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Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, 630 W. 168th St., 10th Flr., Rm. 508, New York, NY 10032. Fax 212-305-6443; e-mail hjw14{at}columbia.edu
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Key Words: indexing terms: hepatitis • primary biliary cirrhosis • metabolic diseases • recombinant DNA • gene therapy
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Introduction |
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The liver is affected by viral, autoimmune, and metabolic disorders
that can be diagnosed and treated with molecular biological methods.
Standard molecular cloning methods have been used to identify the
hepatitis C and hepatitis G viruses (HCV and HGV,
respectively)1
(1)(2). PCR is used to diagnose and
monitor patients with chronic viral hepatitis. Autoantibodies found in
certain liver diseases can be detected with assays that use highly
specific recombinant protein antigens (3). The genes
responsible for inherited disorders of metabolism that affect the
liver, e.g., 
1-antitrypsin deficiency (4),
Wilson disease (5)(6), and hereditary
hemochromatosis (7), have been identified, opening the way
for molecular diagnosis. Liver-specific gene therapy has already been
performed (8). This review will summarize some of the
current and potential molecular biology-based diagnostic methods and
treatments for liver diseases.
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Diagnosis |
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HCV was identified by standard molecular biological techniques; the
strategy is outlined in Fig. 1
. DNA and RNA were extracted from the plasma of chimpanzees
infected with serum from humans with non-A, non-B hepatitis. These
nucleic acids were used to construct a random-primed, complementary DNA
(cDNA) library in bacteriophage lambda that expressed fusion
polypeptides when infected in Escherichia coli. The
expression library was screened with diluted serum from patients with
non-A, non-B hepatitis, and cDNA clones were isolated that encoded
proteins that were recognized by serum antibodies. These investigators
went on to demonstrate that the cDNA clones they isolated were derived
from a positive-stranded RNA virus with a genome of ~10 000
nucleotides. Using a fusion protein expressed from an isolated cDNA
clone, the scientists at Chiron and their colleagues (9)
showed that the large majority of individuals with non-A, non-B chronic
hepatitis had serum antibodies against this newly identified virus.
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After this pioneering work on identification of the virus, the entire
HCV genome was cloned and sequenced in several laboratories
(10)(11)(12)(13). Sequence comparisons showed that HCV was a
member of the Flaviviridae family. The genome is a
positive-stranded RNA of ~9500 nucleotides with a highly conserved 5'
untranslated region followed by a single open-reading frame that
encodes a polyprotein of 3010 to 3033 amino acids, depending on the
isolate (Fig. 2
). The polyprotein is processed by the host cell and virally
encoded proteases into several structural and nonstructural
polypeptides (Fig. 2
). The major structural proteins are the core and
envelope polypeptides. The nonstructural proteins include, among
others, proteases involved in the processing of the polyprotein and an
RNA-directed RNA polymerase.
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The identification, cloning, and sequencing of HCV has led to a new era
in the diagnosis of chronic viral hepatitis. The diagnostic assays that
have resulted from this work can be broadly classified into those that
detect antibodies against HCV polypeptides and those that detect viral
nucleic acids (Table 1
). Each of these assays has a role in the laboratory diagnosis
of HCV infection.
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Because virtually all individuals infected with HCV develop antibodies
against viral polypeptides, antibody detection assays provide the
cornerstone of screening and initial diagnosis. The first ELISA to
detect anti-HCV antibodies utilized one recombinant antigen and
correctly detected ~80% of individuals infected with HCV
(9). Newer ELISAs utilize several recombinant viral
antigens expressed from cDNA clones, and a positive test result in an
individual with risk factors for HCV infection is ~99% sensitive.
Because of their simplicity and high sensitivity, ELISAs can be used to
screen the blood supply for HCV. One limitation of ELISAs in diagnosing
patients is that test results may remain positive in individuals who
have cleared HCV infection, either spontaneously or after treatment
with interferon-. Antibody detection assays are also not useful in
cases of acute HCV infection because of the several weeks it takes for
antibodies to develop (14). ELISAs may also be falsely
positive in individuals with hypergammaglobulinemia—which is often
present in patients with autoimmune hepatitis (15).
