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

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. The sodium-dependent taurocholate co-transporting polypeptide (NTCP)-S267F variant is known to be associated with a reduced risk of HBV infection and disease progression. The NTCP-S267F variant displays diminished function in mediating HBV entry, but its function in HBV infection has not been fully established in more biologically relevant models. We introduced NTCP-S267F variant and tested the infectivity by HBV in genetically edited hepatic cells. HepG2-NTCP clones with both homozygous and heterozygous variants were identified after CRIPSR base editing. NTCP-S267F homozygous clones did not support HBV infection. The heterozygotes clones behaved more or less like wild type clones. We generated genetically edited human stem cells with the NTCP-S267F variant, which differentiated equally well as wild type into hepatocyte like cells (HLC) expressing high levels of hepatocyte differentiation markers. We confirmed that HLC with homozygous variant did not support HBV infection and heterozygous variant clones were infected with HBV equally as well as the wild-type cells. In conclusion, we successfully introduce the S267F variant by CRISPR base editor into the NTCP/SLC10A gene of hepatocytes, and showed the variant is a null mutation. This technology of studying genetic variants and their pathogenesis in a natural context is potentially valuable for therapeutic intervention against HBV. 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.