Hepatitis C Clinical Trial
Official title:
Differential Gene Expression in Liver Tissue and Blood From Individuals With Chronic Viral Hepatitis With or Without a Complicating Hepatoma or Autoimmune Liver Disease
The purpose of this research is to study body materials like blood proteins as well as white
blood cell and liver cellular RNA in individuals with liver diseases such as chronic viral
hepatitis with or without hepatoma and autoimmune liver disease. Presently it is not
understood how infection with chronic viral hepatitis or autoimmune liver disease damages
the liver. This research study enroll patients with either chronic viral hepatitis with or
without hepatoma or autoimmune liver disease.
The purpose of this study is to find the genes that are expressed in both the circulating
white blood cells and the liver of patients with varying degrees of liver damage of
different causes. Genes are biological messengers some of which determine how the body
responds to injury. We anticipate that results from Differential Gene Expression (DGE)
analysis will allow us to make predictions about likelihood of disease progression and/or
response to treatment.
In addition we will test the blood for markers of injury. The blood collected will be
prepared differently from the liver tissue. We will use technologies to express pure
proteins and then we will investigate the functions of these proteins. Nearly all drugs act
on proteins, not genes, so understanding proteins is the key to really effective new
medicines. Similarly the first signs of ill health appear in changes to the body’s blood
proteins, making them the most sensitive diagnostic indicators. The studies we plan are
called proteomics.
We will later correlate the patterns of gene expression in both circulating white blood
cells and the liver tissue with clinical outcome and patterns of proteins measured in blood
and we hope to gain an understanding of how the disease process occurs, which may in turn
help us to make more precise diagnoses and develop new forms of treatment.
These techniques that we use are still experimental and so we do not yet know if they will
be helpful in monitoring changes which may help us to predict the potential severity of your
liver disease or even if they can be used to indicate who will best respond to treatment.
The objective of this study is to identify genes that are specifically up or down regulated
in chronic viral hepatitis and autoimmune liver disease and then to examine how these
expression patterns relate (if at all) to clinical outcome. The expression pattern of
thousands of genes should provide extremely powerful statistical tools to distinguish
between different pathophysiological states. It is well recognized that neither hepatitis B
or hepatitis C is directly cytotoxic, their effect appears to be mediated via an immune
response. Similarly in individuals with autoimmune liver disease e.g. primary biliary
cirrhosis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH) immune
mediated mechanisms appear to be the cause of their liver disease although the eliciting
antigen(s) remain unknown. The pattern of gene expression can give clues as to both the
cause and the pathogenesis of disease. For instance, if a disease is caused by an infection,
a specific cytokine response is observed which will be different if this infection is viral
or bacterial or parasitic. It is also possible that a certain pattern of response would be
elicited if the disease is caused as a response to xenobiotic. In patients with autoimmune
liver disease, it is possible that an endogenous and/or an exogenous antigen initiates the
disease with a subsequent autoimmune response. Finally, it is also possible that a
completely unexpected series of cellular events is responsible for pathogenesis, and the
microarray experiments will enable us to discover these processes.
HCV - The molecular genetics of hepatitis C viral infection HCV molecular biology: HCV is a
positive-stranded RNA Flaviviridae virus with a 9.6kB genome. The genome encodes a
polyprotein of approximately 3000 amino acids which is subsequently cleaved by host and
virus-specific proteases to yield 4 structural and 6 nonstructural polypeptides. The 4
functional proteins include a metalloproteinase (NS5A), serine protease (NS3/NS4A), RNA
helicase (NS3), and a RNA-dependent RNA polymerase (NS5B). Six different genotypes of HCV
have been described, with genotype 1 being the most prevalent worldwide and genotype 4 being
the most prevalent in the Middle East.
The various genotypes interact differently with the host immune system, though the molecular
basis for this is unclear. Chronic infection by genotypes 1 and 4 is notoriously resistant
to treatment with IFN, while genotypes 2 and 3 have relatively better responses. This
difference may be in part due to mutations in the NS5A protein. In cellular models NS5A can
interact with IFN-induced antiviral enzymes such as 2’5’OAS. Other HCV proteins likely
interact with cellular proteins to alter responses to immune and IFN antiviral mechanisms:
HCV core protein dampens lymphocytic Th1 responses and influences IRF, Jak/STAT and iNOS
pathways. Although these results suggest how the virus may alter known cellular pathways,
viral evasion of the immune system is multifactorial and clearly complex.
