therefore, recombinant lentiviruses are being developed for liver-directed gene therapy. However, it has been suggested that recombinant lentiviruses may need the hepatocytes to be in cell cycle for efficient gene transfer in vivo.⁶⁴

Table 2

Advantages and limitations of liver-directed gene therapy methods.

Recombinant Adenovirus

Adenovirus types 5 and 2, are large linear double-stranded DNA viruses that are commonly used for preparing gene transfer vectors. Recombinant adenoviruses can transfer genes into both dividing and quiescent cells with high efficiency. Recombinant adenoviruses localize preferentially to the liver after intravenous administration in rodents.⁶⁵ However, clinical trials in human subjects indicate transduction of human liver cells may not occur with such high efficiency.⁶⁶ It is unclear whether this difference is based on the density of the adenoviral receptor, CAR, on the cell surface of hepatocytes of different species.

Adenoviral vectors are generated by disruption of the early region-1 (E1) by the insertion of the transgene. The E1 region encodes transcription factors required for the expression of adenoviral genes, and its disruption markedly inhibits the expression of the viral proteins. The recombinant virus is generated in packaging cells that provide the E1 gene products in trans. To abolish the possibility of viral gene expression and to increase the space available for insertion of the transgene, all structural genes of the virus have been deleted from the vectors.⁶⁷ These “gene-deleted” vectors require helper adenoviruses to provide the structural proteins.

Cellular and humoral immune responses to adenoviral proteins limit the clinical application of adenovectors. In humans, antibodies are memory cells often exist from previous infections by the adenoviruses, and naïve individuals readily develop humoral and cell-mediated immunity against the viral antigens after the initial injection of the recombinant adenovirus. Neutralizing antibodies block gene transfer following subsequent administrations of the vector. Adenovirus-specific cytotoxic lymphocytes attack the host cells that are infected by the recombinant virus, resulting in liver damage and rapid loss of the transgene after secondary gene transfer.⁶⁸ The helper-dependent, adenovectors, in which all viral genes deleted, retain their immunogenicity because of the viral proteins provided in trans by the packaging cells.⁶⁹ Although these vectors express transgenes for a longer duration than do the first generation adenoviral vectors, secondary or tertiary administration fails to transfer the transgenes.

Development of immune response requires presentation of antigenic peptides by antigen presenting cells. Following docking of the antigen presenting cells with uncommitted T cells, the antigen presenting cells and the T cells costimulate each other via the B7-CD28 and CD40-CD40 ligand interactions. Inhibition of the B7-CD28 costimulation prevents effective immune response. CTLA4-Ig, a soluble inhibitory protein inhibits this costimulation (see Fig. 3). Injecting CTLA4-Ig alone at the time of administration of adenovectors does not prevent antibody formation.⁷⁰ However, coexpression of CTLA4-Ig along with the target transgene permits multiple administration of the recombinant adenovirus.⁷¹ Host tolerance toward adenoviral proteins can be induced by injecting recombinant adenoviruses in utero or in newborn rats,⁶⁵ inoculating adenoviral proteins into the thymus of young adult rats,⁷² or orally administering of small doses of adenoviral proteins.⁷³ Since wildtype adenoviruses are human pathogens, safety concerns persist regarding tolerization of humans to adenoviral antigens remain. Adenoviral proteins may have additional toxic effects. A patient with ornithine transcarbamylase deficiency died shortly after recombinant adenovirus administration. The death was thought to have resulted from massive cytokine release, rather than an immune response to the virus.

Figure 3

Adenoviral vector, coexpressing CTLA4Ig. A recombinant adenovirus, expressing both CTLA4-Ig and human UGT1A1 (Ad-hUGT1A1-CTLA4Ig) was injected intravenously into Gunn rats. 3A. Immunohistochemistry of liver sections: panel a, untreated Gunn rat; panel (more...)