In cases where serum HCV antibody detection may be falsely positive by ELISA, a recombinant immunoblot assay (RIBA) can be helpful. In this assay, several recombinant HCV polypeptides, along with a control protein, are immobilized on a membrane, which is processed like a routine immunoblot (16). The advantage of the RIBA is that reactivity with one or more distinct recombinant HCV polypeptides and lack of reactivity with an unrelated control can be ascertained. Nonspecific reactivity with plastic microtiter wells, e.g., those used for ELISAs, is also eliminated.
Two general methods are currently available to detect HCV nucleic acids in serum. One, the branched DNA (bDNA) assay, utilizes capture of viral RNA by virus-specific nucleotide probes, followed by hybridization to bDNA molecules, which are detected by a chemiluminescent substrate system (17)(18). The bDNA assay can be reliably quantified and is relatively simple to perform in the clinical laboratory. The analytical sensitivity of the bDNA assay for detection of serum HCV RNA, however, is only 10-4 that of assays based on PCR (19).
Assays with the most analytical sensitivity currently routinely available for the detection of HCV are based on reverse transcription followed by PCR (RT-PCR). The discovery by Baltimore (20) and Temin and Mitzutani (21) of retroviral reverse transcriptase, which directs the synthesis of cDNA from a RNA template, was of fundamental significance in modern molecular biology. PCR, conceived of by Mullis (22)(23), has also revolutionized modern molecular biology by allowing the in vitro enzymatic amplification of large amounts of DNA from a small number of molecules. The use of thermostable DNA polymerases, such as Taq from Thermus aquaticus, has made PCR a rapid, reproducible, and semiautomated procedure (24). The area of viral hepatitis diagnosis has not escaped the impact of the discoveries of reverse transcriptase and PCR. These procedures can be combined to detect very low quantities of HCV RNA in serum or tissue. RNA extracted from serum is used as the template for reverse transcription of cDNA, which is then amplified by PCR. The amplified cDNA products are separated by agarose gel electrophoresis and detected by various methods, e.g., ethidium bromide staining or Southern hybridization. The major potential drawback of RT-PCR assays is their extreme sensitivity, which makes contamination a concern. However, standardization of this method in excellent clinical laboratories has made it the gold standard for detecting HCV RNA.
Several techniques, such as competitive inhibition or use of various reaction lengths and cycle numbers with known amounts of template, can make RT-PCR semiquantitative (19)(25)(26)(27). Values for viral load can be used to assess a patient's response to treatment. RT-PCR can also be used to determine the HCV genotype. At least 6 major genotypes and 11 subtypes of HCV have been identified on the basis of genomic sequence differences (28). Different genotypes may differ in the severity of disease they cause and in their response to treatment (29). Several methods exist for determining HCV genotype, including RT-PCR followed by direct sequencing, RT-PCR with genotype-specific primers, restriction fragment length polymorphism (RFLP) analysis of PCR-amplified DNA sequences, and hybridization of the amplified products to oligonucleotide probes immobilized on a solid-phase support (30)(31). Some of these genotyping assays are now performed in clinical laboratories; combined with estimates of serum viral load, knowledge of the infecting genotype may be useful in predicting prognosis and response to therapy (29). Sequence variations more subtle than those between different genotypes, e.g., point mutations that cause amino acid substitution in the NS5A gene product, may also correlate with response to interferon therapy (32).
With regards to chronic hepatitis C, molecular biology has created a
new discipline for the practicing hepatologist. Before 1990, the virus
that causes this disease was not known. Now, infected individuals can
be identified in the clinical laboratory with assays based on molecular
biology and consequently be considered for treatment with
interferon- (produced by recombinant DNA technology) and other
potentially promising agents.
Hepatitis B.
Regarding serological diagnosis, hepatitis
B differs from hepatitis C in that viral protein antigens of HBV can be
readily detected in serum. Hepatitis B surface antigen is detectable in
virtually all infected individuals, and the hepatitis B e antigen is
detected in most patients who have high amounts of viral replication.
Accordingly, nucleic acid tests are not necessary to diagnose viral
infection with HBV in the clinical laboratory. In some instances,
however, amplification of viral nucleic acid sequences from serum may
be helpful in assessing HBV-infected individuals.
