CRISPR/Cas9 ?-globin gene targeting in human haematopoietic stem cells.

ABSTRACT: The ?-haemoglobinopathies, such as sickle cell disease and ?-thalassaemia, are caused by mutations in the ?-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure ?-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult ?-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for ?-haemoglobinopathies.

SUBMITTER: Dever DP 

PROVIDER: S-EPMC5898607 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature