Reactivate expression of fetal hemoglobin In CD34+ primary adult erythroid cells by gene therapy

Abstract

Beta thalassemia patients with the co-inheritance of hereditary persistence of fetal hemoglobin (HPFH), the patients’ red blood cells still express fetal hemoglobin (Hb F) which has been found to alleviate the severity of the symptoms. In our study, CRISPR/Cas9 technique was used to modify the DNA, which mimicked the natural deletion in HPFH patients, in erythroleukemia cell line (K562). Designed CRISPR sgRNA with Cas9, used for cutting specific DNA sequences, could induce DNA double strand break (DSB) and site-specific deletion of 3.5 kb in γ-δ globin gene. Additionally, designed CRISPR sgRNA, which could cut a small region of DNA sequences (69 bp) including binding site of BCL11A, was successfully developed. This binding site is an important transcription factor, which involves in a silencer of fetal hemoglobin. Moreover, we have achieved in using CRISPR-tagged transcriptional activator to impulse an expression of Hb F. These genetically-engineered K562 and designed CRISPR sgRNA would allow the further studies on the controlling mechanism of fetal hemoglobin expression and reactivation, which might eventually become a therapeutic option in will be available for further studies of control mechanism of Beta-thalassemia patients.