Research Hypothesis: The processes that lead to persistence of acute HCV infection are
related, at a molecular level, to those that drive HCV resistance to IFN therapy.
Elucidating the specifics of the host/viral response in both of these contexts will provide
novel targets for small-molecule antiviral therapy which can be applied both to acute and
chronicHCV.
Progress Report: CIHR 106800 We have established a large and growing tissue and RNA registry
for the study of liver disease, and have performed a comprehensive gene array analysis of
chronic HCV. Some of our most important findings are detailed here.
HCV is a complex and progressive disease in which the interaction of the host and virus has
profound effects. The combination of clinical and experimental variability can seriously
confound the results of a gene expression study and mandates that a large number of patients
and samples be examined. The fact that liver pathology can be similar among a number of
different diseases also necessitates that many different patients and diseases are included
in the analysis. Accordingly, we have analyzed large numbers of patients and controls, and
have maintained careful databases of clinical details in order to perform multivariate
analyses of the gene array data. At present we have used a 19K human microarray to determine
hepatic gene expression in 86 HCV patients and have compared these to 24 normal, 20 HBV and
14 PBC patients. The array data are of high quality: our rates of confirmation by real-time
PCR exceed 80%. We have now examined the relative contributions of age, sex, viral genotype
(1 vs 2/3), degree of fibrosis and disease activity on the genes most upregulated by
infection with HCV.The most important determinant of consistent gene expression profiles in
HCV infected livers is viral genotype (genotype 1 vs all others). However, within Genotype 1
samples there is a clear difference in gene expression.
This divergent host-virus interaction is reflected in the subsequent response to PegIFN/Rib
therapy. All of the HCV liver biopsies we have analyzed to date were taken from patients who
went on to treatment with PegIFN/Rib. At present, treatment has been completed in 49 of
these patients. In an observation that may have significant clinical utility, we showed that
the ultimate response to treatment (sustained viral response versus vs
nonresponder/relapser) was reflected in the original gene expression profiles. We identified
18 genes whose expression levels distinguished responders from non-responders/relapsers in
with an accuracy of >90%. Based on this observation we filed a patent to protect the
intellectual property and formed a company to commercialize the genetic test. The results
have also been submitted for publication. Taken together we believe we have described a set
of genes, or perhaps a biological process, that lies at the heart of the host-HCV
interaction.
Section I. A possible role for the UBP43, ISG15 Pathway in the HCV Interferon While our gene
expression profiles have distinguished two types of chronic HCV infection – one which
responds to IFN, and the other that does not - our main objective is to achieve a molecular
understanding of the roles of specific genes and proteins in the host response to HCV.
Though an altered gene expression level does not directly implicate the gene product in the
biological pathway, there are compelling biological reasons to suggest that at least some of
the genes on our list play an important role in viral-IFN responses. Several of the most
up-regulated genes are interferon-sensitive (including OAS, Mx1, ISG15, VIPERIN, IFIT, and
GIP2). Polymorphisms of OAS have been weakly linked to self-limited HCV infection, and
polymorphisms of Mx1 have been weakly linked to response status. Hepatic mRNA levels for
OAS, Mx1, and GIP2 are increased in chronic HCV but none, alone, have been linked to
treatment outcome. The genes that are not directly IFN-responsive may play roles in cellular
pathways important for IFN responses (PI3AP1, DUSP1), and are involved in inflammatory cell
activation and maturation (LAP).
ISG15 and UBP43/USP18, which are components of a newly-recognized IFN regulatory pathway,
are perhaps the most interesting subset of genes that were found to be differentially
expressed in our studies. Both genes are expressed more highly in nonresponder (NR) compared
with responder (R) liver tissue, suggesting that they might interfere with the immune
response in HCV-infected liver ISG15 is a ubiquitin-like (Ubl) protein which is thought to
be important to innate immune functions and is covalently linked to proteins following
interferon activation. How this link is accomplished is controversial and may involve some
overlap with known E2 Ub enzymes. Interestingly, the E2 Ub enzyme UBCH8 was recently
identified as an activator of ISG15,34 and is one of the genes upregulated in response to
chronic HCV in our microarray analysis. The conjugation of ISG15 to its target proteins is
reversed by a highly specific protease, USP18/UBP43. UBP43 belongs to a family of
Ub-specific proteases and is induced by IFN, LPS and viral infection; it is degraded by the
Skp2 Ub ligase. Loss of USP18 in mice leads to IFN hypersensitivity. Our data links UBP43
with HCV, and suggests that the pathway is important in a divergent host/viral response.
