Gene therapy represents an attractive alternative to treat hemophilia B. Here we established three hepatocyte-derived cell lines based on Huh7, PLC/PRF/5, and Hep3B cells stably carrying a mutated canine FIX (cFIXmut) transgene containing a single point mutation in the catalytic domain. Based on these in vitro models resembling a commonly used canine large animal model, the tetracycline-controlled transcriptional activator (Tet-on)-inducible CRISPR/Cas9 system and an optimized donor were used to correct mutated cFIX gene through homology-directed repair (HDR). For efficient delivery of designer nuclease and donor DNA, we produced a high-capacity adenovirus vector type 5 (HCAdV5) containing the Tet-on-inducible cFIX-specific CRISPR/Cas9 system and a single-stranded adeno-associated virus type 2 vector (ssAAV2) containing the modified donor. Moreover, we designed a single HCAdV5 delivering all components for HDR. Our amplification-refractory mutation system based on qPCR analysis (ARMS-qPCR) revealed that the single vector application in Huh7-cFIXmut cells resulted in up to 5.52% HDR efficiencies, which was superior to the two-vector strategy. Furthermore the single vector also resulted in increased phenotypic correction efficiencies assayed by ELISA. We conclude that HDR in combination with viral vector delivery holds great promise for the correction of mutated FIX in disease models.

基因治疗代表了一种治疗B型血友病的有吸引力的替代方法。在这里，我们建立了基于Huh7，PLC / PRF / 5和Hep3B细胞的三种肝细胞衍生细胞系，这些细胞系稳定地携带突变犬FIX（cFIXmut）转基因，该基因中含有单点突变。催化结构域。基于这些体外类似于常用犬大动物模型的模型，四环素控制的转录激活因子（Tet-on）诱导的CRISPR / Cas9系统和优化的供体用于通过同源性定向修复（HDR）校正突变的cFIX基因。为了有效传递设计者核酸酶和供体DNA，我们生产了一种大容量腺病毒载体类型5（HCAdV5），其中包含Tet-on-inducable cFIX特异CRISPR / Cas9系统和单链腺相关病毒2型载体（ ssAAV2）包含修饰的供体。此外，我们设计了单个HCAdV5，可提供HDR的所有组件。我们基于qPCR分析的扩增-难治性突变系统（ARMS-qPCR）显示，在Huh7-cFIXmut细胞中单载体应用导致高达5.52％的HDR效率，优于双载体策略。此外，单个载体还导致通过ELISA测定的增加的表型校正效率。我们得出的结论是，HDR与病毒载体传递相结合对突变的校正具有广阔的前景在疾病模型中修复。