HBV is a partially double-stranded DNA virus. Therefore, viral genomic sequences can be amplified directly by PCR without reverse transcription. Because viral load may be predictive of response to treatment and prognosis, it may sometimes be useful to use semiquantitative PCR to estimate serum viral concentrations. Assays utilizing bDNA can also be used to quantify HBV DNA in serum (17). Sequencing the HBV DNA amplified by PCR can be useful for identifying mutant viruses of clinical significance. For example, mutations in the HBV precore region of the genome have been identified that prevent transcription of the e antigen but allow the continued assembly of infectious virus (33)(34). Early reports associated these precore mutants with severe liver disease in the absence of hepatitis B e antigen (33). A recent report has also shown that surgeons infected with precore mutant strains, without detectable serum e antigen, can transmit HBV to patients (35).
Hepatitis G.
In 1995 and 1996, a new human hepatotropic
virus was identified (2)(36). In initial
studies by investigators at Abbott Labs., representational difference
analysis was used to identify nucleic acid sequences of two related
viruses, GB-A and GB-B, in tamarin marmosets that had been infected
with serum from a surgeon, "GB," who had contracted acute hepatitis
(37). Representational difference analysis is a powerful
method that had previously been used in pioneering work by
investigators at Columbia University to identify a herpesvirus
responsible for Kaposi sarcoma (38). In the present
instance, however, the two putative hepatitis viruses identified by
this method were probably tamarin viruses and not human pathogens
(39). Nonetheless, when the Abbott investigators took RNA
from human serum samples that contained antibodies against GB-A and
GB-B and subjected them to RT-PCR with degenerate oligonucleotide
primers, DNA sequences were amplified that were related to but distinct
from GB-A, GB-B, and HCV; these sequences were determined to belong to
a novel flavivirus the investigators termed GB-C (36).
In independent work, investigators at Genelabs Technologies (2) determined the complete genomic sequences of two isolates of a flavivirus that they called HGV. HGV is essentially identical to the GB-C virus and is related to HCV, GB-B, and GB-A. To isolate HGV, the investigators at Genelabs screened a cDNA expression library constructed from the plasma of a patient with chronic hepatitis C. Immunoscreening of the expression library with the patient's serum identified several cDNA clones encoding HCV polypeptides as well as clones encoding other related but unique polypeptides. From the unique cDNA clones, an anchored PCR method was used to amplify overlapping clones for the entire viral genome. Using RT-PCR, these workers identified HGV sequences in 13% of 38 US patients with non-A, non-B, non-C, non-D, non-E hepatitis and in ~18% of patients with HCV infection.
At present, the significance of HGV/GB-C virus as a cause of acute and chronic liver disease remains controversial. Although it has been associated with acute and chronic hepatitis (2), some investigators argue that this virus may not be a major cause of hepatitis in humans (39)(40). Resolution of this controversy is important, given the presence of HGV/GB-C in the blood supply (2)(39)(40). ELISAs based on HGV/GB-C polypeptides are able to detect antibodies against this virus in the blood supply and in infected individuals.
autoimmune liver diseases
Primary biliary cirrhosis (PBC), autoimmune hepatitis, and some
cases of primary sclerosing cholangitis are associated with the
presence of autoantibodies against intracellular proteins. Testing for
autoantibodies provides a cornerstone of diagnosis of these diseases,
especially PBC. Molecular biological methods have made possible the
identification of some of the intracellular protein antigens and the
predominant epitopes recognized by some of the disease-specific
autoantibodies. This work has led to development of assays that can be
used in the clinical laboratory. Assays utilizing recombinant proteins
should make tissue-based immunofluorescence assays obsolete in cases
where the autoantigen is known. In PBC, cDNAs for several of the major
mitochondrial and nuclear autoantigens have been cloned and sequenced,
and recombinant proteins have been used to detect autoantibodies (Table 2
).
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In almost all cases of PBC, specific autoantibodies are found that are almost never present in individuals with other diseases. For example, >90% of individuals with PBC have autoantibodies directed against the E2 subunits of mitochondrial oxoacid dehydrogenases (3)(41)(42). In ~25% of PBC cases, autoantibodies against gp210, an integral membrane protein of the nuclear pore complex, are detectable (43)(44).