Research Objective I. To establish the roles of the 18 genes – in particular, of ISG15 and
UBP43 - associated with a divergent host/ virus interaction, using a HCV replicon model.
Research Protocols for Section I:
DNA microarray studies shed light on the transcription state of the cell but do not
necessarily link specific gene products with the biological problem being addressed.
Accordingly, it is important to test the roles of individual genes using other approaches.
We will use in vitro assays of viral replication to study the roles of genes whose
expression we have found to be altered in HCV infected liver. These are not straightforward
experiments, since HCV is a difficult virus to study. HCV replicates only in humans and
chimpanzees, and only poorly in isolated human peripheral blood cells, though these do act
as an extrahepatic viral pool. Subgenomic RNA replicons can be transfected into certain cell
lines and maintained, but to date only a few genotypes have been successfully translated
into a replicon model (1b, 1a, and 2a). Recently, an elegant HCV model was described in
Edmonton in which human hepatocytes are transplanted into SCID-beige mice. This model
supports HCV infection and replication; however, since the bulk of HCV effects are due to
the immune response generated by the virus, this model as it exists currently may not be
ideal for studying how the virus leads to liver damage and evades antiviral immune responses
in humans.
We have chosen to explore the role of our genes of interest using the full-length HCV
genotype 1a replicon assay, in collaboration with Dr. Charles Rice (Rockefeller University,
New York). This replicon is a variant of the full-length HCV H77 strain described recently
by Dr. Rice, but has higher levels of replication in cell culture than the previous version.
The replicon model is ideal for rapidly testing the effects of many genes and has been used
extensively to study HCV mechanisms at a cellular level. This approach will be used for all
of the genes identified as of potential interest in the studies above, focusing first on the
ISG15 and UBP43 genes, second on the 16 genes remaining in our response-discriminatory list,
and finally on novel genes identified in the gene expression studies detailed below.
Ia. Effect of siRNA knockdown and upregulation (transfection) of individual genes, including
UBP43 and ISG15, on HCV replicon in Huh7.5 cells.
We will use RNA interference (RNAi) to "knock down" specific genes in order to evaluate
their roles on viral replication. Small double-stranded RNA molecules will be used to induce
sequence-specific degradation of homologous single-stranded RNA. For most of these studies
stock Huh7.5 cells will be compared to Huh7.5 cells stably expressing the full-length
Genotype 1a HCV replicon developed in Dr. Charles Rice’s laboratory. The methodology
detailed below is routine in our laboratory and the laboratories of our collaborators.
Control studies: Baseline gene and protein expression, effect of IFN, effect of replicon:
The replicon system can be used not only to test the effect of a given gene on viral
replication, but also for the effects on the IFN response. Previous studies have
demonstrated that HCV 1b genotype RNA replication is sensitive to IFN-a treatment, even at
low doses. In fact, the roles of two of the genes on our list of eighteen, MxA and
GIP3/IFI6-16, have already been examined. The IFN inhibition of HCV 1b replicon RNA
replication is independent of MxA, and transient transfection of GIP3 does not itself
inhibit the 1b replicon but does enhance the effect of IFNa. We will now study the roles of
the remaining genes in our list using the HCV genotype 1a replicon. The studies described
below outline our approach for the ISG15 and UBP43 genes; however, similar strategies will
be employed for the other genes of interest.
Huh7.5 cells and HCV replicon cells will be incubated for 12 hours with increasing doses of
IFNa2b (0, 1,10 and 100U/ml), IFNb (0,10,100 and 1000 U/ml ), or IFNg (0,10,100 and 1000
U/ml), and ISG 15, UBP43, and HCV mRNA levels determined by real-time PCR. ISG15 protein
levels will be determined by western blot (mAb the kind gift of Dr. E.C. Borden, Scripps
Institute); UBP43 western studies will be performed after we create antibodies or as the
antibody becomes available (currently it is being developed in the lab of Dr. D.E. Zhang,
Scripps Institute). RNA isolation will be performed by standard Trizol extraction, and
protein extraction by lysing the cells in RIPA buffer. These studies will establish mRNA and
protein levels for ISG15 and UBP43 at baseline, and in response to IFN, replicon and
replicon/IFN.
