Department of Hematology, The Second Hospital of Shanxi Medical University, Taiyuan, 030001, Shanxi Province, China.
Institutes of Biomedical Sciences, Shanxi University, Taiyuan, 030006, Shanxi Province, China.
Stem Cell Res Ther. 2022 Jul 26;13(1):353. doi: 10.1186/s13287-022-03036-2.
Hemophilia B is a rare inherited genetic bleeding disorder caused by a deficiency or lack of coagulation factor IX, the gene for which (F9) is located on the X chromosome. Hemophilia B is currently incurable and the standard treatment is coagulation factor replacement therapy. Although gene therapy has the potential to cure hemophilia, significant barriers are still needed to be overcome, e.g., off-target effects and immunoreactivity, so new approaches must be explored. Nonsense mutations account for 8% of all the hemophilia B mutation types and can result in the development of coagulation factor inhibitors. In this study, CRISPR/Cas9 technology was used to construct a mouse embryonic stem cell model with a hemophilia B nonsense mutation (F9 c.223C > T) in humans to investigate the pathogenesis and treatment of nonsense mutations in hemophilia B.
First, a donor plasmid with a mutation (F9 c.223 C > T) and sgRNAs were constructed. Second, both the donor plasmid and the px330-sgRNA were electroporated into mouse embryonic stem cell, and the mutant cells were then screened using puromycin and red fluorescence. Third, the mutant cell lines were tested for pluripotency and the ability to differentiate into three layers. Finally, the effect of mutation on gene function was studied in the differentiation system.
The mutant vector and effective sgRNA were constructed, and the mutant cell line was screened. This mutant cell line exhibited pluripotency and the ability to differentiate into three layers. This point mutation affects F9 expression at both the RNA and protein levels in the differentiation system.
The mutant cell line obtained in the current study had a single-base mutation rather than a base deletion or insertion in the exon, which is more similar to clinical cases. In addition, the mutant has the characteristics of mouse embryonic stem cells, and this point mutation affects F9 gene transcription and translation, which can be used as a disease model for studying the pathogenesis and treatment of hemophilia at the stem cell level.
血友病 B 是一种罕见的遗传性出血性疾病，由凝血因子 IX 缺乏或缺乏引起，该基因（F9）位于 X 染色体上。血友病 B 目前无法治愈，标准治疗是凝血因子替代疗法。尽管基因治疗有治愈血友病的潜力，但仍需要克服重大障碍，例如脱靶效应和免疫原性，因此必须探索新的方法。无义突变占所有血友病 B 突变类型的 8%，可导致凝血因子抑制剂的产生。在这项研究中，使用 CRISPR/Cas9 技术构建了一个人类血友病 B 无义突变（F9 c.223C>T）的小鼠胚胎干细胞模型，以研究血友病 B 无义突变的发病机制和治疗方法。
首先，构建了一个带有突变（F9 c.223C>T）和 sgRNA 的供体质粒。其次，将供体质粒和 px330-sgRNA 电穿孔到小鼠胚胎干细胞中，然后使用嘌呤霉素和红色荧光筛选突变细胞。第三，测试突变细胞系的多能性和向三胚层分化的能力。最后，在分化系统中研究突变对基因功能的影响。
构建了突变载体和有效的 sgRNA，并筛选出突变细胞系。该突变细胞系表现出多能性和向三胚层分化的能力。该点突变在分化系统中影响 RNA 和蛋白质水平的 F9 表达。
目前获得的突变细胞系在外显子中仅有单个碱基突变，而不是碱基缺失或插入，这与临床病例更为相似。此外，突变体具有小鼠胚胎干细胞的特征，该点突变影响 F9 基因的转录和翻译，可作为研究干细胞水平血友病发病机制和治疗的疾病模型。
