Therapy, Vaccine and Model Development in Viral Hepatitis and Liver Diseases

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Despite the development of highly effective HCV treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins E1 and E2 in the entry process with E2 binding to cell receptors and E1 mediating endosomal fusion. The structure of E1E2 has only been partially resolved by the X-ray crystallography of the core domain of E2 protein (E2c) and its complex with various neutralizing antibodies. Structural understanding of the E1E2 heterodimer in its native form can advance the design of candidates for HCV vaccine development. Here we analyze the structure of recombinant HCV E1E2 heterodimer with the aid of well-defined monoclonal anti-E1 and E2 antibodies as well as a small molecule chlorcyclizine-diazirine-biotin that can target and cross-link the putative E1 fusion domain. 3D models were generated after extensive 2D classification analysis with negative-stain single particles datasets. We modeled the available crystal structures of the E2c and Fabs into the 3D volumes of E1E2-Fab complexes based on the shape and dimension of the domain density. The E1E2 heterodimer exists in monomeric form and consists of a main globular body presumably depicting the E1 and E2 stem/transmembrane domain and a protruding structure representing the E2c based on anti-E2 Fab binding. At low resolution, a model generated from negatively stained analysis reveals the unique binding and orientation of individual or double Fabs onto the E1 and E2 components of the complex. Cryo-electron microscopy (cryo-EM) of double Fab complexes resulting in a refined structural model of the E1E2 heterodimer is presented. Since E1 plays an integral role in the structure and function of HCV envelope proteins, it is important to fully characterize this protein in the context of E1E2 heterodimer and HCV virion. In the current study, we generated HCV E1-specific antibodies. Using phage display technology, we identified over 17 clones of specialized single-chain antibodies, or nanobodies, specific to E1. These nanobodies consist of the variable portion of a camelid heavy-chain-only antibody fused to a humanized Fc region. We then characterized 10 of these nanobodies with recombinant E1E2 via ELISA, immunoprecipitation and transmission electron microscopy. ELISA data showed that the selected nanobodies have varying affinities for recombinant E1E2 and E2. Three of the ten examined nanobodies showed high affinity for E1E2 with negligible binding to E2, supporting that they are E1-specific. In addition, all the nanobodies successfully pulled down E1E2 but not E2 alone, through immunoprecipitation. Several of the nanobodies were observed under TEM in negative staining with direct binding to HCV E1E2. Using a combination of anti-E2 antibodies AR1B and AR2A to define domains of E1E2 on TEM (Kanai et al, JV 2023), we showed that the binding sites of the nanobodies are distinct from those of anti-E2 antibodies. These results confirm that these nanobodies are E1-specific and can be valuable tools for the study of HCV E1E2 structure and function. 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. 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. Next we applied the CRISPR-Cas9 system to study HBV infection. 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.