One important issue is confirming that the gene or protein-of-interest is expressed in these
cells prior to the knock-down experiments. In preliminary studies we have determined that
there is baseline expression of both ISG15 and UBP43 mRNA in both Huh7.5 and replicon cells
(real-time PCR) and that IFNa strongly induces protein expression of ISG15 in Huh7.5 cells.
These results argue that the ISG15 and UBP43 pathway is relevant to this model.
siRNA studies: We hypothesize that elimination of UBP43 will lead to less HCV RNA
replication at baseline and will magnify the effect of IFN, and that elimination of ISG15
will increase baseline HCV RNA and decrease the effect of IFN. Silencing and control siRNAs
will be obtained from Ambion. These will be transfected using GenePorter according to the
manufacturer’s instructions; transfection conditions will be optimized using the SV40
promoter/enhancer luciferase control plasmid, pGL2-control, to ensure that increasing
amounts of templates results in proportional increases in luciferase activity. Forty-eight
hours after the transfection, total RNA or protein samples will be prepared as before.
Real-time PCR will be performed to determine mRNA expression in the presence or absence of
siRNA, and protein levels will be determined by Western blot. After these control studies
have been performed, the effect of siRNA inhibition on baseline and IFN-treated HCV RNA
replication will be tested.
Transfection studies:
We hypothesize that upregulation of UBP43 will increase in vitro HCV RNA replication at
baseline and will decrease the effect of IFN, and that upregulation of ISG15 will decrease
baseline HCV RNA and increase the effect of IFN. UBP43 expression will be upregulated using
a UBP43 expression plasmid pcDNA6-UBP43; ISG15 expression plasmid will be obtained by
subcloning ISG15 gene into pcDNA6 vector. As before, HCV RNA levels will be determined in
the presence and absence of IFNa using real-time PCR.
Taken together these studies will define the roles of the genes suggested to play an
important role in the distinction between clinical PegIFN/Rib treatment responders and
nonresponders.
Ib. Mechanism of action of the ISG15/UBP43 pathway:
If we determine that ISG15 and UBP43 play a role in HCV RNA replication in the genotype 1
replicon model we will examine how this effect is mediated. We will determine whether the
effect of UBP43 is dependent on its protease function or on its association with another
cellular protein. We will also identify which ISG15 protein targets might mediate its
effect.
Role of UBP43 protease activity:
If elimination of UBP43 is shown to reduce viral replication or magnify the interferon
response, we hypothesize that inhibition of UBP43 will represent a novel means of treatment
of HCV. UBP43 would be of particular interest because it has protease activity: proteases
are known and validated targets for small molecule therapies. Thus, it would be critical to
establish whether the protease activity of UBP43 is important for its effect on HCV
replication, and not, for instance, its interaction with another cellular protein. To
address this question we will over-express UBP43 protease inactive constructs. An inactive
form of UBP43 will be created by mutation of a critical cysteine residue (Cys61) into serine
using site-directed mutagenesis of pBK/CMV-UBP43 plasmid as described previously. In order
to characterize the protease active and inactive forms of UBP43, wild type and mutant UBP43
will be expressed as GST fusion proteins in the expression vector pGEX-4T-3
(pGEX-4T-3-UBP43), and transformed into E.Coli BL21 (DE3) with a plasmid, pET-ISG15-UBP43-H,
that expresses ISG15-UBP43 fusion protein.35 ISG15 cleavage from the fusion protein will be
shown by Western blot. Once we have confirmed that the protease function is eliminated by
site-directed mutagenesis, wild-type and mutated UBP43 will be overexpressed in Huh7.5 cells
with the HCV replicon and exposed to increasing doses of IFNa. If the protease function is
critical overexpression of the inactive (in a dominant negative fashion) will increase
responsiveness to IFN.
