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Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg -/- /Rag2 -/- /Fah -/- /Aavr -/- (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting., (© 2024. The Author(s).)

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., Competing Interests: The authors declare no conflicts of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Authors.)

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., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Preclinical evaluation of drug candidates in experimental animal models is an essential step in drug development. Humanized mouse models have emerged as a promising alternative to traditional animal models. The purpose of this mini-review is to provide a brief survey of currently available mouse models for studying human xenobiotic metabolism. Here, we describe both genetic humanization and human liver chimeric mouse models, focusing on the advantages and limitations while outlining their key features and applications. Although this field of biomedical science is relatively young, these humanized mouse models have the potential to transform preclinical drug testing and eventually lead to a more cost-effective and rapid development of new therapies., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

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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.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome engineering has revolutionised biomedical science and we are standing on the cusp of medical transformation. The therapeutic potential of this technology is tremendous, however, its translation to the clinic will be challenging. In this article, we review recent progress using this genome editing technology and explore its potential uses in studying and treating diseases of the liver. We discuss the development of new research tools and animal models as well as potential clinical applications, strategies and challenges., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

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.

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.

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.