ABSTRACT
BACKGROUND & AIMS: The accumulation of neutral lipids within hepatocytes underlies non-alcoholic fatty liver disease (NAFLD), which affects a quarter of the world's population and is associated with hepatitis, cirrhosis, and hepatocellular carcinoma. Despite insights gained from both human and animal studies, our understanding of NAFLD pathogenesis remains limited. To better study the molecular changes driving the condition we aimed to generate a humanised NAFLD mouse model. METHODS: We generated TIRF (transgene-free Il2rg -/-/Rag2 -/-/Fah -/-) mice, populated their livers with human hepatocytes, and fed them a Western-type diet for 12 weeks. RESULTS: Within the same chimeric liver, human hepatocytes developed pronounced steatosis whereas murine hepatocytes remained normal. Unbiased metabolomics and lipidomics revealed signatures of clinical NAFLD. Transcriptomic analyses showed that molecular responses diverged sharply between murine and human hepatocytes, demonstrating stark species differences in liver function. Regulatory network analysis indicated close agreement between our model and clinical NAFLD with respect to transcriptional control of cholesterol biosynthesis. CONCLUSIONS: These NAFLD xenograft mice reveal an unexpected degree of evolutionary divergence in food metabolism and offer a physiologically relevant, experimentally tractable model for studying the pathogenic changes invoked by steatosis. LAY SUMMARY: Fatty liver disease is an emerging health problem, and as there are no good experimental animal models, our understanding of the condition is poor. We here describe a novel humanised mouse system and compare it with clinical data. The results reveal that the human cells in the mouse liver develop fatty liver disease upon a Western-style fatty diet, whereas the mouse cells appear normal. The molecular signature (expression profiles) of the human cells are distinct from the mouse cells and metabolic analysis of the humanised livers mimic the ones observed in humans with fatty liver. This novel humanised mouse system can be used to study human fatty liver disease.
ABSTRACT
BACKGROUND & AIMS: Development of new and more effective therapies against hepatitis B virus (HBV) is limited by the lack of suitable small animal models. The HBV transgenic mouse model containing an integrated overlength 1.3-mer construct has yielded crucial insights, but this model unfortunately lacks covalently closed circular DNA (cccDNA), the episomal HBV transcriptional template, and cannot be cured given that HBV is integrated in every cell. METHODS: To solve these 2 problems, we generated a novel transgenic mouse (HBV1.1X), which generates an excisable circular HBV genome using Cre/LoxP technology. This model possesses a HBV1.1-mer cassette knocked into the ROSA26 locus and is designed for stable expression of viral proteins from birth, like the current HBV transgenic mouse model, before genomic excision with the introduction of Cre recombinase. RESULTS: We demonstrated induction of recombinant cccDNA (rcccDNA) formation via viral or transgenic Cre expression in HBV1.1X mice, and the ability to regulate HBsAg and HBc expression with Cre in mice. Tamoxifen-inducible Cre could markedly downregulate baseline HBsAg levels from the integrated HBV genome. To demonstrate clearance of HBV from HBV1.1X mice, we administered adenovirus expressing Cre, which permanently and significantly reduced HBsAg and core antigen levels in the murine liver via rcccDNA excision and a subsequent immune response. CONCLUSIONS: The HBV1.1X model is the first Cre-regulatable HBV transgenic mouse model and should be of value to mimic chronic HBV infection, with neonatal expression and tolerance of HBV antigens, and on-demand modulation of HBV expression. LAY SUMMARY: Hepatitis B virus (HBV) can only naturally infect humans and chimpanzees. Mouse models have been developed with the HBV genome integrated into mouse chromosomes, but this prevents mice from being cured. We developed a new transgenic mouse model that allows for HBV to be excised from mouse chromosomes to form a recombinant circular DNA molecule resembling the natural circular HBV genome. HBV expression could be reduced in these mice, enabling curative therapies to be tested in this new mouse model.
ABSTRACT
New therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P < 0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.