Protein targets of ISG15:
Previous work using thymic tissue has identified several targets for ISGylation by ISG15,
including phospholipase Cg1, Jak1, and ERK1. Whether these are targets in Huh7.5 cells is
not known, though ERK and JAK have been previously linked to antiviral effects in the HCV
replicon model. If we find that ISG15 is implicated in HCV replication, we would seek to
identify substrates for ISG15 modification. We will first perform a series of
immunoprecipitation studies directed specifically at the proteins previously shown found to
be modified by ISG15, and explore the differences between infected and uninfected cells. In
brief, following incubation of Huh7.5 cells +/- replicon with IFNa for 12 hours, cells will
be lysed in RIPA buffer and immunoprecipitation of ISG15 conjugates performed with the
anti-ISG15 mAb used above. After resolution on a reducing gel, western blots will be
performed to determine if the three targets above are conjugated by ISG15, using
commercially-available mAbs. If any or all of the targets are conjugated, further studies
will be performed to elucidate the consequences of ISGylation.
To more fully explore which proteins are modified with ISG15 in cell culture, we will
undertake a more comprehensive screen using mass-spectroscopy – applying a strategy
previously used to identify targets of the ubiquitin-like SUMO protein. TAP-tagging
approaches coupled with MALDI-TOF mass spectrometry will be used. We will transfect cells
with TAP-tagged or SPA-tagged ISG15 and purify the protein and protein complexes from cells
treated or untreated with IFN. Using established procedures, we will then purify and
identify the substrates using gel electrophoresis and mass spectrometry. Gel slices
containing the proteins will be reductively alkylated and subjected to trypsinolysis. The
peptides will be purified and analyzed by MALDI-TOF mass spectrometry using a
cyano-4-hydroxycin-namic acid matrix on a Voyager DE-STR instrument (Applied Biosystems).
Identification of the proteins using these mass fingerprinting data will be carried out
using the ProFound software . Since MS cannot be used to quantify protein amounts, we will
identify proteins of interest by comparing lists of proteins ISGylated under the various
conditions and then perform Western blot studies to compare protein amounts. In doing these
experiments we will use the MS equipment and expertise readily available at the Best
Institute.
Conclusions I: Taken together these studies will clearly define the roles of the ISG15 and
UBP43 pathway in the HCV replicon response to IFN, and will suggest how the effect is
mediated. Importantly, the methodologies described above, though detailed for ISG15 and
UBP43, are generic and can be applied to other candidate genes of interest.
Section II: Gene Expression Profiling of Acute Hepatitis C Viral Infection:
There are similarities in the pathways that we have identified as important in the chronic
disease with those that may be important in acute infection. For example, polymorphisms of
2’5’OAS – a gene found on our list – alter the risk of progressing from acute to chronic
infection.20 Therefore we hypothesize that this same set of genes is likely to play an
important role in determining failure to clear acute infection. We will test this hypothesis
using microarray analysis.
We plan to examine the role of these and other genes in acute HCV infection in both the
systemic and local host/virus interaction. Acutely-infected liver tissue is difficult to
obtain because most acute HCV infections are not identified, and because it is inappropriate
to perform liver biopsies in these cases. Thus we will use two different approaches. First,
we will study the gene expression patterns in peripheral blood mononuclear cells (PBMCs)
collected from Egyptian health care workers with acute HCV (provided by Dr. Sanaa Kamal) and
compare these to PBMCs from patients with genotype-matched chronic HCV and to healthy
volunteers. We will develop predictive models to test which gene subsets are most associated
with clinical outcomes such as the establishment of chronic infection. Second, we will use
the recurrence of HCV post transplantation as a model of acute hepatitis in vivo. We will
take biopsies of the donor liver PRIOR to transplantation and then follow gene expression
levels over time as the virus infects the graft. After transplantation the HCV-naïve liver
rapidly and universally becomes infected with HCV, though only 70% of patients develop
histological evidence of recurrence on liver biopsies taken 3 and/or 6 months
post-transplant. It is these patients who we currently treat for recurrent HCV using
PegIFN/Rib. As before, we will develop predictive models to associate gene subsets with
clinical outcomes. We have achieved more than a 60% response rate by encouraging treatment
adherence despite the even more grueling side effects after transplantation. Thus, as for
the treatment of HCV prior to transplantation a significant fraction of patients do not
respond to therapy and are needlessly exposed to morbid side effects: the key clinical
outcome we will consider is response to therapy with PegIFN/Rib. Another outcome of interest
is the rapid development of cirrhosis within 5 years in 25-30% of patients, and the
development of an aggressive fibrosing cholestatic state in 7-9% within 2 years of
transplant. Together these data will point to the genes important for the immunological
response that results in disease persistence or elimination (acute HCV in health care
workers) and progression of liver disease (post-transplantation patients). Highly up- and
down-regulated genes will be further studied in the HCV replicon model.
