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系统已成为基因组编辑的强大而有效的工具，我们正在将该技术应用于病毒性肝炎和肝脏疾病的模型开发。 CRISPR-Cas9系统的重要缺点之一是当Cas9内切核酸酶及其sgRNA共表达时的组成型内切核酸酶活性。 这种组成型核酸内切酶活性导致不期望的脱靶效应，这阻碍了使用CRISPR-Cas9系统的研究，例如理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，该方法允许通过将Cas9基因表达的转录调控和Cas9蛋白的蛋白质稳定性控制相结合来暂时控制CRISPR-Cas9活性。 为了实现这种双重控制，我们联合收割机用于转录调控的强力霉素诱导型系统和融合至Cas9的FKBP 12衍生的去稳定化结构域组合用于蛋白质稳定性调控。 我们表明，Cas9基因表达及其蛋白质稳定性受到多西环素和合成配体（Shield 1）的严格调控。 我们还证实，当仅使用两种对照中的一种时，观察到约10%的Cas9基因表达。 通过组合两种可调控的系统，我们能够显著降低基线Cas9基因表达并限制Cas9核酸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、磷酸酶和张力蛋白同源物（PTEN）和腺瘤性结肠息肉病（APC）来评估我们的系统在人干细胞（hESC或hiPSC）中的敲除效率。 对于我们的系统的体内应用，产生诱导型p53基因敲除SW iPSC克隆并皮下移植到无胸腺裸鼠中。 目前，我们正在通过替换Cas9基因表达的启动子来改善Cas9基因在体内的表达，因为我们观察到Cas9基因在体内的低水平表达。 我们预计，我们的新型条件性CRISPR-Cas9系统将成为系统表征和鉴定各种病理过程基因的宝贵工具，并为开发更安全的方法在人类中临床使用CRISPR-Cas9系统铺平道路。 在HBV和HCV合并感染的患者中，使用不含干扰素（IFN）的直接作用型抗病毒药物（DAA）治疗HCV感染时，已报告HBV再激活导致重度肝炎。在这里，我们研究了HBV和HCV的相互作用和分子机制，导致HBV再激活后，在HBV和HCV合并感染的HCV消除。在原代人肝细胞（PHH），HBV复制抑制HCV共同感染相比，HBV单一感染。这种抑制被sofosbuvir和Janus激酶抑制剂减弱，这减少了在共感染培养物中IFN刺激的基因表达，但对HBV单一感染没有影响。在PHH移植的Alb-UPA/SCID小鼠中，HBV和HCV共感染的HBV病毒血症显著低于HBV单感染小鼠。与HCV单感染相似，IFN应答在共感染小鼠中被激活。在共感染小鼠中用组合DAA治疗有效地清除了HCV，并导致与HBV再活化一致的HBV病毒血症增加。DAA治疗的HBV-HCV合并感染患者研究了与HBV再激活相关的潜在循环免疫生物标志物。治疗前血浆C-X-C基序趋化因子10（CXCL 10）水平较高，是IFN活化的血清标志物，在HBV再活化患者中观察到。基于CXCL 10的倍数变化（基线值超过第1周值），我们开发了一个具有高灵敏度和特异性的HBV再激活预测模型（AUC，0.86; 95%CI，0.74-0.98）。在这项研究中，我们表明，HCV感染抑制HBV感染在体外和体内。这种抑制是由HCV感染诱导的内源性IFN应答介导的。在共感染的人源化小鼠和接受DAA的患者中HCV的快速清除导致肝脏IFN活性降低，这解除了HBV复制的抑制并导致HBV再活化。定量CXCL 10血清水平可用于鉴定接受DAA治疗的HBV-HCV合并感染患者，这些患者具有HBV再激活的高风险。 非酒精性脂肪肝（NAFLD）是一种日益严重的公共卫生负担。基因组研究揭示了NAFLD进展与PNPLA 3中的I148 M变体之间的强关联。我们使用PNPLA 3基因敲除（PNPLA 3 KO）或I148 M变体（PNPLA 3 I148 M）的等基因人诱导多能干细胞（hiPSC）系来模拟PNPLA 3相关的NAFLD。将hiPSC分化成肝细胞，用游离脂肪酸处理以诱导NAFLD样表型，并通过各种功能、转录组学和脂质组学测定来表征。PNPLA 3 KO肝细胞表现出更高的脂质蓄积和对脂质诱导的应激反应的改变模式。有趣的是，脂肪变性增加和脂质代谢改变导致PNPLA 3 KO细胞对乙醇诱导的毒性更敏感。PNPLA 3 I148 M细胞表现出介于野生型和PNPLA 3 KO细胞之间的中间表型。总之，这些结果表明I148 M变体诱导功能丧失，导致脂肪变性并增加对肝毒素的易感性。