Autoantibodies against the mitochondrial oxoacid dehydrogenase E2 subunits are virtually 100% specific for PBC (41)(42). Their detection is of central importance in the diagnosis of PBC, which depends on a combination of clinical, biochemical, immunological, and histological findings (45). Autoantibodies that recognize these proteins have been traditionally been called anti-mitochondrial antibodies and are detected in most clinical laboratories by indirect immunofluorescence microscopy. Indirect immunofluorescence microscopy is fairly simple to perform in the clinical laboratory but has several limitations. First, the precise nature of the autoantigen cannot be established, and antibodies that label other mitochondrial proteins, not necessarily associated with PBC, will be detected. Second, antibodies against nonmitochondrial cytoplasmic antigens, including those against the cytochrome P450 isoforms found in patients with type II autoimmune hepatitis, produce labeling patterns similar to anti-mitochondrial antibodies and are often misinterpreted as such. Third, ELISAs utilizing recombinant proteins or synthetic polypeptides are easier to automate than are tissue-based immunofluorescence assays, which usually require subjective human interpretation.
Screening of bacteriophage 
cDNA expression libraries with
autoantibodies from affected patients identified the E2 subunits of the
pyruvate dehydrogenase complex, the branched-chain 2-oxoacid
dehydrogenase complex, and the 2-oxoglutarate dehydrogenase complex as
the major mitochondrial autoantigens in PBC
(41)(42)(46)(47)(48). ELISAs utilizing
expressed recombinant proteins or designer polypeptides have been
devised that are sensitive and specific for detection of antibodies
against these proteins
(3)(49)(50). Determination of the
immunodominant epitopes on these three proteins has allowed
construction of a "designer hybrid" clone that contains the major
epitope of each (50). An immunoassay utilizing a designer
polypeptide expressed from this clone is highly sensitive and specific
for the diagnosis of PBC (50).
In addition to antibodies against mitochondrial oxoacid dehydrogenase E2 subunits, some individuals with PBC also have other specific autoantibodies, e.g., as mentioned above, against gp210. Gp210 was first identified as a common autoantigen in PBC by immunoblotting assays utilizing the protein purified from cells (51). The cDNA cloning of gp210 made possible the identification of its immunodominant epitope, which was mapped to a stretch of 15 amino acids (52). Two ELISAs have been developed for detection of autoantibodies against the predominant autoepitope of gp210, one utilizing a recombinant fusion protein expressed in E. coli (53), the other a synthetic polypeptide (54).
In addition to oxoacid dehydrogenases and gp210 autoantigens in PBC, several other proteins have been identified as autoantigens in liver diseases, and for some the immunodominant epitopes have also been determined. Examples include Sp100 (55) and LBR (56)(57) in PBC and nuclear lamins (58) and cytochrome P450 isoforms in type II autoimmune hepatitis (59)(60). Future work with expression cloning may also identify other protein autoantigens in liver diseases, such as the "atypical-ANCA" or "x-ANCA" autoantigen in primary sclerosing cholangitis. ELISAs that utilize recombinant proteins or synthetic polypeptides to detect autoantibodies in individuals with liver diseases should be useful in the clinical laboratory.
inherited metabolic diseases
The Human Genome Project and the emerging discipline of genomics
is changing the way in which much of clinical medicine is practiced.
The identification of disease genes by positional cloning, in which the
inheritance of linked genetic markers at known chromosomal locations is
examined, and other methods has already made a tremendous impact. The
genes responsible for many of the major inherited metabolic diseases
that affect the liver have now been identified (Table 3
). These discoveries have paved the way for molecular diagnostic
methods for these disorders. Molecular genetics assays will replace
traditional chemical measurements and invasive procedures such as
biopsy to diagnose inherited liver diseases. The molecular biology of
some of the major inherited liver disorders is briefly reviewed here,
followed by a discussion of some feasible molecular diagnostic methods.
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1-Antitrypsin deficiency.
The human gene
encoding 1-antitrypsin is localized to chromosome
14q31–32 (61). Mutations in the
1-antitrypsin gene that cause the disease are of several
types and include those that produce a deficiency of enzyme action,
null mutations, and altered function of the gene product. Mutations
producing deficiency or absence of enzyme action lead to an increased
risk of developing emphysema (62). Mutations that result
in defective secretion of the protein from hepatocytes cause liver
disease. Cloning and sequencing of cDNAs has demonstrated that human
1-antitrypsin is a protein of 394 amino acids
(63)(64). The ZZ phenotype, the one most
commonly associated with the development of liver disease, has a lysine
substituted for a glutamic acid residue at position 342
(65)(66). In ZZ homozygotes, the protein is
not properly processed in the secretory pathway, and 85% of it
accumulates in the endoplasmic reticulum. The Z mutation causes an
interaction between the reactive center loop of one molecule and a
portion of another (67). The resulting protein aggregates
are not secreted and their accumulation can lead to liver disease.