Research Objective 2. To determine the gene expression profiles which are most highly
associated with clinical outcomes in acute hepatitis, both in circulating immune cells and
in the liver.
Research Protocols for Section II:
We propose three major analyses. First, we will perform gene expression profiling in PBMC
preparations provided by Dr. Kamal from patients with acute HCV. These PBMC preparations are
being performed as part of ongoing studies headed by Dr. Kamal. Second, we will use
microarray analysis to describe the hepatic genes which are altered by HCV infection in the
post-transplant liver graft in response to infection by HCV. For this we will use portions
of liver biopsies already being taken as part of routine post-transplant clinical care.
Third, we will prepare PBMCs from patients with chronic HCV and from these determine gene
expression profiles. Not only are these a critical comparison to acute HCV, but they will
also act as the basis for predicting outcomes of treatment in chronic HCV.
IIa. Patient recruitment, study population and collection of samples and data:
In all cases, patients will be identified by clinical staff as potential candidates for the
study and approached by a study nurse. Consent will be obtained for the research use of
tissue and blood samples, and for the collection of patient clinical data.
Acute HCV: systemic PBMC response Dr. Kamal follows a population of Egyptian health care
workers who become infected acutely with HCV, and she routinely isolates PBMCs from them.
This clinical population is composed primarily of Genotype 1 and Genotype 4 infected
individuals, and is thoroughly described for patient demographics, RNA titer at the time of
blood collection, and progression to chronic infection. Given the relative rarity of
Genotype 4 in our local population our initial analysis will focus on Genotype 1 acute HCV,
but we intend to study genotype 4 as well, since this genotype has great relevance to HCV
worldwide. Based on our previous work we anticipate needing between 10-20 patients in a
given group in order to clearly define consistent gene expression changes across a spectrum
of clinical variability. We expect to collect and study PBMC samples from at least 20 acute
genotype 1 patients per year. Samples from 20 healthy control volunteers will also be
obtained over the 3 year study period (recruited by poster).
Acute HCV: Hepatic response to HCV recurrence post-transplantation Our center performs 30-40
liver transplants each year in persons infected with HCV. All clinical data in our liver
transplant population is entered into an Oracle-based database. Liver biopsies are routinely
taken from the HCV-naïve graft prior to transplantation and at 12, 24, and 52 weeks
post-transplantation. Based on previous experience we expect an accrual rate of at least
80%; thus, we will be able to collect data and specimens from roughly 24-32 patients per
year. Biopsies of normal liver are taken as part of an ongoing study in our living donor
population (PI: I. McGilvray). 30-40 such biopsies are taken every year and represent a
unique source of control liver tissue.
Chronic HCV: systemic PBMC response Dr. Heathcote follows a large population of patients
with chronic HCV. Blood samples will be taken from patients in whom treatment with
PegIFN/Rib is being considered and PBMCs will be isolated. Based on our experience with the
liver biopsy studies outlined in the Progress Report above, we expect to obtain PBMC
preparations from at least 50 patients each year. Roughly 2/3 of these are Genotype 1
patients.
IIb. Data Analysis:
Acute HCV: Gene expression changes in response to acute HCV in circulating PBMCs Gene
expression profiles from PBMCs from patients with acute Genotype 1 HCV will be determined
and compared to those from normal healthy controls and chronic genotype 1 HCV. Genes will be
identified by statistical and fold differences between groups (p value of <0.01, fold change
of at least 1.5). This comparison will define which gene expression alterations are specific
to acute HCV infection. In order to associate individual genes with the progression to
chronic disease, subgroup analyses will be done in which gene expression in acutely infected
individuals is compared between those who went on to chronic infection and those who
spontaneously eliminated the virus. After real-time PCR confirmation of these genes we will
use a number of unsupervised (hierarchical clustering, principal components) and supervised
(nearest neighbours, linear discriminants) analyses to develop predictive gene sets which
accurately classify patients who go on to develop chronic infection. Taken together these
results will identify those genes whose expression is altered in the systemic immunologic
response to acute HCV and how these relate to the establishment of chronic disease. Genes
which are of interest as possible therapeutic targets will be further studied using the HCV
replicon model, as described earlier. By the end of the second year of the study we expect
to have analyzed 40 acute genotype 1 patients, and in the third year we will analyze 20
genotype 4 patients.