Subject(s)
DNA, Circular/genetics , DNA, Viral/genetics , Genetic Engineering/methods , Hepatitis B virus/genetics , Hepatitis B/virology , Plasmids/genetics , DNA, Circular/chemistry , DNA, Recombinant/chemistry , DNA, Recombinant/genetics , DNA, Viral/chemistry , Genome, Viral , Hepatitis B Surface Antigens/genetics , Hepatitis B Surface Antigens/metabolism , Hepatitis B virus/chemistry , Hepatitis B virus/metabolism , Humans , Integrases/metabolism , Plasmids/metabolism , TransfectionABSTRACT
BACKGROUND & AIMS: Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts. METHODS: In this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product. RESULTS: We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse. CONCLUSIONS: This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.
Subject(s)
Argininosuccinate Lyase/genetics , CRISPR-Associated Protein 9/administration & dosage , CRISPR-Cas Systems/genetics , Gene Editing/methods , Liver/enzymology , ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics , Animals , Disease Models, Animal , Hepatocytes/enzymology , Hepatocytes/physiology , Mice , Mice, Knockout , Oxidoreductases/genetics , Phenotype , Plasmids/geneticsABSTRACT
BACKGROUND: Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection. METHODS: We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human liver chimeric mice. RESULTS: HBsAg-CAR T cells recognized HBV-positive cell lines and HBsAg particles in vitro as judged by cytokine production. However, HBsAg-CAR T cells did not kill HBV-positive cell lines in cytotoxicity assays. Adoptive transfer of HBsAg-CAR T cells into HBV-infected humanized mice resulted in accumulation within the liver and a significant decrease in plasma HBsAg and HBV-DNA levels compared with control mice. Notably, the fraction of HBV core-positive hepatocytes among total human hepatocytes was greatly reduced after HBsAg-CAR T cell treatment, pointing to noncytopathic viral clearance. In agreement, changes in surrogate human plasma albumin levels were not significantly different between treatment and control groups. CONCLUSIONS: HBsAg-CAR T cells have anti-HBV activity in an authentic preclinical HBV infection model. Our results warrant further preclinical exploration of HBsAg-CAR T cells as immunotherapy for HBV.
Subject(s)
Antiviral Agents/immunology , Chimera/immunology , Hepatitis B Surface Antigens/metabolism , Hepatitis B virus/physiology , Liver/immunology , Liver/virology , T-Lymphocytes/immunology , Animals , Hep G2 Cells , Hepatitis B, Chronic , Humans , Immunotherapy/methods , Mice , Receptors, Antigen, T-Cell/immunology , Receptors, Chimeric Antigen/metabolism , Virion/metabolismABSTRACT
Current therapies against hepatitis B virus (HBV) do not reliably cure chronic infection, necessitating new therapeutic approaches. The T cell response can clear HBV during acute infection, and the adoptive transfer of antiviral T cells during bone marrow transplantation can cure patients of chronic HBV infection. To redirect T cells to HBV-infected hepatocytes, we delivered plasmids encoding bispecific antibodies directed against the viral surface antigen (HBsAg) and CD3, expressed on almost all T cells, directly into the liver using hydrodynamic tail vein injection. We found a significant reduction in HBV-driven reporter gene expression (184-fold) in a mouse model of acute infection, which was 30-fold lower than an antibody only recognizing HBsAg. While bispecific antibodies triggered, in part, antigen-independent T cell activation, antibody production within hepatocytes was non-cytotoxic. We next tested the bispecific antibodies in a different HBV mouse model, which closely mimics the transcriptional template for HBV, covalently closed circular DNA (cccDNA). We found that the antiviral effect was noncytopathic, mediating a 495-fold reduction in HBsAg levels at day 4. At day 33, bispecific antibody-treated mice exhibited 35-fold higher host HBsAg immunoglobulin G (IgG) antibody production versus untreated groups. Thus, gene therapy with HBsAg/CD3-bispecific antibodies represents a promising therapeutic strategy for patients with HBV.
