CRISPR/Cas9-Mediated Knockin of Human FIX into Swine FIX Locus Effectively Alleviated Bleeding in Hemophilia B Pigs

Abstract

Background: Hemophilia B (HB) is a genetic disease resulting from a deficiency of blood coagulation factor IX (FIX). Without prophylactic treatment, patients experience frequent spontaneous bleeds. Well-characterized animal models of hemophilia are necessary for developing factor products and gene therapy. And in recent years, there has been an increasing research focus on the permanent cure of the coagulation disorder caused by single-gene defects with gene therapy. Methods: We generated a porcine model of HB and an HB gene-therapy model carrying human FIX (hFIX) knocked into the porcine FIX (pFIX) locus using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. Then the bleeding phenotype of the hemophilia pigs and the gene-therapy pigs were further observed. Findings: The hemophilia pigs showed severe spontaneous bleeding. In contrast to the HB pigs that showed a serious bleeding tendency and joint damage, the bleeding in the transgenic pigs carrying hFIX exhibited a partial improvement. Interpretation: Our study not only offers a more accurate HB model for testing the efficacy of innovative therapeutic strategies but also lays a foundation for the permanent correction of hemophilia by genome editing in situ in the future. Funding Statement: This work was financially supported by the National Natural Science Foundation of China (Grant No. 31472053 and 31572345). The Program for JLU Science and Technology Innovative Research Team (JLUSTIRT, No. 2017TD-28), the Fundamental Research Funds for the Central Universities. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal experiments and surgical procedures were carried out in accordance with guidelines approved by the Animal Welfare and Research Ethics Committee of Jilin University. All surgeries were performed under anesthesia to minimize animal suffering.