Dataset Information

A genome-editing strategy to treat ?-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.

ABSTRACT: Disorders resulting from mutations in the hemoglobin subunit beta gene (HBB; which encodes ?-globin), mainly sickle cell disease (SCD) and ?-thalassemia, become symptomatic postnatally as fetal ?-globin expression from two paralogous genes, hemoglobin subunit gamma 1 (HBG1) and HBG2, decreases and adult ?-globin expression increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (referred to as HbF or ?2?2) to adult (referred to as HbA or ?2?2) form. These disorders are alleviated when postnatal expression of fetal ?-globin is maintained. For example, in hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition, mutations attenuate ?-globin-to-?-globin switching, causing high-level HbF expression throughout life. Co-inheritance of HPFH with ?-thalassemia- or SCD-associated gene mutations alleviates their clinical manifestations. Here we performed CRISPR-Cas9-mediated genome editing of human blood progenitors to mutate a 13-nt sequence that is present in the promoters of the HBG1 and HBG2 genes, thereby recapitulating a naturally occurring HPFH-associated mutation. Edited progenitors produced RBCs with increased HbF levels that were sufficient to inhibit the pathological hypoxia-induced RBC morphology found in SCD. Our findings identify a potential DNA target for genome-editing-mediated therapy of ?-hemoglobinopathies.

SUBMITTER: Traxler EA

PROVIDER: S-EPMC5706766 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.

Traxler Elizabeth A EA Yao Yu Y Wang Yong-Dong YD Woodard Kaitly J KJ Kurita Ryo R Nakamura Yukio Y Hughes Jim R JR Hardison Ross C RC Blobel Gerd A GA Li Chunliang C Weiss Mitchell J MJ Weiss Mitchell J MJ

Nature medicine 20160815 9

Disorders resulting from mutations in the hemoglobin subunit beta gene (HBB; which encodes β-globin), mainly sickle cell disease (SCD) and β-thalassemia, become symptomatic postnatally as fetal γ-globin expression from two paralogous genes, hemoglobin subunit gamma 1 (HBG1) and HBG2, decreases and adult β-globin expression increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (referred to as HbF or α2γ2) to adult (referred to as HbA or α2β2) form. These disorders are allevia ...[more]

PMID: 27525524

Similar Datasets

Project description:BackgroundSickle cell disease (SCD) and β-thalassemia patients with elevated gamma globin (HBG1/G2) levels exhibit mild or no symptoms. To recapitulate this natural phenomenon, the most coveted gene therapy approach is to edit the regulatory sequences of HBG1/G2 to reactivate them. By editing more than one regulatory sequence in the HBG promoter, the production of fetal hemoglobin (HbF) can be significantly increased. However, achieving this goal requires precise nucleotide conversions in hematopoietic stem and progenitor cells (HSPCs) at therapeutic efficiency, which remains a challenge.MethodsWe employed Cas9 RNP-ssODN-mediated homology-directed repair (HDR) gene editing to mimic two naturally occurring HBG promoter point mutations; -175T > C, associated with high HbF levels, and -158 C > T, a common polymorphism in the Indian population that induces HbF under erythropoietic stress, in HSPCs.ResultsAsymmetric, nontarget ssODN induced high rates of complete HDR conversions, with at least 15% of HSPCs exhibiting both the -175T > C and -158 C > T mutations. Optimized conditions and treatment with the small molecule AZD-7648 increased this rate, with up to 57% of long-term engrafting human HSPCs in NBSGW mice containing at least one beneficial mutation. Functionally, in vivo erythroblasts exhibited high levels of HbF, which was sufficient to reverse the cellular phenotype of β-thalassemia. Further support through bone marrow MSC co-culture boosted complete HDR conversion rates to exceed 80%, with minimal InDels, improved cell viability, and induced fetal hemoglobin levels similar to those of Cas9 RNP-mediated indels at BCL11A enhancer and HBG promoter.ConclusionsCas9 RNP-ssODN-based nucleotide conversion at the HBG promoter offers a promising gene therapy approach to ameliorate the phenotypes of β-thalassemia and SCD. The developed approach can simplify and broaden applications that require the cointroduction of multiple nucleotide modifications in HSPCs.

Dataset Information

A genome-editing strategy to treat ?-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.

Publications

A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets