Lara-Navarro Irving Jair, Jave-Suárez Luis Felipe, Marchal Juan Antonio, Jaloma-Cruz Ana Rebeca
Doctorado en Genética Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico.
División de Inmunología, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara 44340, Jalisco, Mexico.
Life (Basel). 2024 Dec 11;14(12):1640. doi: 10.3390/life14121640.
Hemophilia B is a genetic disorder characterized by clotting factor IX deficiency and bleeding in joints and muscles. Current treatments involve intravenous infusion of plasma-derived products or recombinant proteins, which have limited efficacy due to the short half-life of infused proteins. Recently, gene therapy for bleeding disorders has offered a potential solution. This study aimed to develop an in vitro gene therapy model using the CRISPR/Cas9 system to incorporate the cDNA in human mesenchymal stem cells (hMSCs) to produce clotting factor IX. RNA guide sequences targeting the promoter-exon 1 region of the gene were designed to incorporate a wild-type cDNA into the cells. Knockin was performed with the CRISPR/Cas9 system and pDONOR-CMV/cDNAF9/IRES/EGFP vector template recombination in Lenti-X HEK293 cells and MSCs. A lentiviral cDNA vector was designed as a FIX secretor model to validate the CRISPR/Cas9 system. Results showed successful gene editing and expression in both cell models, although editing efficiency was lower in hMSCs. Future investigations will focus on improving gene editing efficiency using different transfection conditions or hybrid methodologies. This study demonstrates the potential of CRISPR/Cas9-based gene therapy in hMSCs as a target for hemophilia B. Further optimizations are required to translate these findings into clinical applications.
乙型血友病是一种遗传性疾病，其特征是凝血因子IX缺乏以及关节和肌肉出血。目前的治疗方法包括静脉输注血浆源性产品或重组蛋白，但由于输注蛋白的半衰期较短，其疗效有限。最近，针对出血性疾病的基因治疗提供了一种潜在的解决方案。本研究旨在利用CRISPR/Cas9系统开发一种体外基因治疗模型，将凝血因子IX的互补DNA（cDNA）整合到人间充质干细胞（hMSCs）中以产生凝血因子IX。设计靶向该基因启动子-外显子1区域的RNA引导序列，以将野生型cDNA整合到细胞中。在慢病毒包装细胞（Lenti-X HEK293细胞）和间充质干细胞中，利用CRISPR/Cas9系统和pDONOR-CMV/cDNAF9/IRES/EGFP载体模板重组进行基因敲入。设计一种慢病毒cDNA载体作为FIX分泌模型，以验证CRISPR/Cas9系统。结果表明，在两种细胞模型中均成功实现了基因编辑和表达，尽管在hMSCs中的编辑效率较低。未来的研究将集中于使用不同的转染条件或混合方法来提高基因编辑效率。本研究证明了基于CRISPR/Cas9的基因治疗在hMSCs中作为乙型血友病治疗靶点的潜力。需要进一步优化，以便将这些研究结果转化为临床应用。
