CRISPR/Cas9-mediated BCL11A Gene Editing In CD34+Hematopoietic Stem Cells Increases HbF Expression

Thalassemia,also known as Mediterranean anemia,is a group of hereditary hemolytic anemia that res?lts from one or more defective hemoglobin production caused by genetic defects.Due to the genetic defects res?lting in one or more globin chain absent or insufficient synthesis,the patient suffering from anemia and hemolysis pathological state.Reduction and deletion globin chain synthesis of hemoglobin leads to abnormal structure of hemoglobin,which res?lts in decreased deformability of the red blood cells,shorten life-span,hemolysis in the bone marrow and destruction in liver and spleen when into the peripheral blood circ?lation,and finally evolved to anemia,iron deposition and developmental abnormalities.Treatment for ?-thalassemia includes general support,high doses of blood transfusion and iron chelator,augmentation of fetal hemoglobin synthesis,gene therapy,and hematopoietic stem cell transplantation,which is the only curative treatment.After the first transplantation success achieved by Thomas in 1981,the hematopoietic stem cell transplantation was carried out in m?ltiple centres around the world and replaced the previous classical blood transfusion and iron chelation therapy in recent 30 years.However,transplantation application is limited by paucity of HLA-matched donors,fixed risk of graft versus host disease(GVHD),and transplant-related mortality.Many experiments have been conducted to find new treatment drugs similar to hydroxyurea,which is currently the only FDA-approved medicine for sickle cell disease treatment.Hydroxyurea mainly induces fetal hemoglobin expression to alleviate disease symptoms.Hydroxyurea can cause several side effects,and its clinical efficacy is unstable because the gene reg?latory mechanisms of fetal hemoglobin expression remain unclear.GWAS data showed that loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor,BCL11 A,are causative for HbF elevation.This study provides a fundamental novel approach to develop therapies by using data from GWAS as guide to create a disease-ameliorating genotype in patient's own cells in a targeted manner.In this experiment,we apply CRISPR / cas9 system to perform the genome editing in +55,+58,+62 erythroid specific enhancer in HEK293 T cells and the res?lts indicate that the efficiency of gene editing is low due to the use of Cas9D10 A.According to the latest reports,We select +58 erythroid specific enhancer as target to apply the genome editing using wild type Cas9 combined with single gRNA in K562 cells an achieve the 24% of mutation frequency.By screened sgRNA and CRISPR/Cas9 system,we achieved 10% gene editing efficiency at the same site in bone marrow derived CD34+ cells in patients with thalassemia.In vitro erythroid differentiation of genome editing CD34+ hematopoietic stem and progenitor cells was carried out and the expression of fetal hemoglobin levels were detected in mRNA level and F-cells level.The res?lts indicated that the expression of mRNA in the experimental group was 1.9 times higher than that of the control group,and the proportion of the experimental group was 50% higher than that of the control group.In order to verify hematopoietic stem cell engraftment after gene editing,we inject cells into NPG mice exposed to sublethal doses irradiation.Human CD45+ cells were detected at 8 weeks after transplantation in mice which indicat gene editing hematopoietic stem cells still have the capacity of hematopoietic reconstruction.For off-target analysis,We choose six highest possible off-target sites predicted by the software and design primers flanking the potential sites.By cloning analysis of six targets,there is no gene mutation in the target sites.We established for the first time a genome-editing method in bone marrow CD34+ hematopoietic stem and progenitor cells of thalassemia patients.Increase in fetal hemoglobin expression in cells of erythroid differentiation was observed after genome-editing of Bcl11 A erythroid enhancer with CRISPR/Cas9.In vivo transplantation indicate the genome editing cells still have the ability to reconstruct hemotoposis.This study aims to develop a new treatment strategy and establish a readily available clinical method for thalassemia children without suitable hematopoietic stem cell transplantation donor.