Abstract
β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.
Highlights
Β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB)
Best electroporation settings were chosen for both K-562 cells (1450 V, 10 ms, 3 pulses) and CD34+ hematopoietic stem and progenitor cells (HSPCs) (1650 V, 10 ms, 3 pulses) where high transfection efficiency and viability were achieved (>90%; Supplementary Fig. S1a)
Varying levels of mean indel frequencies were observed for KLF1 (T1: 36.2 ± 6.5%; T2: 34.9 ± 5.1%), BCL11A (T1: 22.2 ± 2.2%; T2: 17.0 ± 1.4%), and HBG1/2 (T1: 30.9 ± 14.4%; T2: 21.1 ± 6.0%; Supplementary Fig. S1b)
Summary
Β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). All three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies. To attain a clinical profit for β-hemoglobinopathies, several attempts were made to re-establish the expression of HbF either by lentiviral transfer of the γ-globin gene or by CRISPR/Cas9-mediated gene disruption of specific regulators[14,15,16,17,18,19]. In the present study, we compared all these targets in parallel for their impact on HbF resurgence and performed safety measurements by molecular analyses in order to select the best candidate for clinical translation
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call