Rare diseases have always been a big challenge in the field of medical science. Not only do these diseases have complex mechanisms of occurrence and different types, but importantly, there are no suitable animal models for studying the underlying pathogenesis and evaluating efficacy of drugs. Driven by the urgent demands, Cyagen has launched the "Ten Deadly Sins of Rare Diseases" column, aiming to explore the causes of the occurrence and development of rare diseases through current therapeutic research, from multiple aspects such as pathogenesis, preclinical animal models, and gene therapy methods.

In the previous issue, we have discussed how RHO achieves "1+1=1" gene therapy for ophthalmology diseases. This issue, we will focus on hemophilia, which is a rare blood disease caused by mutations in the FⅧ (F8) and FⅨ(F9) genes. Patients with hemophilia have a lifelong tendency of spontaneous bleeding and prolonged bleeding time after trauma due to impaired blood clotting ability. Even minor collisions in daily life might cause serious bleeding and endanger their lives. Therefore, hemophiliacs are also known as "glass people". Finding out how to cure hemophilia and expand its research depth are big concerns for many people.

The pathogenesis of FⅧ and FⅨ

Hemophilia A (HA) and hemophilia B (HB) are X-linked recessive inherited diseases caused by mutations in FVIII and FIX genes, which result in clotting disorders. As an X-linked recessive disease, it only affects XX females when both parents carry the disease-causing gene. However, for XY males who only have one X chromosome, they have the potential to be affected if one of their parents carries the disease-causing gene. Therefore, hemophilia is more commonly seen in males.

The clotting factors FVIII and FIX are essential components in the coagulation process. When these genes undergo mutations, the body is unable to produce sufficient clotting factors, leading to patients being unable to stop bleeding normally after injury or surgery.

More than 3000 mutations have been reported in the FVIII gene, with around 57.4% being missense mutations and nonsense mutations. FVIII is mainly expressed in the liver, with high genetic heterogeneity and no mutation hotspots. However, an inversion in intron 22 of FVIII gene is the main cause of severe hemophilia A; wherein the FVIII gene breaks and inverts at intron 22, thereby separating exons 1-22 from exons 23-26 of the FVIII gene, preventing normal transcription and leading to a severe deficiency of FVIII factor, resulting in the development of severe hemophilia A[1].

Over 3000 mutations have been reported in the FIX gene, with point mutations making up the largest proportion at 60.8%, while large deletions account for about 7.5%, and there are no reported mutation hotspots (similar to FVIII).

Gene therapy for FA and FB

The incidence of hemophilia A (about 1/5000) is higher than hemophilia B (about 1/25000). For a long time, hemophilia A has been considered an ideal target for gene therapy, since 1%-5% FVIII from healthy blood serum donors can significantly improve patients' quality of life. The most common enzyme replacement therapy is Spark's Spk-9001, which uses a FVIII variant expressed specifically in the liver to improve coagulation function.

To cure hemophilia, the Intellia company is targeting the insertion of the FIX gene in the liver of non-human primates by CRISPR-Pro, which can produce normal or even higher levels of FIX. This is currently in the IND application phase[2]. There are also many other CRISPR-based therapies that have been reported by multiple studies[3-4]. It is important to note that CRISPR-based therapies have a bright future in the treatment of hemophilia, which also set higher requirements for superior mouse models to better evaluate the efficacy. The pathogenic mutations of FVIII and FIX genes are numerous and highly heterogeneous, however, most commonly used mouse models are knockout (KO) mice, which cannot meet the requirements of preclinical animal models for gene therapies.

Next-Generation Humanized Mouse Models For Hemophilia

Driven by urgent demands, Cyagen has launched the Next-Generation Humanized Mouse Model Construction Program: HUGO-GTTM (Humanized Genomic Ortholog for Gene Therapy) Program to construct the whole genomic DNA humanized mouse models. We have successfully constructed HUGO-GTTM models of hemophilia: hFVIII and hFIX mice, by replacing mouse gene with human FVIII and FIX gene (whole genomic DNA) on the X chromosome, that enables consistent homology with human disease pathogenesis.

In addition to hemophilia, other diseases are also caused by X chromosome gene mutations. For example, Rett syndrome is caused by mutations in the MeCP2 gene, and Duchenne muscular dystrophy is caused by mutations in the DMD gene. Cyagen could provide whole genomic DNA humanized HUGO-GTTM mice for hFVIII, hFIX, FXI, hMeCP2, and hDMD, and more. Furthermore, we could also construct hotspot mutations on these models, which will be more suitable for genetic disease research and gene therapy drug development.

Blood Disease Target Gene Target Type
Diamond-Blackfan anemia, DBA Flvcr1 KO、CKO
Fanconi anemia, FA
Fanca CKO
Glanzmann's thrombasthenia, GT Itga2b KO、CKO、MU
Hemophilia A, FA FⅧ(F8) KO、CKO、Humanization(WT、MU)
Hemophilia B, FB FⅨ(F9) KO、CKO、Humanization(WT、MU)
Hemophilia C, FC FⅪ(F11) KO、CKO、Humanization(WT)
β-thalassemia Hbb-bs+Hbb-bt KO

Next-Generation Humanized Mouse Model Construction Program


1. Han JP, Song DW, Lee JH, Lee GS, Yeom SC. Novel Severe Hemophilia A Mouse Model with Factor Ⅷ Intron 22 Inversion. Biology (Basel). 2021 Jul 23;10(8):704. doi: 10.3390/biology10080704. PMID: 34439937; PMCID: PMC8389204.

2. https://www.intelliatx.com/pipeline/

3. Luo S , Li Z , Dai X ,et al.CRISPR/Cas9-Mediated in vivo Genetic Correction in a Mouse Model of Hemophilia A[J].Frontiers in Cell and Developmental Biology, 2021, 9:672564.DOI:10.3389/fcell.2021.672564.

4. Han J P , Kim M J , Choi B S ,et al.In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy[J].Science advances, 2022, 8(3):eabj6901.DOI:10.1126/sciadv.abj6901.