Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B
Viral Vector-Based Delivery of CRISPR/Cas9 and Donor DNA for Homology-Directed Repair in an In Vitro Model for Canine Hemophilia B
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DOI：
10.1016/j.omtn.2018.12.008
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发表时间：
2019-03-01
影响因子：
8.8
通讯作者：
Ehrhardt, Anja
中科院分区：
文献类型：
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作者：
Gao, Jian;Bergmann, Thorsten;Ehrhardt, Anja
基因治疗是治疗血友病B的一种有吸引力的替代方法。在这里，我们建立了三个肝细胞衍生的细胞系的基础上Huh 7，PLC/PRF/5，和Hep 3B细胞稳定携带突变的犬FIX（cFIXmut）转基因，含有一个单一的点突变的催化结构域。基于这些类似于常用犬大型动物模型的体外模型，使用四环素控制的转录激活因子（Tet-on）诱导的CRISPR/Cas9系统和优化的供体通过同源定向修复（HDR）来校正突变的cFIX基因。为了有效递送设计者核酸酶和供体DNA，我们产生了含有Tet-on诱导的cFIX特异性CRISPR/Cas9系统的高容量5型腺病毒载体（HCAdV 5）和含有经修饰的供体的单链腺病毒相关病毒2型载体（ssAAV 2）。此外，我们设计了一个单一的HCAdV 5，为HDR提供所有组件。我们的基于qPCR分析的扩增难治性突变系统（ARMS-qPCR）显示，在Huh 7-cFIXmut细胞中的单载体应用导致高达5.52%的HDR效率，这上级于双载体策略。此外，单一载体还导致通过ELISA测定的表型校正效率增加。我们的结论是，HDR与病毒载体递送相结合，对疾病模型中突变FIX的校正具有很大的希望。
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 ad-eno-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.