B6-hATP7B*H1069Q Mice

Catalog Number: C001610

Strain Name: C57BL/6NCya-Atp7btm2(hATP7B*H1069Q)/Cya

Genetic Background: C57BL/6NCya

Reproduction: Homozygous Males x Heterozygous Females.

One of Cyagen's HUGO-GT™ (Humanized Genomic Ortholog for Gene Therapy) Mouse Strains

 

Strain Description

Hepatolenticular degeneration (HLD), also known as Wilson disease (WD), is an autosomal recessive copper transport disorder that can lead to liver failure. The incidence rate is about 1:30,000 [1]. The clinical manifestations of HLD mainly include chronic liver damage, and neurological and psychiatric symptoms, and can occasionally cause acute liver failure and hemolytic anemia. Its typical manifestation is the combination of liver disease and movement disorders in adolescence or early adulthood, but there is a large variation in phenotypic differences among patients, and up to 60% of patients have neurological or psychiatric symptoms [2]. Studies have shown that mutations in the ATP7B gene are associated with HLD. The characteristic feature is that with the loss of functional ATP7B protein, the clearance of excess copper is affected, leading to copper accumulation to toxic levels, damaging tissues and organs such as the liver and brain [1,3-4]. The copper ion transport ATPase β-peptide encoded by the ATP7B gene is a member of the P-type cation transport ATPase family. This family uses the energy stored in ATP to transport metals into and out of cells. The ATP7B protein consists of multiple transmembrane domains, an ATPase consensus sequence, a hinge domain, a phosphorylation site, and at least two putative copper-binding sites [5]. This protein mainly exists in the liver, with small amounts found in the kidneys and brain. Its function as a copper transport ATPase plays a role in transporting copper from the liver to other parts of the body. More than 900 pathogenic mutations of the ATP7B gene have been reported, with the mutation types mainly concentrated in missense, nonsense, or frameshift mutations, and other mechanisms include exon skipping, large deletions, and intron variations. The most common mutation in patients from Northern and Eastern Europe is H1069Q, but its frequency varies greatly among countries [2].

Hepatolenticular degeneration (HLD) treatments are mainly categorized into pharmacotherapy and surgical intervention. Pharmacotherapy is aimed at alleviating symptoms, preventing disease progression, and preventing complications, while surgery is typically liver transplantation. With the continuous exploration of the genetic etiology of Wilson’s disease, targeted gene therapy is expected to become the next "star therapy." Currently, multiple biotechnology companies and research institutions, including Prime Medicine and LogicBio Therapeutics, are developing a variety of gene editing therapies based on Targeted Gene Editing/Cas9, Prime Editor, or other technologies to correct mutations in the ATP7B gene or replace the mutated ATP7B gene as a whole. These highly promising therapies are currently in preclinical studies [7-14]. Given that these gene editing therapies require precise targeting of the human ATP7B gene, humanizing mouse genes will help accelerate the entry of gene therapy into the clinical stage. This strain is a humanized point mutation model constructed by introducing the common pathogenic mutation p.H1069Q (CAC>CAA) into the humanized ATP7B gene of B6-hATP7B mice (Catalog No.: I001130). This model is suitable for studying the pathogenic mechanisms of Wilson's disease, and homozygous animals are viable and fertile. In addition, based on the independently developed TurboKnockout fusion BAC recombination technology, Cyagen can also generate hot mutation models based on this strain and provide customized services to meet the experimental needs.

Strain Strategy

  • Gene editing strategy of B6-hATP7B mice: The mouse Atp7b endogenous domain (aa.1-1462) was replaced with the human ATP7B domain (aa.1-1465).

  • Gene editing strategy of B6-hATP7B*H1069Q mice: The p.H1069Q (CAC to CAA) was introduced into exon 14 of human ATP7B.


Application

  • Research on hepatolenticular degeneration (HLD);
  • Preclinical evaluation of ATP7B-targeted drugs.

 

Validation Data

1. RT-qPCR

Figure 1. Gene expression analysis of liver and lung tissues from 6-week-old male homozygous B6-hATP7B*H1069Q mice, B6-hATP7B mice, and wild-type (WT) mice (n=4). RT-qPCR results indicate significant expression of the human ATP7B gene in liver and lung tissues of B6-hATP7B*H1069Q mice and B6-hATP7B mice, with no expression of the mouse Atp7b gene. In contrast, WT mice only exhibit expression of the mouse Atp7b gene.

(ND: Not detected; Bars represent mean ± SEM)

 

2. Western Blot

Figure 2. Protein expression analysis* in the liver tissues of 6-week-old male homozygous B6-hATP7B*H1069Q mice, B6-hATP7B mice, and wild-type (WT) mice (n=3). Western blot results show significant expression of the human ATP7B protein in the liver tissues of B6-hATP7B mice. However, B6-hATP7BH1069Q mice exhibit lower levels of human protein compared to B6-hATP7B mice, and WT mice do not express human ATP7B protein. Based on RT-qPCR results, the mRNA levels in B6-hATP7BH1069Q mice are either elevated or normal, while the protein levels are reduced. This discrepancy is primarily due to the H1069Q mutation leading to protein misfolding and instability. The mutation does not affect transcription but may reduce post-transcriptional regulation or translation efficiency. Additionally, the abnormal protein is prone to degradation via the ubiquitin-proteasome pathway, resulting in significantly lower protein levels. This inconsistency between mRNA and protein levels reflects the impact of the H1069Q mutation on protein stability and degradation pathways.

*The antibody used for detection binds to the region of the human protein from Human ATP7b aa.1400 to the C-terminus (C terminal), excluding the H1069Q mutation (aa.1070).

