Mechanisms of Therapy and Model Development in Viral Hepatitis and Liver Diseases

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs) and the technology to developmentally program these cells to various cell lineages offer great promise for cell therapy of various diseases. Recently, different protocols to differentiate them into hepatocytes-like cells (HLC) have been described. In this context, we hypothesize that this approach could be useful in developing a relevant model for HCV infection in vitro. Moreover, the hiPSCs approach could allow the generation of patient-specific hepatocytes with promising opportunity for cell therapy of viral liver diseases. We have generated hiPSCs from primary fibroblasts using lentiviruses or Sendai virus vectors, and characterized them in comparison to hESCs. Human pluripotent stem cells were efficiently differentiated into HLC, as demonstrated by induction of the expression of hepatic markers and the secretion of hepatic proteins (AFP and albumin) in the supernatants. Moreover these cells recapitulate hepatocyte-specific metabolic functions like lipid and glycogen accumulation, and indocyanin green metabolism. We also successfully engrafted, via intra-splenic injection, 2-4 millions HLCs into the liver parenchyma of immune-deficient transgenic mice carrying the urokinase-type plasminogen activator gene driven by the major urinary protein promoter (MUP-uPA/SCID/Bg). CRISPR-Cas9 system has emerged as a powerful and efficient tool for genome editing. However, one of the important drawbacks of CRISPR-Cas9 system is the constitutive endonuclease activity when Cas9 endonuclease and its sgRNA are co-expressed. This constitutive endonuclease activity results in undesirable off-target effects that hinders studies using CRISPR-Cas9 system such as understanding gene functions or its therapeutic use in humans. Here, we describe a novel method that allows temporal control of CRISPR-Cas9 activity by combining transcriptional regulation of Cas9 gene expression and protein stability control of Cas9 protein. To achieve this dual controls, we combine the doxycycline-inducible system for transcriptional regulation and FKBP12-derived destabilizing domain fused to Cas9 for protein stability regulation. We showed that Cas9 gene expression and its protein stability are tightly regulated by a doxycycline and a synthetic ligand (Shield1). We also confirmed that approximately 10% of Cas9 gene expression was observed when only one of the two controls was used. By combining two regulatable systems, we were able to markedly lower the baseline Cas9 gene expression and limit the exposure time of Cas9 endonucleases in the cell, resulting in little or no off-target effects. We assess knock-out efficiency of our system in human stem cells (hESC or hiPSC) by targeting several tumor suppressor genes such as p53, phosphatase and tensin homolog (PTEN), and adenomatous polyposis coli (APC). For in vivo application of our system, an inducible p53 gene knock-out SW iPSC clone was generated and engrafted subcutaneously into the athymic nude mice. Currently, we are improving Cas9 gene expression in vivo by replacing the promoter for Cas9 gene expression because we observed low level of Cas9 gene expression in vivo. We anticipate that our novel conditional CRISPR-Cas9 system will serve as a valuable tool for the systematic characterization and identification of genes for various pathological processes as well as paving the way to develop safer method for clinical use of CRISPR-Cas9 system in humans. In patients with HBV and HCV co-infection, HBV reactivation leading to severe hepatitis has been reported with the use of interferon (IFN)-free direct-acting antiviral agents (DAAs) to treat HCV infection. Here we study the interplay of HBV and HCV and molecular mechanisms leading to HBV reactivation after HCV elimination in HBV-HCV co-infection. In primary human hepatocytes (PHHs), HBV replication was suppressed by HCV co-infection as compared to HBV mono-infection. This suppression was attenuated by sofosbuvir and inhibitor of Janus kinase, which reduced IFN-stimulated genes expression in co-infected cultures but had no impact on HBV mono-infection. In PHH-transplanted Alb-UPA/SCID mice co-infected with HBV and HCV, HBV viremia was significantly lower than that in HBV mono-infected mice. Similar to HCV mono-infection, IFN response was activated in co-infected mice. Treatment with combined DAAs in co-infected mice efficiently cleared HCV and resulted in increased HBV viremia that are consistent with HBV reactivation. DAA-treated HBV-HCV coinfected patients were studied for potential circulating immune biomarkers associated with HBV reactivation. A higher pre-treatment plasma C-X-C motif chemokine 10 (CXCL10) level, a serum marker of IFN activation, was observed in patients with HBV reactivation. Based on fold-changes of CXCL10 (baseline over week 1 values), we developed a predictive model of HBV reactivation with high sensitivity and specificity (AUC, 0.86; 95%CI, 0.74-0.98). In this study, we showed that HCV infection suppresses HBV infection in vitro and in vivo. This suppression is mediated by endogenous IFN responses induced by HCV infection. Rapid clearance of HCV in co-infected humanized mice and patients receiving DAAs leads to a reduction of hepatic IFN activity, which de-represses HBV replication and leads to HBV reactivation. Quantifying CXCL10 serum levels may be used to identify HBV-HCV co-infected patients on DAA treatment with a high risk of HBV reactivation.