NIDA Center Genomics Projects Kathie Walters October 2008

NIDA Center: Functional Genomics Approach to Study HCV-associated Liver Disease Systems biology approach using multiple model systems to gain insight into pathogenic mechanisms of chronic HCV infection Integration of gene expression, proteomic, virological, histological data Detailed molecular portrait of host response to HCV Relationship of host response to disease progression Molecular mechanisms of liver injury/fibrogenesis markers that predict fibrosis development markers of fibrosis progression potential therapeutic targets

HCV Model Systems Liver Transplant Model most clinically relevant ONLY model of liver disease (fibrosis/cirrhosis) extremely complex (mixed cell population) high patient variation SCID-beige/uPA Mouse Model small animal model of HCV infection/replication role of host factors in host response control certain variables (HCV genotype/ length of infection) absence of adaptive immune response lack of liver disease (evidence of liver injury) HCV 2a (JFH) in vitro infection system Complete virus life cycle useful for biological validation/functional studies

Specific Aims/Questions Are there differences in the host transcriptional response between people who develop fibrosis post-transplant vs those who do not? Can these differences be detected prior to disease development? Does the host response to HCV infection change over time? What are the contributing roles of viral and host factors in liver disease development? Is liver injury mediated exclusively by immune cells? Accelerated liver disease progression during immuno-suppression? Does HCV replication significantly impact hepatocyte cellular pathways? What role does HCV viral load play in liver disease progression? Relationship between serum and intrahepatic levels?

Advantages of using liver transplant model to study HCV-associated liver disease Time of HCV infection is known Study gene expression changes during the early phase of HCV infection Known patient outcome associate gene expression patterns/cellular processes with specific disease states (fibrosis development/protective response) Comparison of progression vs non-progression Identification of markers predict early progression/ rapidly progressive fibrosis liver disease progression “protective” response

Liver transplant (LT) model of HCV infection and disease progression 2-year and further time points 3 months post-operation 6 months post-operation 12 months post-operation Microarrays * All biopsies were compared to a common reference (Pool of 10 normal liver samples) Liver transplant

Transplant Study ~ 125 microarray experiments from 70 liver transplant patients Serial biopsies from 21 HCV patients with no evidence of fibrosis (4-5 years post-transplant) Serial biopsies from 41 HCV patients with fibrosis (Stage 2-4) Biopsies from 8 non-HCV patients (autoimmune, EtOH, Tylenol, PBC) Extensive clinical information (pathology, liver enzymes, medications, etc) Intrahepatic HCV RNA quantitation (early time-points, kinetics, fibrosis progression) All biopsies have been graded/staged by single liver pathologist Scott Emerson provides statistical support

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Nature Medicine (Mercer et al, 2001) SCID-Alb-uPA (urokinase plasminogen activator) Mouse hepatocytes carrying uPA transgene uPA activates plasmin and induces proteolytic damage within endoplasmic reticulum Mouse hepatocytes are progressively depleted Transplanted human hepatocytes have selective growth advantage over mouse hepatocytes re-populate mouse liver Mouse with human-mouse chimeric liver is then infected with HCV HCV Mouse Model

Alb-uPA/SCID Mouse Model of HCV Hepatocyte transplantation via splenic injection Red Nodules = Human liver

Why the Mouse? Identify HCV-host interactions in the absence of a HCV- specific immune response Investigate the host genetic component of the response to HCV infection – different HCV genotypes in single donor-transplanted mice – single HCV genotype in multiple donor-transplanted mice Opportunity to manipulate system ex. IFN treatment Serial biopsies?? Potential to study HCV-mediated cytopathic effect: evidence of apoptosis in infected livers

HCV 2a Infection System HCV interaction with innate antiviral response Gene expression data suggests RIG-I, TLR3-independent pathway regulating ISG induction HCV and Type I IFN signaling pathway Anti-HCV activity of IFN-α versus IFN-β HCV-mediated cytopathic effect Gene expression data suggests potential role of cell cycle delay in viral-mediated apoptosis Cell sorting demonstrates HCV infection delays cell cycle progression Integration with transplant study gene expression data suggests that cell cycle dysregulation contributes to liver disease progression On-going studies to determine mechanism of cell arrest