利用CRISPR/Cas9基因编辑技术实现β-globin基因在造血干细胞中高效敲入治疗地中海贫血的研究

β-thalassaemia is caused by mutations in the β-globin (HBB) gene and affects millions of people worldwide. Allogeneic hematopoietic stem cell transplantation as a curative option is currently recommended for β-thalassaemia if a human leukocyte antigen (HLA)-matched sibling donor is available. However, fewer than 25% of patients have a suitable intrafamilial donor. Severe adverse events include graft failure, graft-versus-host disease (GVHD), early or late side effects from conditioning regimens that are both myeloablative and immunosuppressive (infections, hemorrhages, secondary malignancies), and aggravation of preexisting organ damage. For patients who lack a suitable HLA-matched donor, ex vivo gene therapy using autologous HSCs brings hope as a potential curative treatment option. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-thalassaemia. But the gene mutation type of globin in β-thalassaemia is very diverse, preventing the design for therapies to target every single mutation. To solve this problem, we propose an alternative strategy- improve the gene editing efficiency of CRISPR/ Cas9 in hematopoiesis stem cells by using a double cutting template system to precisely insert the HBB sequence (about 1.5 kb) into the HBB locus. We expect that the survival rate of hematopoietic stem cells after nucleofection and editing will be improved by adding anti-apoptotic proteins. The targeting efficiency and specificity will also be improved by screening multiple sgRNAs. This project, if successful, would lead to the development of novel hematopoietic stem cell gene therapy strategy for clinical treatment of hereditary diseases such as thalassemia.

地中海贫血是世界上最常见的一种常染色体遗传病，由于珠蛋白HBB基因缺失或突变引起。异基因造血干细胞移植能够治愈地贫，但MHC完全匹配率低而且有GVHD等并发症的风险。β-珠蛋白的基因突变类型多样化，如果采用基因修复的方式针对每个突变进行修复，工作量极大，难以开发出标准的临床治疗方案。我们将开发一种适用于所有中国地中海贫血患者替代方案-用病人自身的造血干细胞通过CRISPR基因编辑技术导入没有变异的β-珠蛋白HBB序列（大约1.5kb)。将利用我们开发的双切模板引导同源重组插入，有望提高HSC基因编辑效率5-10倍。还将通过设计精准高效且低脱靶率的sgRNA来提高打靶效率和特异性；通过在电转过程中加入抗凋亡因子提高造血干细胞转染后存活率。我们预期，本项目研究成功后，将为造血干细胞基因治疗在地中海贫血等遗传性疾病中的临床应用开辟先河。

地中海贫血是珠蛋白HBB基因缺失或突变引起的一种常染色体遗传病。通过造血干细胞基因编辑治疗有望彻底治愈该疾病。我们研究了一系列优化造血干细胞基因编辑的方法包括：加入抗凋亡基因BCL-XL提高细胞存活率和基因编辑效率；使用组蛋白去乙酰化酶抑制剂HDACi在基因组的非开放位点实现高效的基因编辑；设计精准高效且低脱靶率的sgRNA来提高打靶效率和特异性；利用CRISPR-Cas9靶向内含子进行基因编辑，并建立报告细胞系以扩大CRISPR-Cas9基因编辑技术的应用范围；优化sgRNA长度和骨架序列，促进SaCas9基因编辑工具的高效体内外基因敲入。最终成功实现在造血干细胞中高效进行基因编辑，并设计了一种截短型HBB表达载体用于地贫基因治疗。