State Key Laboratory of Experimental Hematology, Tianjin, 300020, China.
Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300020, China.
Genome Biol. 2019 Dec 16;20(1):276. doi: 10.1186/s13059-019-1907-9.
Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%).
We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects.
These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.
血友病 A 是一种由 F8 基因突变引起的出血性疾病，只能通过基因治疗来治愈。一种很有前途的策略是利用 CRISPR-Cas9 介导在高度表达的基因座（如白蛋白 (Alb)）中将 F8 精确插入肝细胞。不幸的是，长插入物的精确体内整合效率非常低（约 0.1%）。
我们报告说，使用双切割供体可将肝脏编辑效率提高 10 到 20 倍，从而在用 Cas9-sgAlb 和 B 结构域缺失 (BDD) F8 供体质粒进行 hydrodynamic 注射后，完全重建血友病 A 小鼠模型中的血清 F8 活性。我们发现，双切割供体在小鼠肝脏中 Alb 基因座的整合主要是通过非同源末端连接 (NHEJ) 介导的敲入。然后，我们将 BDD F8 靶向到内含子 11 和 13 的多个位点，发现 NHEJ 介导的 BDD F8 插入可恢复止血。最后，使用 3 个 AAV8 载体递送基因组编辑组件，包括 Cas9、sgRNA 和 BDD F8 供体，我们观察到相同的治疗效果。对 100 只小鼠进行为期 1 年的随访未发现不良反应。
这些发现为通过 AAV 介导的编辑组件递送后在 Alb 内含子中进行 NHEJ 敲入 BDD F8 来治愈血友病 A 奠定了基础。
Zotero 插件
