Novel Severe Hemophilia A Mouse Model with Factor VIII Intron 22 Inversion

Abstract

Hemophilia A (HA) is an X-linked recessive blood coagulation disorder, and approximately 50% of severe HA patients are caused by F8 intron 22 inversion (F8I22I). However, the F8I22I mouse model has not been developed despite being a necessary model to challenge pre-clinical study. A mouse model similar to human F8I22I was developed through consequent inversion by CRISPR/Cas9-based dual double-stranded breakage (DSB) formation, and clinical symptoms of severe hemophilia were confirmed. The F8I22I mouse showed inversion of a 391 kb segment and truncation of mRNA transcription at the F8 gene. Furthermore, the F8I22I mouse showed a deficiency of FVIII activity (10.9 vs. 0 ng/mL in WT and F8I22I, p < 0.0001) and severe coagulation disorder phenotype in the activated partial thromboplastin time (38 vs. 480 s, p < 0.0001), in vivo bleeding test (blood loss/body weight; 0.4 vs. 2.1%, p < 0.0001), and calibrated automated thrombogram assays (Thrombin generation peak, 183 vs. 21.5 nM, p = 0.0012). Moreover, histological changes related to spontaneous bleeding were observed in the liver, spleen, and lungs. We present a novel HA mouse model mimicking human F8I22I. With a structural similarity with human F8I22I, the F8I22I mouse model will be applicable to the evaluation of general hemophilia drugs and the development of gene-editing-based therapy research.

In vivo genome editing using 244-cis LNPs and low-dose AAV achieves therapeutic threshold in hemophilia A mice

“…This study utilized the C57BL/6. F8 intron 22 inversion (F8 I22I ) mice, 35 which mimic the most common and severe bleeding clinical phenotype in patients with hemophilia A. Targeted DSB and the delivery of donor templates are necessary to achieve KI.…”

“…The F8 I22I mouse model develops spontaneous liver and lung bleeding and kidney edema. 35 Therefore, restoring blood coagulation ability through AT inhibition and hFVIII production is expected to alleviate the clinical symptoms of hemophilia. First, liver and lung tissues were embedded in paraffin, followed by the preparation of tissue slides.…”

“…The C57BL/6 mice (B6, wild type; WT) were purchased from KoaTech (Pyeongtaek, Gyeonggi, Korea), whereas F8 I22I mice were generated using CRISPR-Cas9-based gene editing. 35 Eight- to 10-week-old WT and F8 I22I male mice were subjected to in vivo delivery of genetic material. The WT and F8 I22I mice were divided randomly into the WT control, F8 I22I control, LNP-CRISPR, AAV-donor, and AAV-LNP groups.…”

In vivo delivery of CRISPR-Cas9 using lipid nanoparticles enables antithrombin gene editing for sustainable hemophilia A and B therapy

“…B6.FVIII intron 22 inversion (F8 I22I ) and B6.FIX knockout (F9 Mut ) were generated by CRISPR-Cas9–based gene editing (fig. S7) ( 56 ). Seven- to 9-week-old male mice were subjected to further experiments.…”

Frequency of Intron 22 Inversion in Severe Hemophilia A Patients

“…The recombination of chromosomes between extragenic copy, Int1h-2, and Int1h-1 results in factor FVIII Inv1 [4]. Due to this inversion change, messenger RNA (mRNA) for FVIII does not form, resulting in the absence of FVIII, causing severe HA [5][6][7][8][9]. Both Inv22 variants have been postulated to have a higher likelihood of inhibitor formation, making treatment more difficult [5].…”