Functional study of EGLN1 gene in β-thalassemia hematopoietic stem and progenitor cells using CRISPR/Cas9

Abstract

Reactivation of fetal hemoglobin (HbF, α2γ2) potentially alleviates clinical presentations of β-thalassemia. Prolyl hydroxylase domain enzymes (PHDs) play roles in the canonical oxygen-sensing pathway and maintain the stability of cellular hypoxia-inducible factor α (HIF-α) in response to low oxygen levels or hypoxia. Pharmacological inhibition of PHDs has been shown to increase HbF production in erythroid progenitors derived from non-thalassemia individuals. This study demonstrated the relationship between PHD2, the main PHD isoform, and clinical phenotypes in β0-thalassemia/HbE disease. Although exome sequencing annotated three insignificance variants in EGLN1, the PHD2 encoding gene, CRISPR-mediated EGLN1 silencing led to increased HBG (γ-globin) mRNA and HbF expression in β0-thalassemia/HbE hematopoietic stem/progenitor cells. Moreover, HbF induction after EGLN1 silencing was independent to the two well-known HbF regulators including BCL11A and GATA1. These findings introduce an additional mechanism for HbF regulation in β-thalassemia and propose that targeting the canonical oxygen-sensing pathway, particularly PHD2, may offer a promising novel therapeutic strategy to β-thalassemia diseases.