 

Expanded Information: The Rare Disease Data Center (RDDC)

1. Basic information about the ATP7B gene

2. ATP7B clinical variants


3. Disease introduction

Hepatolenticular degeneration (HLD), also known as Wilson disease (WD), is an autosomal recessive copper transport disorder that can lead to liver failure. The incidence rate is about 1:30,000 [1]. The clinical manifestations of HLD mainly include chronic liver damage, and neurological and psychiatric symptoms, and can occasionally cause acute liver failure and hemolytic anemia. Its typical manifestation is the combination of liver disease and movement disorders in adolescence or early adulthood, but there is a large variation in phenotypic differences among patients, and up to 60% of patients have neurological or psychiatric symptoms [2]. Studies have shown that mutations in the ATP7B gene are associated with HLD. The characteristic feature is that with the loss of functional ATP7B protein, the clearance of excess copper is affected, leading to copper accumulation to toxic levels, damaging tissues and organs such as the liver and brain [1,3-4].

4. ATP7B gene and mutations

The copper ion transport ATPase β-peptide encoded by the ATP7B gene is a member of the P-type cation transport ATPase family. This family uses the energy stored in ATP to transport metals into and out of cells. The ATP7B protein consists of multiple transmembrane domains, an ATPase consensus sequence, a hinge domain, a phosphorylation site, and at least two putative copper-binding sites [5]. This protein mainly exists in the liver, with small amounts found in the kidneys and brain. Its function as a copper transport ATPase plays a role in transporting copper from the liver to other parts of the body. More than 900 pathogenic mutations of the ATP7B gene have been reported, with the mutation types mainly concentrated in missense, nonsense, or frameshift mutations, and other mechanisms include exon skipping, large deletions, and intron variations. The most common mutation in patients from Northern and Eastern Europe is H1069Q, but its frequency varies greatly among countries [2].

5. ATP7B-targeted gene therapy

With the continuous exploration of genetic pathogenesis, targeted gene therapy is expected to become the next “star therapy”. VTX-801 from Vivet Therapeutics has received U.S. FDA Fast Track Designation for the treatment of Wilson’s Disease. VTX-801 is a gene therapy vector based on rAAV, which is used to deliver small ATP7B transgenes. The functional protein encoded by the transgene can restore copper balance, reverse liver pathological changes, and reduce copper accumulation in the brain of Wilson’s disease mice models [6]. UX701 is a gene therapy drug based on AAV9 developed by Ultragenyx. It treats HLD by delivering modified ATP7B genes. Its preclinical studies have shown that UX701 can reduce copper accumulation in the liver, increase ceruloplasmin levels, and improve liver pathology [15].

In summary, the ATP7B gene is a significant pathogenic gene for hepatolenticular degeneration (HLD). ATP7B gene humanized mice from Cyagen can be used for preclinical research on HLD and customized services can also be provided for different point mutations.

 

References
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[2]Shribman S, Poujois A, Bandmann O, Czlonkowska A, Warner TT. Wilson's disease: update on pathogenesis, biomarkers and treatments. J Neurol Neurosurg Psychiatry. 2021 Oct;92(10):1053-1061.
[3]Ferenci, P. Regional distribution of mutations of the ATP7B gene in patients with Wilson disease: impact on genetic testing. Hum Genet 120, 151–159 (2006).
[4]Fatemi N, Sarkar B. Molecular mechanism of copper transport in Wilson disease. Environ Health Perspect. 2002 Oct;110 Suppl 5(Suppl 5):695-8. doi: 10.1289/ehp.02110s5695.
[5]Cater MA, Forbes J, La Fontaine S, Cox D, Mercer JF. Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites. Biochem J. 2004 Jun 15;380(Pt 3):805-13.
[6]Pfizer. (2021, August 12). VTX-801 Receives U.S. FDA Fast Track Designation for the Treatment of Wilson Disease. Retrieved January 22, 2022, from https://www.pfizer.com/news/press-release/press-release-detail/vtx-801-receives-us-fda-fast-track-designation-treatment
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[8]Prime Medicine. (2024). AASLD WD Talk. Retrieved February 21, 2025, from https://primemedicine.com/wp-content/uploads/2024/12/2024-11-18-AASLD-WD-Talk-v3_Final_PDF.pdf
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[10]Liu L, Cao J, Chang Q, Xing F, Yan G, Fu L, Wang H, Ma Z, Chen X, Li Y, Li S. In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System. Hum Gene Ther. 2019 Sep;30(9):1079-1092.
[11]Wei R, Yang J, Cheng CW, Ho WI, Li N, Hu Y, Hong X, Fu J, Yang B, Liu Y, Jiang L, Lai WH, Au KW, Tsang WL, Tse YL, Ng KM, Esteban MA, Tse HF. Targeted Gene Editing-targeted genome editing of human induced pluripotent stem cell-derived hepatocytes for the treatment of Wilson's disease. JHEP Rep. 2021 Oct 30;4(1):100389.
[12]Padula A, Spinelli M, Nusco E, Bujanda Cundin X, Capolongo F, Campione S, Perna C, Bastille A, Ericson M, Wang CC, Zhang S, Amoresano A, Nacht M, Piccolo P. Genome editing without nucleases confers proliferative advantage to edited hepatocytes and corrects Wilson disease. JCI Insight. 2023 Nov 8;8(21):e171281.
[13]Pöhler M, Guttmann S, Nadzemova O, Lenders M, Brand E, Zibert A, Schmidt HH, Sandfort V. Targeted Gene Editing/Cas9-mediated correction of mutated copper transporter ATP7B. PLoS One. 2020 Sep 30;15(9):e0239411.
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[15]Ultragenyx. (n.d.). UX701 for Wilson Disease. Retrieved January 22, 2022, from https://www.ultragenyx.com/our-research/pipeline/ux701-for-wilson-disease/