The Z allele results from a single amino acid substitution created by a
G to A transition in the gene for 1-antitrypsin.
Synthetic oligonucleotide probes have been used to develop a sensitive
and direct assay for the presence or absence of this mutation
(68). This assay has even been used to diagnose the
mutation prenatally (69).
Wilson disease.
In 1993, the gene for Wilson disease on
chromosome 13 was cloned (5)(6). The gene
encodes a copper-transporting ATPase homologous to the protein that is
mutated in Menkes disease, another inherited disorder of copper
metabolism (5)(6). At least 25 different
mutations in the Wilson disease gene have been described, including
small insertions, deletions, missense, nonsense, and splice-site
mutations (70). These different mutations may explain in
part the wide phenotypic variation in individuals with Wilson disease:
e.g., age of onset, neurological vs liver disease, and severity
(70).
Wilson disease is at present diagnosed by measurements of 24-h urine copper and serum ceruloplasmin concentrations and by the presence of excessive copper in liver tissue obtained at biopsy. Given the wide variety of mutations that can cause this disease, molecular biological diagnosis in a random individual could require sequencing the entire gene. If a particular mutation has been characterized in one subject, however, it can be readily detected in a family member. As methods for rapid sequencing and the simultaneous detection of multiple mutations are developed, genetic tests for the laboratory diagnosis of Wilson disease should be produced.
Hereditary hemochromatosis.
Hereditary hemochromatosis
is the most common inherited disease in persons of European descent,
the prevalence of homozygosity being 3–5 per 1000 and a carrier
frequency of 1 in 10 (71)(72). Hemochromatosis
is inherited as an autosomal recessive trait, and homozygous
individuals have increased absorption of dietary iron resulting in
excess deposition in the liver, heart, joints, and some endocrine
organs. Phlebotomy is an effective treatment; however, the disease
often goes undiagnosed. Increases in serum ferritin and transferrin
saturation are suggestive of hemochromatosis, and diagnosis is made by
measuring hepatic iron contact in liver tissue obtained at biopsy.
In the 1970s, a linkage of hereditary hemochromatosis to the HLA-A locus on chromosome 6 was established (73). This linkage has been useful clinically in the analysis of risk in the relative of a patient. In 1996, investigators at Mercator Genetics (7) identified a candidate gene for hemochromatosis on chromosome 6. They termed this gene HLA-H because of its homology to other MHC class I family members (recently, the name HFE has been recommended for this gene). These investigators found a G to A transition at nucleotide 845 of this gene that resulted in a cysteine to tyrosine substitution at amino acid residue 282 in the protein. Of 178 patients examined, 148 were homozygous for this mutation, 9 were heterozygous, and 21 carried only the normal allele. Several other groups have confirmed the frequency of this mutation in individuals with hereditary hemochromatosis (74)(75)(76). A C to G transversion that results in a histidine to aspartic acid substitution at amino acid residue 63 has also been identified in fewer individuals with hereditary hemochromatosis (7); however, some investigators have not been able to establish a relationship between this mutation and the disease (76). The finding that only ~85% of patients carry the cysteine to tyrosine mutation suggests that other mutations in the HFE gene or in different genes may also cause hemochromatosis.
Hereditary hemochromatosis is a common disorder, for which effective treatment is available. The fact that ~85% of affected individuals appear to have a single mutation should make genetic testing for the majority of cases relatively simple. For these reasons, hereditary hemochromatosis may be an ideal disorder for genetic screening of the entire population.
Hereditary hyperbilirubinemias.
Hereditary
hyperbilirubinemias are generally divided into those that cause
increases in unconjugated serum bilirubin concentrations and those that
cause increases in conjugated serum bilirubin. Two disorders of the
former type are Crigler–Najjar syndrome and Gilbert syndrome. Patients
with type I Crigler–Najjar have a complete absence of activity of the
isoform of UDP-glucuronosyltransferase that conjugates bilirubin to
mono- and diglucuronides in hepatocytes. Individuals with type I
Crigler–Najjar syndrome have severe childhood disease. Patients with
type II disease have partial deficiency of this enzyme and generally
survive to adulthood without significant problems. Several different
mutations in the gene that encodes the bilirubin-conjugating isoform of
UDP-glucuronosyltransferase, UGT1 on chromosome 2, have been
shown to cause type I and type II Crigler–Najjar
(77)(78)(79)(80)(81)(82). Gilbert syndrome is a common cause of hereditary
hyperbilirubinemia. The condition is benign and of little clinical
significance except that its presence can cause physicians to search
for other liver diseases in patients. Mutations in the promoter region
of UGT1 have been described in individuals with Gilbert
syndrome (83). These and other mutations in
UGT1 and possibly other genes may be responsible for the
condition.