As discussed above, the hepatotropism of recombinant adenoviruses that is seen in rodents was not found in human trials.⁷⁴ Therefore, attempts are being made to modify the viral surface proteins to change their tropism, so that they can be internalized by hepatocytes through receptors that are normally present on the hepatocyte surface.⁷⁵

Herpes Simplex Virus-1

Herpes simplex virus-1 (HSV-1) is a 150 kb double-stranded DNA virus with a broad host range⁷⁶ that can infect both dividing and non-dividing cells. However, long-term gene expression in the liver has not been achieved with the currently available HSV vectors.

Recombinant Baculovirus

The Autographa californica nuclear polyhedrosis virus (AcNPV), which is usually used to generate recombinant proteins in insect cells,⁷⁷ can infect hepatocytes, but not other mammalian cells and can express transgenes that are driven by mammalian or viral promoters.

Recombinant Adeno-associated Virus

The wildtype adeno-associated virus-2 (AAV-2) is a small (4.7 kb) single-stranded DNA parvovirus that integrates preferentially on the q13.4-ter arm of human chromosome 19.⁷⁸ The AAV can remain latent for long periods, but can cause lytic infection following infection with a “helper virus,” such as adeno- or herpes simplex virus, or when the cell is exposed to genotoxic stimuli, e.g., UV light or irradiation.⁷⁹ AAV type 2 is internalized following binding to a cell surface heparin sulfate proteoglycan. The 145-bp inverted terminal repeats (ITR) that flank the AAV genome are needed for integration into the host genome. Following internalization, AAV vectors may remain as episomes or may integrate into the host genome over several weeks. The viral Rep protein directs the site specificity of AAV integration into chromosome 19. Recombinant vectors that lack this gene, do not exhibit the site-specificity of integration and in fact may remain in the nucleus as episomes for prolonged periods.⁸⁰

Infusion of high doses of the recombinant virus into the portal vein of factor IX-deficient dogs resulted in the appearance of 5% of normal levels of factor IX activity in plasma.⁸¹ Efficiency of AAV-mediated gene transfer to tumors could be augmented when used in combination with tumor irradiation or chemotherapy.⁸² Recombinant AAV causes a humoral immune response, which may be a problem if the vector needs to be readministered.

Simian Virus 40—based Vectors

Simian virus 40 (SV40) is a non-enveloped 5.2 kb double-stranded DNA papova virus. The large (Tag) and small (tag) T antigens are transcription factors required for the expression of the viral structural genes, VP1, VP2 and VP3. The recombinant vector is generated by replacing the Tag genes of the viral genome by the target transgene. All three structural genes can be deleted to make additional space for inserting transgenes. The recombinant virus is generated by transfecting the recombinant viral genome into a helper cells (e.g., COS cells) that provide Tag in trans.⁸³ Absence of the Tag gene makes the recombinant virus replication deficient and markedly reduces its immunogenicity. Up to 4.7 kb of exogenous DNA. can be insertd into SV40 vectors. The recombinant vector can be generated at 10⁹ infectious units (IU)/ml and concentrated to 10¹² IU/ml.^84,85 Recombinant SV40 integrates into the genome of both dividing and non-dividing cells (see Fig. 4). The range of target cells is broad and includes hepatocytes, neural cells and bone marrow cells.⁸³⁸⁵

Figure 4

Generation of a recombinant SV40 vector. The wildtype viral genome is a circular DNA, containing the origin, promoter and the viral assembly signal, the coding region for the transcription factors large (Tag) and small (tag) antigens, the coding regions (more...)

Non-Viral Vectors

Acknowledgments

This work was upported in part by NIH grants: RO1-DK 46057 (to JRC), RO1-DK 39137 (to NRC), the Liver Research Core Center grant DK-P30-41296 (Director, David A. Shafritz), The Gene Therapy Core of the Institute of Human Genetics of Albert Einstein College of Medicine and an American Cancer Society grant, RPG-00-066-01-CCE (to CG). The authors acknowledge the important contributions by many investigators in the fields of hepatocyte transplantation and liver-directed gene therapy that were not cited in this limited review.

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