Induction of Fetal Hemoglobin by Introducing Natural Hereditary Persistence of Fetal Hemoglobin Mutations in the γ-Globin Gene Promoters for Genome Editing Therapies for ß-Thalassemia.

Reactivation of γ-globin expression is a promising therapeutic approach for ß-hemoglobinopathies. Here, we propose a novel Cas9/AAV6-mediated genome editing strategy for the treatment of ß-thalassemia: Natural HPFH mutations -113A > G, -114C > T, -117G>A, -175T > C, -195C > G, and -198T > C were introduced by homologous recombination following disruption of BCL11A binding sites in HBG1/HBG2 promoters. Precise on-target editing and significantly increased γ-globin expression during erythroid differentiation were observed in both HUDEP-2 cells and primary HSPCs from ß-thalassemia major patients. Moreover, edited HSPCs maintained the capacity for long-term hematopoietic reconstitution in B-NDG hTHPO mice. This study provides evidence of the effectiveness of introducing naturally occurring HPFH mutations as a genetic therapy for ß-thalassemia.

CRISPR/Cas9-mediated ß-globin gene knockout in rabbits recapitulates human ß-thalassemia.

ß-thalassemia, an autosomal recessive blood disorder that reduces the production of hemoglobin, is majorly caused by the point mutation of the HBB gene resulting in reduced or absent ß-globin chains of the hemoglobin tetramer. Animal models recapitulating both the phenotype and genotype of human disease are valuable in the exploration of pathophysiology and for in vivo evaluation of novel therapeutic treatments. The docile temperament, short vital cycles, and low cost of rabbits make them an attractive animal model. However, ß-thalassemia rabbit models are currently unavailable. Here, using CRISPR/Cas9-mediated genome editing, we point mutated the rabbit ß-globin gene HBB2 with high efficiency and generated a ß-thalassemia rabbit model. Hematological and histological analyses demonstrated that the genotypic mosaic F0 displayed a mild phenotype of anemia, and the heterozygous F1 exhibited typical characteristics of ß-thalassemia. Whole-blood transcriptome analysis revealed that the gene expression was altered in HBB2-targeted when compared with WT rabbits. And the highly expressed genes in HBB2-targeted rabbits were enriched in lipid and iron metabolism, innate immunity, and hematopoietic processes. In conclusion, using CRISPR-mediated HBB2 knockout, we have created a ß-thalassemia rabbit model that accurately recapitulates the human disease phenotype. We believe this tool will be valuable in advancing the investigation of pathogenesis and novel therapeutic targets of ß-thalassemia and associated complications.

Generation of induced pluripotent stem cell GZLSL-i001-A derived from urine-derived cells of Hemophilia A patient with Inv22 mutation.

Hemophilia A (HA), is a X-linked recessive congenital bleeding disorder, caused by deficiency of the coagulation factorVIII (FVIII) which is encoded by coagulation factor 8 (F8). HA affects 1 of every 5,000 males worldwide. The intron 22 inversion (Inv22) mutation of F8 causes about 45% of severeHA cases.Here, we generated induced pluripotent stem cells (iPSCs) from a HA patient with Inv22 mutation by electroporation of urine-derived cells (UCs) with episomal plasmids under feeder-free, virus-free, serum-free condition and without oncogene c-MYC. This iPSCs line could facilitate future applications of human iPSCs by provide a valuable cell model.