Dubin–Johnson syndrome is characterized by conjugated hyperbilirubinemia and deposition of a dark pigment in the liver. Although rare in most of the world, Dubin–Johnson syndrome occurs with a frequency of ~1 in 1300 among Iranian Jews (84). The disorder results from the inability of conjugated bilirubin to be secreted from hepatocytes. A cDNA for rat cMOAT, a protein homologous to the multidrug resistance proteins located in the canalicular membrane of hepatocytes, has recently been characterized (85). A single basepair deletion in the cMOAT gene, which results in a truncated protein and is associated with the impaired secretion of organic acids from hepatocytes, has been described in the TR- rat, an animal model of Dubin–Johnson syndrome (85). Cloning and sequencing the human homolog of rat cMOAT may establish the genetic defect in Dubin–Johnson syndrome.
Diagnostic methods.
Knowledge of the genes and mutations
that cause inherited metabolic liver diseases will revolutionize the
manner in which these conditions are diagnosed. Screening tests can be
developed for common diseases such as hemochromatosis. Genetic tests
can replace invasive procedures, such as liver biopsy, to rule out
rarer conditions. Some molecular diagnostic methods that can be used,
depending on the nature of the mutation, are given in Table 4
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It is relatively easy to detect a specific mutation that causes a
disease. Examples are the G to A transition that most commonly causes
the Z-variant of 1-antitrypsin deficiency or the
nucleotide change in HLA-H associated with 85% of cases of
hereditary hemochromatosis. Detection of a mutation in a family member
when the mutation is already known in a relative is also fairly simple.
In these instances, RFLP analysis or specific oligonucleotide probes
can be used to detect the mutation in total genomic DNA or DNAs
amplified by PCR.
It is more difficult to diagnose a genetic disorder if multiple mutations can be responsible, as in Wilson disease, and if a mutation has not been characterized in a family member. If a truncated protein is produced by most mutations, in vitro translation and examination of the product can detect an abnormal protein. An assay based on in vitro translation has been developed to detect mutations in the APC gene in individuals with familial adenomatous polyposis (86). Amplification of cDNA or genomic regions by PCR followed by standard DNA sequencing methods can also be used to analyze genes in which many different mutations can cause disease; however, this is very time consuming.
High-density oligonucleotide arrays, such as those produced by light-directed, spatially addressable parallel chemical synthesis, can be used for rapid DNA sequence determinations (87)(88)(89)(90). Oligonucleotide probes synthesized and bound in ordered arrays to glass or nylon chips are used to hybridize to fluorescently labeled target DNAs. The extent and pattern of fluorescence on the chips after washing can be used to determine DNA sequences. Oligonucleotide arrays on chips have been used to simultaneously monitor the expression of multiple genes in a cell type (91), analyze the entire human mitochondrial genome (92), and simultaneously screen for 24 different heterozygous mutations or polymorphisms in a portion of the human BRCA1 gene (93). With regard to inherited liver diseases, oligonucleotide arrays can be used to simultaneously detect the various possible mutations in the genes for disorders such as Wilson disease.
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). The use of recombinant interferons to treat viral hepatitis
and the use of recombinant vaccines to protect against HBV infection
are currently used widely by medical practitioners. Liver-directed gene
therapy is not yet routine; however, pioneering human trials have
already been performed (8). The use of recombinant
polypeptides to obtain critical protein structural information for the
rational design of drugs has also been applied to liver diseases
(94)(95). DNA vaccines (96),
antisense oligonucleotides (97), and ribozymes
(98) are being developed in many laboratories and may hold
promise as agents for the treatment and prevention of liver diseases.
