| Objective:Hemoglobin H disease(HbH disease)which is the most common type of thalassemia in China belongs to intermediateα-thalassemia.The clinical treatment of HbH disease is limited.Its pathogenesis is due to disorder in the synthesis ofα-globin chain and the imbalance betweenα-globin andβ-globin chain caused byα-globin gene deletion or mutation.Excessβ-globin chains are self-polymerized intoβ-chain tetramer(HbH inclusion body),which resulting in ineffective intramedullary hematopoiesis and hemolysis.The level of HbH is an important factor to determine the severity of the disease,therefore it may be an effective strategy to treat HbH disease by appropriately reducing the synthesis ofβ-globin chain and improving the imbalance betweenα-globin andβ-globin chain.We used CRISPR/Cas9 technology to edit the promoter region ofβ-globin gene in order to down-regulate the expression ofβ-globin gene,reduce the synthesis ofβ-globin chain,correct the imbalance betweenα-globin andβ-globin chain and then reduce the formation of HbH inclusion bodies,which provides a new idea for gene therapy ofα-thalassemia.Methods1.The two-stage liquid culture system of erythroid cells in vitro was established.The human CD34~+cells were separated by immunomagnetic beads,then amplified and differentiated into erythroid cells in the two-stage culture system.The morphological characteristics of erythroid cells were observed by Gemisa staining,the expression of CD71 and CD235a on the surface of erythroid cells and the terminal enucleation were detected by Flow cytometry,and the expression of globin gene and globin chain were detected by RT-PCR and HPLC.2.The HUDEP-2 cell line stably expressing Cas9 protein was constructed and a set of sgRNA sequences were designed in the promoter region ofβ-globin gene.The cell line was edited by delivering sgRNA through lentivirus.By evaluating the editing efficiency and detecting the mRNA expression of various globin genes by RT-PCR,a group of sgRNA sequences which could effectively down-regulate the expression ofβ-globin gene were screened out in the promoter region ofβ-globin gene.3.The sgRNA sequences were verified and screened in normal human CD34~+hematopoietic stem and progenitor cells.Firstly,the editing system was optimized by analyzing the effects of different sgRNA synthesis methods,Cas9concentrations and Cas9:sgRNA molar ratios on editing efficiency and cell viability.Then CD34~+hematopoietic stem and progenitor cells were edited by delivering RNP complex through electroporation to evaluate the editing efficiency.Taking the unedited group as the control group,the expression of globin genes and globin chains were detected by RT-PCR and HPLC,meanwhile the process on differentiation of erythroid cells and the off-target effects were monitored so as to further screen out the sgRNA sequences that could targeted down-regulate the expression ofβ-globin gene.4.The sgRNA sequences were further verified in CD34~+hematopoietic stem and progenitor cells of patients with HbH disease.The sgRNA-37 and sgRNA-48 with appropriate down-regulation were selected according to the ratios ofβ/α-globin mRNA in patients.Gene editing was performed by electroporation.The editing effects and its down-regulation on expression ofβ-globin gene were detected,the deposition of excessβ-globin chain was observed under electron microscope and the distribution of HbH inclusions in erythrocytes was observed with by brilliant cresyl blue staining.5.To verify the long-term editing effect of CRISPR/Cas9 technology in NCG-X mice,we edited normal CD34~+hematopoietic stem and progenitor cells by the two effective sgRNA sequences successfully verified in cell experiments and transplanted into the NCG-X mice.After 16 weeks,the level of humanization,the reconstruction of the cell lineage,editing efficiency and long-term effect of gene editing in vivo were evaluated.6.The homozygous mutant cell line of HbCS was constructed by homologous recombination method,and the mutation of HbCS was corrected by single base editor.Results1.The CD34~+cells sorted by MACS have high purity.Erythroid cells could expand hundreds or thousands of times in the two-stage culture system,and finally differentiate into a high proportion of mature erythrocytes.2.The HUDEP-2 cell line stably expressing Cas9 protein was successfully constructed,and then it was edited by delivering sgRNA through lentivirus.As a result,a group of sgRNA sequences which down-regulated the expression ofβ-globin gene with different degrees were screened out in the promoter region ofβ-globin gene.3.The editing system was optimized in normal CD34~+hematopoietic stem and progenitor cells.It was clear that the modified synthetic-sgRNA,Cas9protein concentration of 5μM and Cas9/sgRNA molar ratio of 1:2 were three important factors for efficient editing.Furthermore,eight selected sgRNA sequences were verified.Compared with the unedited group,the ratios ofβ/α-globin mRNA and globin chain in the edited group was significantly down-regulated,and there were no off-target effects at the predicted sites and no adverse effects on the differentiation of erythroid cells.4.The sequences of sgRNA-37 and sgRNA-48 were further verified in CD34~+hematopoietic stem and progenitor cells of patients with HbH disease.After editing,the ratios ofα/β-globin mRNA and globin chain in patients were significantly up-regulated.The deposition ofβ-globin chain was improved under electron microscope,and HbH inclusion bodies were significantly reduced.At the same time,no off-target effects and inhibition of erythroid differentiation were detected.5.Normal CD34~+hematopoietic stem and progenitor cells edited by sgRNA-37 and sgRNA-48 were transplanted into NCG-X mice.After 16 weeks,the editing efficiency was detected.There was no significant reduction in editing efficiency between the two sgRNA edited groups and pre-transplantation group.More than 90%of humanized cells were detected in both unedited group and two sgRNA edited groups,and there were no statistic differences between the above groups.There was no significant difference in the proportion of B-lineage and myeloid reconstitution between the unedited group and the edited groups.Nevertheless the proportion of erythroid cells in the two sgRNA edited groups was statistically lower than that in the unedited group.6.In the transplantation experiment,the mRNA expression ofβ-globin gene was also down-regulated in NCG-X mice,which was consistent with the results of cell experiments in vitro.7.The homozygous mutant cell line of HbCS was successfully constructed,but the single base editor failed to correct the HbCS mutation.Conclusion1.The two-stage liquid culture system of erythroid cells in vitro has efficient expansion and directed differentiation on erythroid cells.2.The HUDEP-2 cell line stably expressing Cas9 protein was efficiently edited by delivering sgRNA through lentivirus,so that a group of sgRNA sequences which could effectively down-regulate the expression ofβ-globin gene with different degrees were screened out in the promoter region ofβ-globin gene.3.CRISPR/Cas9 technology was used to edit the promoter region ofβ-globin gene in CD34~+cells,which could down-regulate the expression ofβ-globin gene with different degrees.The editing efficiency was related to factors such as sgRNA synthesis mode,Cas9 protein concentration and Cas9/sgRNA molar ratio.There were no off-target effects at the predicted site and no adverse effects on the differentiation of erythroid cells.4.By editing the promoter region ofβ-globin gene in CD34~+cells of HbH patients with CRISPR/Cas9 technology,the expression ofβ-globin gene can be appropriately down-regulated,the synthesis ofβ-globin chain can be reduced,and the imbalance betweenαandβ-globin chain can be improved.5.The editing effect of CRISPR/Cas9 technology on CD34~+cells and the regulation ofβ-globin gene can exist effectively in NCG-X mice for a long time.The humanization level of mice is high,meanwhile B lymphocytes,myeloid cells and erythroid cells can be well reconstructed.6.Single base editors have not yet been able to correct HbCS mutation. |
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