Genome Editing Mediated Reactivation Of ?-globin Expression To Treat ?-thalassemia

Gene editing technologies have been widely used in the researches of single-gene genetic diseases because of their highly capable of manipulating genomic sequences.?-thalassemia is caused by?-globin gene mutation,which can be alleviated by increasing the expression of?-globin in erythrocytes.A proof-of-concept study demonstrated it was effective to mimic a naturally occurring 13bp deletion on?-globin promoter identified in patients with hereditary of persistence of fetal hemoglobin by using the lentiviral delivery of the Cas9/sgRNA.However,this strategy is not suitable for clinical applications because of two important reasons.First,CD34~+hematopoietic stem/progenitor cells are not susceptible for lentivirus transduction.Second,the random integration characteristic of lentivirus vector may pose risks of iatrogenic pathology and cause the constitutive expression of Cas9protein which increases the risk of off-target events.In order to establish the?-thalassemia gene editing treatment technology that could be clinically transformed,we explored the conditions for efficient editing of Cas9 protein in human CD34~+hematopoietic stem/progenitor cells.We found that we synthesized and chemically modified sgRNAs targeting the promoter region of?-globin gene-102 to-114,and then delivered Cas9 protein and chemically modified sgRNA-1 ribonucleoprotein complex to CD34~+hematopoietic stem/progenitor cells from?-thalassemia patients by electroporation.We achieved an average editing efficiency of over 80%without sorting the positively transduced cells.After erythroid differentiation,the erythroid progeny of edited cells exhibited higher level of?-globin expression.The mRNA of?-globin relative to?-globin increased to 126%.The?-globin chain relative to?-globin chain could be increased to 64%.The enucleation rate was also increased to be close to that of normal human CD34~+hematopoietic stem/progenitor cells after erythroid differentiation without off target events.These results indicated our strategy had therapeutic effects and avoided the risks of the lentiviral vector.The essence of gene editing mediated by the CRISPR/Cas9 system is to produce double-strand breaks.We hoped to advance the base editor strategy which caused much less DSBs to treat?-thalassemia.We tested whether the BCL11A binding site could be disrupted by base editing.We delivered the codon-optimized hA3A-BE3 protein and sgRNA-2 to CD34~+hematopoietic stem/progenitor cells derived from?-thalassemia patients by electroporation.We demonstrated that the base editor hA3A-BE3 was able to simultaneously mutate-114C and-115C on the?-globin promoter.Although the editing efficiency was only about 15%,the average level of the mRNA of?-globin relative to?-globin in the edited cells was at least 6.5 times higher than unedited cells.In summary,we optimized the CRISPR/cas9 system targeting?-globin gene promoter to treat?-thalassemia,and proved that base editing technology could be used to induce the expression of?-globin,which provided a new strategy for gene therapy of ?-thalassemia.