ABSTRACT
An incorrect version of the Supplementary Information was inadvertently published with this Article where the wrong file was included. The HTML has been updated to include the correct version of the Supplementary Information.
ABSTRACT
Only one out of 10 drugs in development passes clinical trials. Many fail because experimental animal models poorly predict human xenobiotic metabolism. Human liver chimeric mice are a step forward in this regard, as the human hepatocytes in chimeric livers generate human metabolites, but the remaining murine hepatocytes contain an expanded set of P450 cytochromes that form the major class of drug-metabolizing enzymes. We therefore generated a conditional knock-out of the NADPH-P450 oxidoreductase (Por) gene combined with Il2rg - /- /Rag2 - /- /Fah - /- (PIRF) mice. Here we show that homozygous PIRF mouse livers are readily repopulated with human hepatocytes, and when the murine Por gene is deleted (<5%), they predominantly use human cytochrome metabolism. When given the anticancer drug gefitinib or the retroviral drug atazanavir, the Por-deleted humanized PIRF mice develop higher levels of the major human metabolites than current models. Humanized, murine Por-deficient PIRF mice can thus predict human drug metabolism and should be useful for preclinical drug development.Human liver chimeric mice are increasingly used for drug testing in preclinical development, but express residual murine p450 cytochromes. Here the authors generate mice lacking the Por gene in the liver, and show that human cytochrome metabolism is used following repopulation with human hepatocytes.
Subject(s)
Atazanavir Sulfate/metabolism , Cytochrome P-450 Enzyme System/metabolism , Quinazolines/metabolism , Animals , Antineoplastic Agents/metabolism , Chimera , Cytochrome P-450 Enzyme System/genetics , Cytochromes/metabolism , Female , Gefitinib , Genotype , HIV Protease Inhibitors/metabolism , Humans , Liver/enzymology , Liver/metabolism , Male , Mice , Mice, Inbred StrainsABSTRACT
Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and ß-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and ß-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.
Subject(s)
Amniotic Fluid/cytology , Fetal Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Animals , Antigens, Surface/genetics , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , Female , Humans , Kruppel-Like Factor 4 , Mice , Nanog Homeobox Protein/genetics , Octamer Transcription Factor-3/genetics , Pregnancy , Pregnancy Trimester, Second , Proteoglycans/genetics , TransfectionABSTRACT
Many metabolic liver disorders are refractory to drug therapy and require orthotopic liver transplantation. Here we demonstrate a new strategy, which we call metabolic pathway reprogramming, to treat hereditary tyrosinaemia type I in mice; rather than edit the disease-causing gene, we delete a gene in a disease-associated pathway to render the phenotype benign. Using CRISPR/Cas9 in vivo, we convert hepatocytes from tyrosinaemia type I into the benign tyrosinaemia type III by deleting Hpd (hydroxyphenylpyruvate dioxigenase). Edited hepatocytes (Fah(-/-)/Hpd(-/-)) display a growth advantage over non-edited hepatocytes (Fah(-/-)/Hpd(+/+)) and, in some mice, almost completely replace them within 8 weeks. Hpd excision successfully reroutes tyrosine catabolism, leaving treated mice healthy and asymptomatic. Metabolic pathway reprogramming sidesteps potential difficulties associated with editing a critical disease-causing gene and can be explored as an option for treating other diseases.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Genetic Therapy/methods , Metabolic Networks and Pathways/genetics , Tyrosinemias/genetics , Animals , Cell Line , Cell Proliferation/genetics , Cyclohexanones/therapeutic use , Disease Models, Animal , Enzyme Inhibitors/therapeutic use , Exons/genetics , Hepatocytes/metabolism , Humans , Hydrolases/genetics , Liver/cytology , Liver/pathology , Mice , Mice, Inbred Strains , Mice, Knockout , Nitrobenzoates/therapeutic use , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/genetics , Oxidoreductases/metabolism , Phenotype , Tyrosinemias/metabolism , Tyrosinemias/pathology , Tyrosinemias/therapyABSTRACT
