B6-hF9 Mice

Catalog Number: C001644

Strain Name: C57BL/6NCya-F9tm2(hF9)/Cya

Genetic Background: C57BL/6NCya

Reproduction: Homozygote x Homozygote

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

 

Strain Description

Hemophilia is a group of inherited bleeding disorders primarily caused by deficiency or dysfunction of coagulation factor VIII or IX, leading to impaired coagulation.  Patients typically present with prolonged clotting time, easy bleeding even after minor trauma, and in severe cases, spontaneous bleeding, commonly occurring in joints and deep tissues. Hemophilia is mainly classified into three types: type A (factor VIII deficiency), type B (factor IX deficiency), and type C (factor XI deficiency). Among these, types A and B are the most prevalent. Hemophilia A is caused by mutations in the F8 gene, resulting in factor VIII deficiency, while hemophilia B is caused by mutations in the F9 gene, leading to factor IX deficiency. Coagulation factor IX, encoded by the F9 gene, is activated to FIXa during coagulation and works in concert with FVIIIa, Ca2+, and membrane phospholipids to activate factor X. Hemophilia A and B are both X-linked recessive genetic disorders with a higher incidence in males. The incidence of hemophilia B is approximately 1/25,000 to 1/30,000, accounting for about 15%-20% of all hemophilia cases [1].

Currently, coagulation factor replacement therapy is the primary treatment for hemophilia [1]. For hemophilia A, the treatment is intravenous injection of factor VIII concentrates; for hemophilia B, factor IX concentrates are injected to maintain normal levels of coagulation factors in patients. However, this therapy is a supplementary approach, requiring lifelong regular injections, which may not only cause side effects but also impose a substantial economic burden on patients. Therefore, gene therapy, particularly for hemophilia B, is considered a highly promising research direction. Etranacogene dezaparvovec (brand name Hemgenix) is the first gene therapy for hemophilia B approved by the U.S. FDA [2-3]. This therapy utilizes adeno-associated virus vector AAV5 to deliver the coagulation factor IX gene to patient hepatocytes, thereby increasing FIX activity in vivo and reducing bleeding events [2]. Gene therapy is considered a potential curative approach for hemophilia B. Considering the genetic differences between animals and humans, and that most gene therapies target human genes, humanizing mouse genes will help accelerate the drug pipeline of gene therapies into the clinical stage.

This strain is a humanized mouse F9 gene model, which can be used for preclinical evaluation of hemophilia B pathogenesis and therapeutic drugs. Homozygotes of this model are viable and fertile. In addition, based on the independently developed TurboKnockout fusion BAC recombination technology innovation, Cyagen can also provide disease models of popular point mutations based on this model, and can also provide customized services for different point mutations to meet the experimental needs of researchers in the pharmacodynamics of hemophilia B.

 

Strain Strategy


Figure 1. Gene editing strategy of B6-hF9 mice. The mouse F9 protrin endogenous domain (aa. 29~471) will be replaced with the human protrin F9 domain (aa. 29~461). The murine protrin signal peptide (aa. 1~28) will be remained.

 

Application

  • Research on Hemophilia B.

 

Validation Data

1. Detection of Human F9 Gene Expression


Figure 2. Detection of human and murine F9 gene expression in the liver of wild-type (WT) and B6-hF9 (hF9) mice at 7 weeks of age. RT-qPCR results revealed that human F9 expression was significantly detected in the liver of male B6-hF9 mice, whereas mouse F9 expression was undetectable. In contrast, mouse F9 expression was significantly detected in the liver of male wild-type mice, whereas human F9 expression was undetectable (n=3, Bars represent mean ± SEM).

 

2. Detection of human Factor IX protein expression


Figure 3. Detection of human Factor IX expression in the plasma of wild-type (WT) and B6-hF9 (hF9) mice at 6 weeks of age. ELISA results demonstrated that human Factor IX expression in the plasma of female and male B6-hF9 mice was significantly higher than that in wild-type mice (n=5, Bars represent mean ± SD).

 

3. Coagulation Assay


Figure 4. Comparative analysis of coagulation parameters in wild-type (WT) and B6-hF9 (hF9) mice at 6 weeks of age. The levels of Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT), Thrombin Time (TT), and Fibrinogen (FIB) in female and male B6-hF9 mice showed no statistically significant difference compared to wild-type mice (nB6-hF9=5, nWT=4, Bars represent mean ± SEM).

