Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII
Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII
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DOI：
10.1038/s41598-019-53198-y
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发表时间：
2019-11-14
影响因子：
4.6
通讯作者：
Chen-Tsai, Ruby Yanru
中科院分区：
文献类型：
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作者：
Chen, Hainan;Shi, Mi;Chen-Tsai, Ruby Yanru
血友病A是一种单基因疾病，由凝血因子VIII（F8）基因突变引起凝血因子VIII（FVIII）缺乏。目前和新兴的治疗如FVIII蛋白注射和通过游离体中的AAV递送的F8转基因的基因疗法是昂贵的和非永久性的。在这里，我们描述了一种基于CRISPR/Cas9的体内基因组编辑方法，结合非同源末端连接，使修饰的人B结构域缺失-F8（BDDF 8）在肝细胞中的白蛋白（Al B）基因座处的永久染色体整合成为可能。为了在小鼠中测试该方法，C57 BL/6小鼠接受两种载体的尾静脉注射，靶向Alb内含子13的AAV 8-SaCas 9-gRNA和AAV 8-BDD-F8。这导致BDD-F8在Alb基因座处插入，并且FVIII蛋白在载体处理的小鼠（而非媒介物处理的小鼠）的肝脏中表达。在血友病小鼠中使用这种方法，BDD-F8在肝细胞中表达为功能性人FVIII，导致FVIII的血浆水平升高，凝血特性以剂量依赖性方式恢复至少7个月，未检测到肝毒性或有意义的脱靶效应。基于这些发现，我们的BDD-F8基因组编辑方法可能为血友病A患者提供有效，长期和安全的治疗。
Hemophilia A is a monogenic disease with a blood clotting factor VIII (FVIII) deficiency caused by mutation in the factor VIII (F8) gene. Current and emerging treatments such as FVIII protein injection and gene therapies via AAV-delivered F8 transgene in an episome are costly and nonpermanent. Here, we describe a CRISPR/Cas9-based in vivo genome editing method, combined with non-homologous end joining, enabling permanent chromosomal integration of a modified human B domain deleted-F8 (BDDF8) at the albumin (Alb) locus in liver cells. To test the approach in mice, C57BL/6 mice received tail vein injections of two vectors, AAV8-SaCas9-gRNA, targeting Alb intron 13, and AAV8-BDD-F8. This resulted in BDD-F8 insertion at the Alb locus and FVIII protein expression in the liver of vector-, but not vehicle-, treated mice. Using this approach in hemophilic mice, BDD-F8 was expressed in liver cells as functional human FVIII, leading to increased plasma levels of FVIII and restoration of blood clotting properties in a dose-dependent manor for at least 7 months, with no detectable liver toxicity or meaningful off-target effects. Based on these findings, our BDD-F8 genome editing approach may offer an efficacious, longterm and safe treatment for patients with hemophilia A.