BACKGROUND & AIMS: Pediatric liver cancer is a rare but serious disease whose incidence is rising, and for which the therapeutic options are limited. Development of more targeted, less toxic therapies is hindered by the lack of an experimental animal model that captures the heterogeneity and metastatic capability of these tumors. METHODS: Here we established an orthotopic engraftment technique to model a series of patient-derived tumor xenograft (PDTX) from pediatric liver cancers of all major histologic subtypes: hepatoblastoma, hepatocellular cancer and hepatocellular malignant neoplasm. We utilized standard (immuno) staining methods for histological characterization, RNA sequencing for gene expression profiling and genome sequencing for identification of druggable targets. We also adapted stem cell culturing techniques to derive two new pediatric cancer cell lines from the xenografted mice. RESULTS: The patient-derived tumor xenografts recapitulated the histologic, genetic, and biological characteristics-including the metastatic behavior-of the corresponding primary tumors. Furthermore, the gene expression profiles of the two new liver cancer cell lines closely resemble those of the primary tumors. Targeted therapy of PDTX from an aggressive hepatocellular malignant neoplasm with the MEK1 inhibitor trametinib and pan-class I PI3 kinase inhibitor NVP-BKM120 resulted in significant growth inhibition, thus confirming this PDTX model as a valuable tool to study tumor biology and patient-specific therapeutic responses. CONCLUSIONS: The novel metastatic xenograft model and the isogenic xenograft-derived cell lines described in this study provide reliable tools for developing mutation- and patient-specific therapies for pediatric liver cancer. LAY SUMMARY: Pediatric liver cancer is a rare but serious disease and no experimental animal model currently captures the complexity and metastatic capability of these tumors. We have established a novel animal model using human tumor tissue that recapitulates the genetic and biological characteristics of this cancer. We demonstrate that our patient-derived animal model, as well as two new cell lines, are useful tools for experimental therapies.
Subject(s)
Liver Neoplasms , Animals , Carcinoma, Hepatocellular , Cell Line, Tumor , Child , Disease Models, Animal , Heterografts , Humans , Mice , Neoplasm Transplantation , Xenograft Model Antitumor AssaysABSTRACT
Diseases of lipid metabolism are a major cause of human morbidity, but no animal model entirely recapitulates human lipoprotein metabolism. Here we develop a xenograft mouse model using hepatocytes from a patient with familial hypercholesterolaemia caused by loss-of-function mutations in the low-density lipoprotein receptor (LDLR). Like familial hypercholesterolaemia patients, our familial hypercholesterolaemia liver chimeric mice develop hypercholesterolaemia and a 'humanized' serum profile, including expression of the emerging drug targets cholesteryl ester transfer protein and apolipoprotein (a), for which no genes exist in mice. We go on to replace the missing LDLR in familial hypercholesterolaemia liver chimeric mice using an adeno-associated virus 9-based gene therapy and restore normal lipoprotein profiles after administration of a single dose. Our study marks the first time a human metabolic disease is induced in an experimental animal model by human hepatocyte transplantation and treated by gene therapy. Such xenograft platforms offer the ability to validate human experimental therapies and may foster their rapid translation into the clinic.