Acute HCV: local hepatic gene expression in post-transplantation HCV infection Gene
expression profiles will be determined from liver biopsies at organ retrieval, prior to
infection by HCV, and at 3, 6 and 12 months after transplantation. Gene changes can
therefore be followed in the same patient and between patients; genes levels will be
compared to normal liver tissue and to baseline gene expression in HCV-naïve grafts, using
statistical methods developed in our lab and at the EBI. Currently 70% of the patients
transplanted with HCV in our center are treated for recurrent HCV post-transplantation after
evidence of recurrent HCV on liver biopsy, and in these patients quantitative HCV titers are
done at 3 or 6 months post-transplantation. Roughly 80% of our patients complete therapy
over a 12 month course. Thus, by the end of our 3 year study we will know the response to
treatment of between 28-36 patients (we expect a 60% response rate, thus roughly 17-22
responders and 11-14 nonresponders), and eventually will have data on the 42-54 patients
expected to complete therapy. With this sample size we can determine which gene subsets are
predictive of response to treatment (using the methods noted above). Equally we will be able
to relate gene expression to HCV RNA titer in 50-60 patients. By the end of the three year
study we will have clinical and genomic data on 72 to 90 patients. Ultimately we will relate
gene expression to the development of recurrent cirrhosis, and we anticipate that roughly
18-30 patients will have gone on to this state within 5 years of transplantation. In order
to control for the effects of the many confounding factors that could influence hepatic gene
expression post-transplantation (eg. acute rejection, immunosuppression) we will perform
subgroup analyses using our extensive clinical database. We will compare HCV treatment
responders to nonresponders, rejection to no rejection, and different immunosuppressive
regimens, in addition to comparing gene expression profiles from acute hepatic HCV to those
already determined for chronic HCV. These analyses will allow us to distinguish and describe
the effects of acute HCV infection on hepatic gene expression.
Chronic HCV: circulating PBMC response and prediction of treatment outcome:
Over the first 2 years of our study we will use gene expression profiling to study PBMCs
from roughly 30 patients with chronic genotype 1 HCV. This data will serve as a crucial
control for our studies of acute HCV in PBMCs. However, all of these samples are taken from
patients who will go on to treatment with PegIFN/Rib. Therefore we will use the gene
expression data from these studies not only for comparison with the acutely infected PBMCs,
but also to determine which gene subsets are predictive of treatment outcome. These results
will form the basis for a noninvasive prognostic test.
Conclusions II: These studies will provide a comprehensive description of the gene
expression changes that accompany acute HCV infection at both the systemic and the local
level. They will clearly identify genes which are important to the progression to chronic
disease and to adverse clinical outcomes, thereby suggesting novel prognostic and
therapeutic targets.
;
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Phase 4 | |
Recruiting |
NCT04405024 -
Pilot Study on the Feasibility of Systematic Hepatitis C Screening of Hospitalized Patients
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N/A | |
Completed |
NCT04525690 -
Improving Inpatient Screening for Hepatitis C
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N/A | |
Completed |
NCT04033887 -
Evaluation Study of RDTs Detecting Antibodies Against HCV
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Withdrawn |
NCT04546802 -
HepATocellular Cancer Hcv Therapy Study
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Phase 3 | |
Active, not recruiting |
NCT02961426 -
Strategic Transformation of the Market of HCV Treatments
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Phase 2/Phase 3 | |
Completed |
NCT02992184 -
PoC-HCV Genedrive Viral Detection Assay Validation Study
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N/A | |
Completed |
NCT03186313 -
A Study to Evaluate the Safety and Efficacy of the Combined Single Dose of Dactavira Plus Or Dactavira in Egyptian Adults With Chronic Genotype 4 HCV Infection
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Phase 3 | |
Completed |
NCT02683005 -
Study of Hepatitis C Treatment During Pregnancy
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Phase 1 |