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Coagulation and its Disorders
CRISPR/Cas9-mediated knockin of human factor IX into swine factor IX locus effectively alleviates bleeding in hemophilia B pigs
Ferrata Storti Foundation
Haematologica 2021 Volume 106(3):829-837
Jiahuan Chen,1* Beiying An,2* Biao Yu,1 Xiaohuan Peng,1 Hongming Yuan,1 Qiangbing Yang,1 Xue Chen,1 Tingting Yu,1 Lingyu Wang,1 Xinwei Zhang,1 He Wang,1 Xiaodong Zou,1 Daxin Pang,1 Hongsheng Ouyang1 and Xiaochun Tang1
1College of Animal Sciences, Jilin University, Changchun and 2Department of Medical Laboratory, The First Hospital of Jilin University, Changchun, China
*JC and BA contributed equally as co-first authors.
ABSTRACT
Hemophilia B is an X-linked recessive bleeding disorder caused by abnormalities in the coagulation factor IX gene. Without prophy- lactic treatment, patients experience frequent spontaneous bleed- ing episodes. Well-characterized animal models are valuable for deter- mining the pathobiology of the disease and for testing novel therapeutic innovations. Here, we generated a porcine model of hemophilia B (HB) using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. We also tested the possibility of HB therapy by gene insertion. Frequent spontaneous joint bleeding episodes that occurred in HB pigs allowed a thorough investigation of the pathological process of hemophilic arthropathy. In contrast to the HB pigs, which showed a severe bleeding tendency and joint damage, the transgenic pigs carrying human coagula- tion factor IX exhibited a partial improvement in bleeding. In summary, this study not only offers a translational HB model for exploring the pathological process of hemophilic arthropathy, but also provides a pos- sibility for the permanent correction of hemophilia in the future by genome editing in situ.
Introduction
Factor IX (FIX) is a vitamin K-dependent plasma protein that participates in the intrinsic blood coagulation pathway by converting factor X to its active form in the presence of Ca2+, phospholipids, and factor VIIIa.1 FIX is primarily synthesized in the liver and secreted into plasma. Defects in F9 will cause hemophilia B (HB), which is an inherited X-linked bleeding disorder. Based on the residual FIX plasma levels, the disease is classified as mild (5-40%), moderate (1-5%), and severe (<1%).2 The mor- bidity of HB is 1:25,000 male live births, and according to the latest global report from the World Federation of Hemophilia there are 28,775 patients worldwide with HB.3,4 Recurrent spontaneous bleeding mainly occurs in the synovial joints, particularly in the knees, ankles, and elbows.5 These bleeds may lead to chronic pain, immobility and a significant reduction in quality of life. This debilitating condition is the most frequent complication of hemophilia, known as hemophilic arthropathy.6
The current management for HB requires lifelong intravenous protein replacement therapy with FIX, which is costly, inconvenient and not curative. HB, which is caused by a single gene abnormality, is an attractive target for gene therapy. Gene therapy using adeno-associated viral (AAV) vectors has shown successful amelioration of severe bleeding phenotypes in animal experiments and clinical trials.7-11 However, because of the non-integrating nature of the AAV vector and the high hepatocyte cel- lular turnover, in some cases the expression of FIX decreases over time.11,12 Genome editing can allow stable transgene expression by site-specific gene integration, and the CRISPR/Cas9 gene editing tool shows promise for efficient correction of mono- genic diseases.13
Animal models of hemophilia and related diseases are important for evaluating novel therapeutic strategies and understanding some pathological processes that are
Correspondence:
XIAOCHUN TANG
xiaochuntang@jlu.edu.cn
https://doi.org/10.3324/haematol.2019.224063
©2021 Ferrata Storti Foundation
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haematologica | 2021; 106(3)
829
Received: April 11, 2019. Accepted: January 22, 2020. Pre-published: January 23, 2020.
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