Subject(s)
Disease Models, Animal , Genetic Therapy , Hyperlipoproteinemia Type II/therapy , Receptors, LDL/genetics , Animals , Child , Dependovirus , Female , Hepatocytes/transplantation , Heterografts , Humans , Hyperlipoproteinemia Type II/genetics , Hyperlipoproteinemia Type II/metabolism , Lipoproteins/metabolismABSTRACT
BACKGROUND: Prostate cancer is the most common cancer among elderly men in the US, and immunotherapy has been shown to be a promising strategy to treat patients with metastatic castration-resistant prostate cancer. Efforts to identify novel prostate specific tumor antigens will facilitate the development of effective cancer vaccines against prostate cancer. Prostate-specific G-protein coupled receptor (PSGR) is a novel antigen that has been shown to be specifically over-expressed in human prostate cancer tissues. In this study, we describe the identification of PSGR-derived peptide epitopes recognized by CD8(+) T cells in an HLA-A2 dependent manner. METHODOLOGY/PRINCIPAL FINDINGS: Twenty-one PSGR-derived peptides were predicted by an immuno-informatics approach based on the HLA-A2 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from either HLA-A2(+) healthy donors or HLA-A2(+) prostate cancer patients. The recognition of HLA-A2 positive and PSGR expressing LNCaP cells was also tested. Among the 21 PSGR-derived peptides, three peptides, PSGR3, PSGR4 and PSGR14 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and prostate cancer patients. Importantly, these peptide-specific T cells recognized and killed LNCaP prostate cancer cells in an HLA class I-restricted manner. CONCLUSIONS/SIGNIFICANCE: We have identified three novel HLA-A2-restricted PSGR-derived peptides recognized by CD8(+) T cells, which, in turn, recognize HLA-A2(+) and PSGR(+) tumor cells. The PSGR-derived peptides identified may be used as diagnostic markers as well as immune targets for development of anticancer vaccines.
Subject(s)
Antigens, Neoplasm/immunology , Cancer Vaccines/immunology , Neoplasm Proteins/immunology , Peptide Fragments/immunology , Prostatic Neoplasms/metabolism , Receptors, Odorant/immunology , Amino Acid Sequence , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines/chemical synthesis , Cell Line, Tumor , HLA Antigens/metabolism , Humans , Immunotherapy, Active , Interferon-gamma/metabolism , Male , Neoplasm Proteins/metabolism , Peptide Fragments/chemical synthesis , Prostatic Neoplasms/immunology , Prostatic Neoplasms/therapy , Receptors, Odorant/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
Stringent control of the NF-kappaB and type I interferon signaling pathways is critical to effective host immune responses, yet the molecular mechanisms that negatively regulate these pathways are poorly understood. Here, we show that NLRC5, a member of the highly conserved NOD-like protein family, can inhibit the IKK complex and RIG-I/MDA5 function. NLRC5 inhibited NF-kappaB-dependent responses by interacting with IKKalpha and IKKbeta and blocking their phosphorylation. It also interacted with RIG-I and MDA5, but not with MAVS, to inhibit RLR-mediated type I interferon responses. Consistent with these observations, NLRC5-specific siRNA knockdown not only enhanced the activation of NF-kappaB and its responsive genes, TNF-alpha and IL-6, but also promoted type I interferon signaling and antiviral immunity. Our findings identify NLRC5 as a negative regulator that blocks two central components of the NF-kappaB and type I interferon signaling pathways and suggest an important role for NLRC5 in homeostatic control of innate immunity.
Subject(s)
Immunity, Innate , Interferon Type I/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , NF-kappa B/metabolism , Signal Transduction , Animals , Cloning, Molecular , DEAD-box RNA Helicases/metabolism , Humans , I-kappa B Kinase/metabolism , Inflammation , Intracellular Signaling Peptides and Proteins/chemistry , Ligands , Mice , Phosphorylation , Toll-Like Receptors/metabolismABSTRACT
The complete sequence of a type-1 metacaspase from Acanthamoeba castellanii is reported comprising 478 amino acids. The metacaspase was recovered from an expression library using sera specific for membrane components implicated in stimulating encystation. A central domain of 155 amino acid residues contains the Cys/His catalytic dyad and is the most conserved region containing at least 30 amino acid identities in all metacaspases. The Acanthamoeba castellanii metacaspase has the most proline-rich N-terminus so far reported in type-1 metacaspases with over 40 prolines in the first 150 residues. Ala-Pro-Pro is present 11 times. Phylogenies constructed using only the conserved proteolytic domains or the complete sequences show identical branching patterns, differing only in the rates of change.