Kao Yung-Tsung, Chen Yen-Ting, Fan Hueng-Chuen, Tsai Tung-Chou, Cheng Shin-Nan, Lai Ping-Shan, Chen Jen-Kun, Chen Chuan-Mu
Department of Pediatrics, Tungs' Taichung Metroharbor Hospital, Taichung 435, Taiwan.
Department of Life Sciences, Ph.D. Program in Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan.
Biomedicines. 2021 Aug 30;9(9):1116. doi: 10.3390/biomedicines9091116.
Hemophilia A is a bleeding disease caused by loss of coagulation factor VIII (FVIII) function. Although prophylactic FVIII infusion prevents abnormal bleeding, disability and joint damage in hemophilia patients are common. The cost of treatment is among the highest for a single disease, and the adverse effects of repeated infusion are still an issue that has not been addressed. In this study, we established a nonviral gene therapy strategy to treat knockout ( KO) mice. A novel gene therapy approach was developed using dipalmitoylphosphatidylcholine formulated with iron oxide (DPPC-FeO) to carry the B-domain-deleted (BDD)- plasmid, which was delivered into the KO mice via tail vein injection. Here, a liver-specific albumin promoter-driven BDD- plasmid was constructed, and the binding ability of circular DNA was confirmed to be more stable than that of linear DNA when combined with DPPC-FeO nanoparticles. The KO mice that received the DPPC-FeO plasmid complex were assessed by staining the ferric ion of DPPC-FeO nanoparticles with Prussian blue in liver tissue. The bleeding of the KO mice was improved in a few weeks, as shown by assessing the activated partial thromboplastin time (aPTT). Furthermore, no liver toxicity, thromboses, deaths, or persistent changes after nonviral gene therapy were found, as shown by serum liver indices and histopathology. The results suggest that this novel gene therapy can successfully improve hemostasis disorder in KO mice and might be a promising approach to treating hemophilia A patients in clinical settings.
甲型血友病是一种由于凝血因子VIII（FVIII）功能丧失而导致的出血性疾病。尽管预防性输注FVIII可防止血友病患者出现异常出血，但残疾和关节损伤仍很常见。单一疾病的治疗成本位居最高之列，反复输注的不良反应仍是一个尚未解决的问题。在本研究中，我们建立了一种非病毒基因治疗策略来治疗基因敲除（KO）小鼠。开发了一种新型基因治疗方法，使用与氧化铁配制的二棕榈酰磷脂酰胆碱（DPPC-FeO）携带缺失B结构域（BDD）的质粒，通过尾静脉注射将其导入KO小鼠体内。在此，构建了一种肝脏特异性白蛋白启动子驱动的BDD质粒，并证实环状DNA与DPPC-FeO纳米颗粒结合时的结合能力比线性DNA更稳定。通过用普鲁士蓝对肝脏组织中的DPPC-FeO纳米颗粒的铁离子进行染色，对接受DPPC-FeO质粒复合物的KO小鼠进行评估。通过评估活化部分凝血活酶时间（aPTT）表明，KO小鼠的出血情况在几周内得到改善。此外，血清肝脏指标和组织病理学显示，非病毒基因治疗后未发现肝毒性、血栓形成、死亡或持续性变化。结果表明，这种新型基因治疗可以成功改善KO小鼠的止血障碍，可能是临床治疗甲型血友病患者的一种有前途的方法。