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recombinant drugs and vaccines
Recombinant DNA technology has led to the production of drugs and
products for the treatment and prevention of liver diseases. Notable
among these products are interferons for the treatment of viral
hepatitis and vaccines to prevent hepatitis B. Recombinant interferon
-2b is effective in treating chronic hepatitis B, C, and D
(99). Interferon -2b was the first recombinant drug
approved by the US FDA for treatment of hepatitis and has been widely
used (99). Other forms of recombinant interferons are also
now available.
Human HBV vaccines have been produced by recombinant DNA technology (100). Vaccines composed of hepatitis B surface antigen particles expressed from recombinant DNA in budding yeast have repeatedly been demonstrated to be effective (101). Almost all healthy individuals younger than age 40 develop protective serum titers of anti-hepatitis B surface antigen antibodies after a series of three injections of commercially available preparations. Response rates are lower in immunocompromised and older individuals. Recombinant vaccines that protect against other hepatitis viruses may soon be developed. Preliminary results in cynomolgus monkeys suggest that vaccination with a recombinant protein representing part of the viral capsid antigen may be protective against hepatitis E (102).
rational drug design
The ability to express portions of proteins from recombinant cDNA
clones provides the opportunity to determine their structures.
Structure determination can lead to the rational development of drugs
that can, for example, inhibit an enzyme or bind to a receptor. The
first steps towards rational drug design have recently been reported
for the NS3 protease of HCV. This protease cleaves nonstructural
polypeptides from the HCV polyprotein and is essential for viral
replication. Workers at Vertex Pharmaceuticals (94) and
Agouron Pharmaceuticals (95) have used x-ray
crystallography to determine the structure of the NS3 protease.
Standard molecular biological methods were used to obtain sufficient
quantities of protein for crystallization. Based on the
three-dimensional structures determined in these studies, drugs can be
rationally designed to inhibit this enzyme, which is essential for
viral replication. Similar methods should be useful in developing other
antiviral drugs and possibly even drugs that can stimulate defective
enzymes in inherited diseases.
gene therapy
Gene therapy holds promise as future treatment for liver diseases,
including viral hepatitis, inherited metabolic diseases, and cancer. A
detailed discussion of hepatic gene therapy is beyond the scope of this
paper; however, this topic has recently been elegantly reviewed by
Wilson and Askari (103). In brief, two types of gene
therapy strategies can be used to target the liver: ex vivo and in
vivo.
In ex vivo gene therapy (Fig. 3
), hepatocytes are removed from the patient and cultured in
vitro. The desired expression vector is introduced into the cultured
hepatocytes. Several methods can be used to get the gene into the
cultured cells, including transduction with a recombinant retrovirus.
The transduced hepatocytes are then introduced into the portal vein and
lodge in the patient's liver. Hepatocyte-directed ex vivo gene therapy
has already been performed in human subjects with familial
hypercholesterolemia (8), a disease that results from a
defect in the LDL receptor gene. In these human studies, hepatocyte
cultures established from patients' cells were transduced with
recombinant retrovirus vectors that contained a functional LDL receptor
gene. Once high-efficiency gene transfer was evident, the cells were
introduced into the portal veins of the patients. Three of five
patients treated in this fashion had persistent notable reductions in
serum concentrations of cholesterol and LDL.
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In liver-directed in vivo gene therapy, genetic material is introduced into hepatocytes by gene transfer vectors that function after being introduced directly into the patient. Vectors for liver-directed in vivo gene therapy can be recombinant viruses taken up by hepatocytes, DNA complexed with proteins taken up by hepatocytes, or naked DNA. To develop effective in vivo gene therapies for liver diseases, investigators must devise vectors that are specific for hepatocytes and must induce hepatocytes to readily take up these vectors.
Besides the transfer of human genes, other nucleic acid-based therapies currently under experimental development may be useful in the treatment of liver diseases. Antisense oligonucleotides (97) can be used to inhibit the expression of (e.g.) genes essential for the replication of hepatitis viruses. Ribozymes are catalytic RNA molecules that can be used for similar purposes as antisense oligonucleotides (98). DNA transfer technology can also be used for vaccines (96).
In conclusion, molecular biology has already made a large impact on the diagnosis and treatment of liver diseases. In the next millennium, molecular biological methods will become increasingly important in clinical and laboratory medicine. Creative research and hard work should lead to inexpensive diagnostic tests and effective treatments for many of the liver diseases that affect people throughout the world.
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