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高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病的模型开发。CRISPR-Cas9系统的一个重要缺陷是当Cas9内切酶及其sgRNA共表达时，其结构性内切酶活性较高。这种结构性的核酸内切酶活性导致了不受欢迎的脱靶效应，阻碍了使用CRISPR-Cas9系统的研究，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蛋白的蛋白质稳定性控制来实现对CRISPR-Cas9活性的时间控制。为了实现这一双重控制，我们将多西环素诱导的转录调控系统与FKBP12衍生的不稳定结构域融合到Cas9中，以调节蛋白质的稳定性。我们发现Cas9基因的表达及其蛋白稳定性受到强力霉素和合成配体(Shield1)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。通过结合两个可调控的系统，我们能够显著降低基线Cas9基因的表达，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。我们评估了我们的系统在人类干细胞(hESC或hiPSC)中的敲除效率，方法是针对几个肿瘤抑制基因，如p53、磷酸酶和紧张素同源基因(PTEN)以及腺瘤性结肠息肉病(APC)。为了在体内应用我们的系统，我们获得了一个可诱导的p53基因敲除的Sw iPSC克隆，并将其移植到裸鼠皮下。目前，我们正在通过替换Cas9基因表达的启动子来提高Cas9基因在体内的表达，因为我们在体内观察到了低水平的Cas9基因表达。我们期待我们的新的条件性CRISPR-Cas9系统将成为一种有价值的工具，用于系统地表征和鉴定各种病理过程的基因，并为开发更安全的方法在临床上使用CRISPR-Cas9系统铺平道路。 在乙肝病毒和丙型肝炎病毒混合感染的患者中，有报道称使用不含干扰素的直接作用抗病毒药物(DAAs)治疗丙型肝炎病毒感染时，乙肝病毒的重新激活会导致重型肝炎。在这里，我们研究了在乙肝病毒和丙型肝炎病毒混合感染中，病毒与丙型肝炎病毒的相互作用以及在丙型肝炎病毒消除后导致乙肝病毒重新激活的分子机制。在原代人肝细胞(PHH)中，与单独感染HBV病毒相比，混合感染可抑制HBVDNA复制。这种抑制作用可被索莫布韦和Janus激酶抑制剂减弱，后者减少了干扰素刺激的基因在混合感染的培养物中的表达，但对单一感染的乙肝病毒没有影响。在PHH移植的Alb-uPA/SCID小鼠中，乙肝病毒和丙型肝炎病毒混合感染的小鼠的病毒血症明显低于单一感染的小鼠。类似于单一感染丙型肝炎病毒，干扰素反应在混合感染的小鼠中被激活。在合并感染的小鼠中，联合使用DAA有效地清除了丙型肝炎病毒，并导致了与乙肝病毒重新激活相一致的增加的乙肝病毒血症。对经DAA治疗的乙肝病毒-丙型肝炎病毒混合感染患者进行了研究，以寻找与乙肝病毒重新激活相关的潜在循环免疫生物标志物。乙肝病毒复活者治疗前血浆C-X-C基序趋化因子10(CXCL10)水平明显高于治疗前，CXCL10是干扰素活化的血清标志。基于CXCL10的倍数变化(基线超过第1周的值)，我们建立了一个灵敏度和特异度高的预测模型(AUC，0.86；95%CI，0.74-0.98)。在这项研究中，我们发现在体外和体内，丙型肝炎病毒感染抑制了乙肝病毒的感染。这种抑制是由丙型肝炎病毒感染诱导的内源性干扰素反应介导的。在合并感染的人源化小鼠和接受DAA的患者中，丙型肝炎病毒的快速清除会导致肝脏干扰素活性降低，从而抑制乙肝病毒的复制，导致乙肝病毒的重新激活。定量CXCL10血清水平可用于确定接受DAA治疗的乙肝病毒-丙型肝炎病毒混合感染患者的乙肝病毒重新激活的高风险。 非酒精性脂肪性肝病(NAFLD)是一个日益严重的公共卫生负担。基因组研究显示，NAFLD的进展与PNPLA3的I148M变异有很强的相关性。我们使用具有PNPLA3基因敲除(PNPLA3KO)或具有I148M变体(PNPLA3I148M)的等基因人诱导多能干细胞(HiPSC)株来建立PNPLA3相关NAFLD的模型。将HiPSCs分化为肝细胞，用游离脂肪酸诱导NAFLD样表型，并进行各种功能、转录和脂组分析。PNPLA3KO肝细胞表现出较高的脂质蓄积和对脂质诱导的应激反应模式的改变。有趣的是，脂肪变性加剧和脂质代谢改变导致PNPLA3KO细胞对乙醇诱导的毒性更敏感。PNPLA3I148M细胞的表型介于野生型和PNPLA3KO细胞之间。综上所述，这些结果表明，I148M变异导致功能丧失，导致脂肪变性和对肝毒素的易感性增加。