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

病毒性肝炎和肝病的治疗机制和模型开发

• 批准号: 10248152
• 负责人: T. Jake Liang
• 金额: $ 100.81万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10248152
• 关键词: Adenomatous Polyposis Coli; Antiviral Agents; Attenuated; Biological Assay; Biological Models; CRISPR/Cas technology; CXCL10 gene; Cell Line; Cells; Clinical; Disease Progression; Doxycycline; Ethanol; Exhibits; Gene Expression; Gene Proteins; Genes; Genomics; Hepatic; Hepatitis; Hepatitis B Therapy; Hepatitis B Virus; Hepatitis C; Hepatitis C Therapy; Hepatitis C co-infection; Hepatitis C virus; Hepatocyte; Hepatocyte transplantation; Human; Immunologic Markers; In Vitro; Infection; Interferon Activation; Interferons; Janus kinase; Knock-out; Ligands; Lipids; Liver diseases; Mediating; Methods; Modeling; Molecular; Mus; Nonesterified Fatty Acids; Nude Mice; PTEN gene; Pathologic Processes; Patients; Pattern; Phenotype; Plasma; Predisposition; Proteins; Public Health; Regulation; Reporting; SCID Mice; Sensitivity and Specificity; Serum; Serum Markers; Stress; System; TP53 gene; Tacrolimus Binding Protein 1A; Technology; Therapeutic Uses; Time; Toxic effect; Transcriptional Regulation; Tumor Suppressor Genes; Variant; Viral hepatitis; Viremia; Virus Replication; base; chemokine; endonuclease; gene function; genome editing; hepatotoxin; high risk; human embryonic stem cell; human stem cells; humanized mouse; improved; in vivo; in vivo Model; induced pluripotent stem cell; inhibitor/antagonist; lipid metabolism; loss of function; model development; non-alcoholic fatty liver disease; novel; predictive modeling; promoter; response; subcutaneous; therapy development; therapy resistant; tool; transcriptomics; treatment response

CRISPR-Cas9 system has emerged as a powerful and efficient tool for genome editing and we are applying this technology for model development in viral hepatitis and liver disease. 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 HCVco-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. Non-alcoholic fatty liver disease (NAFLD) is a growing public health burden. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in PNPLA3. We used isogenic human induced pluripotent stem cell (hiPSC) lines with either a knock-out (PNPLA3KO) of the PNPLA3 gene or with the I148M variant (PNPLA3I148M) to model PNPLA3-associated NAFLD. The hiPSCs were differentiated into hepatocytes, treated with free fatty acids to induce NAFLD-like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3KO hepatocytes showed higher lipid accumulation and an altered pattern of response to lipid-induced stress. Interestingly, increased steatosis and altered lipid metabolism led PNPLA3KO cells to be more susceptible to ethanol-induced toxicity. The PNPLA3I148M cells exhibited an intermediate phenotype between the wild type and PNPLA3KO cells. Together, these results indicate that the I148M variant induces a loss of function causing steatosis and increased susceptibility to hepatotoxins.

CRISPR-Cas9系统已经成为一种强大而有效的基因组编辑工具，我们正在将这项技术应用于病毒性肝炎和肝脏疾病的模型开发。CRISPR-Cas9系统的一个重要缺陷是Cas9内切酶与其sgRNA共表达时的组成型内切酶活性不高。这种组成型内切酶活性导致不良的脱靶效应，阻碍了使用CRISPR-Cas9系统的研究，例如了解基因功能或其在人类中的治疗用途。在这里，我们描述了一种新的方法，通过结合Cas9基因表达的转录调控和Cas9蛋白的稳定性控制，可以对CRISPR-Cas9活性进行时间控制。为了实现这种双重控制，我们将强力霉素诱导的转录调控系统和fkbp12衍生的不稳定结构域融合到Cas9中进行蛋白质稳定性调控。我们发现Cas9基因表达及其蛋白稳定性受到强力霉素和合成配体（shield d1）的严格调控。我们还证实，当只使用两个对照中的一个时，观察到大约10%的Cas9基因表达。通过结合两种可调节系统，我们能够显著降低Cas9基因的基线表达，并限制Cas9内切酶在细胞中的暴露时间，导致很少或没有脱靶效应。我们通过靶向几种肿瘤抑制基因，如p53、磷酸酶和紧张素同源物（PTEN）以及大肠腺瘤性息肉病（APC），评估我们的系统在人类干细胞（hESC或hiPSC）中的敲除效率。为了在体内应用我们的系统，我们生成了一个可诱导的p53基因敲除的SW iPSC克隆，并将其皮下移植到胸腺裸鼠体内。目前，由于我们在体内观察到Cas9基因表达水平较低，我们正在通过替换启动子来提高Cas9基因在体内的表达。我们预计我们的新型条件CRISPR-Cas9系统将成为系统表征和鉴定各种病理过程基因的有价值的工具，并为CRISPR-Cas9系统在人类临床应用的更安全方法铺平道路。