Expanded Information: The Rare Disease Data Center (RDDC)

1. Basic information about the F9 gene

 

 

2. F9 clinical variants

 

3. Disease introduction

Hemophilia is a group of bleeding disorders with inherited coagulation disorders. The common features of this disease are impaired generation of active coagulation factors, prolonged clotting time, lifelong tendency to bleed after minor trauma, and "spontaneous" bleeding in severe cases without obvious trauma. This disease is mainly divided into two types: Hemophilia A and Hemophilia B. Hemophilia A patients lack the eighth coagulation factor (Factor VIII) due to F8 (FVIII) gene mutation, while Hemophilia B is caused by F9 (FIX) gene mutation, leading to a deficiency in the ninth coagulation factor (Factor IX). Hemophilia is an X-linked congenital bleeding disorder that occurs more frequently in males. The incidence of Hemophilia B caused by F9 gene mutation is about 1/25,000, accounting for approximately 15-20% of all cases of hemophilia.

 

4. F9 gene and mutations

The F9 gene is smaller and structurally simpler than the F8 gene, with a length of approximately 34,000 base pairs and containing 8 exons, the longest of which is only 1,935 base pairs. The F9 gene encodes a vitamin K-dependent serine protease (Factor IX) that plays a key role in the intrinsic coagulation pathway. Factor IX circulates in the blood as an inactive zymogen and is converted into an active form by cleavage of peptide by factor XIa. Coagulation factor IX activates factor X by interacting with Ca2+ ions, membrane phospholipids, and factor VIII in the coagulation cascade reaction. Deficiency of Factor IX due to F9 gene deletion can cause bleeding disorders, leading to X-linked recessive hemophilia B. Pathogenic mutations in the F9 gene include large segment deletions, nonsense and frameshift mutations, with approximately 80% being single-base mutations. The common mutation R29X produces inhibitory antibodies against Factor IX [3].

 

5. Function of non-coding DNA sequences

A mutation at 10,389 A>G in the intron 3 can cause Hemophilia B [4].

 

6. F9-targeted gene therapy

Currently, the main treatment for hemophilia is enzyme replacement therapy [1]. Maintaining the normal level of FIX in patients through intravenous injection of coagulation factor IX is a “stopgap measure” that may cause side effects and impose a huge economic burden on patients. Therefore, finding a “once-and-for-all” treatment has become a key challenge in the field of blood diseases. Gene therapy that “cures the disease from the source” for Hemophilia B may be the next highly anticipated research direction. “Etranacogene Dezaparvovec” is the world’s first gene therapy for Hemophilia B that has been approved by the US FDA. This therapy uses an AAV5 vector to deliver coagulation factor IX into the patient’s body, which can help effectively increase FIX activity in patients [2]. In addition, “Spk-9001”, a candidate gene therapy developed by Spark Therapeutics and Pfizer, has made significant progress. It uses AAV to deliver normal gene copies to the patient’s liver cells to maintain stable levels of coagulation factor IX [5]. Since most gene therapies act on human genes, considering the genetic differences between animals and humans, humanizing mouse genes will help accelerate the drug pipeline of gene therapy into clinical stages.

 

7. Summary

The F9 gene is an important pathogenic gene for Hemophilia B. F9 whole-genome humanized mice from Cyagen can be used for preclinical research on Hemophilia B, and customized services can also be provided for different point mutations.

 

 

 

References
[1]Liu G, Sun J, Li Z, Chen Z, Wu W, Wu R. F9 mutations causing deletions beyond the serine protease domain confer higher risk for inhibitor development in hemophilia B. Blood. 2023 Feb 9;141(6):677-680.
[2]Pipe SW, Leebeek FWG, Recht M, Key NS, Castaman G, Miesbach W, Lattimore S, Peerlinck K, Van der Valk P, Coppens M, Kampmann P, Meijer K, O'Connell N, Pasi KJ, Hart DP, Kazmi R, Astermark J, Hermans CRJR, Klamroth R, Lemons R, Visweshwar N, von Drygalski A, Young G, Crary SE, Escobar M, Gomez E, Kruse-Jarres R, Quon DV, Symington E, Wang M, Wheeler AP, Gut R, Liu YP, Dolmetsch RE, Cooper DL, Li Y, Goldstein B, Monahan PE. Gene Therapy with Etranacogene Dezaparvovec for Hemophilia B. N Engl J Med. 2023 Feb 23;388(8):706-718
[3]https://www.sciencedirect.com/topics/medicine-and-dentistry/hemophilia-therapy
[4]Cao DH, Liu XL, Mu K, Ma XW, Sun JL, Bai XZ, Lin CK, Jin CL. Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China. Turk J Haematol. 2014 Sep 5;31(3):226-30. doi: 10.4274/tjh.2013.0275. PMID: 25330515; PMCID: PMC4287022.
[5]https://hemophilianewstoday.com/spk-9001/