LRF Binding Site as Potential Therapeutic Target for β-Hemoglobinopathies

The binding site for LRF, a transcriptional repressor of the fetal hemoglobin (HbF) γ-globin genes, has been identified as a potential therapeutic target for β-hemoglobinopathies, according to research presented by Annarita Miccio, PhD, of the Imagine Institute and University of Paris in France, at the virtual 62nd American Society of Hematology (ASH) Annual Meeting and Exposition.¹

In a previous study, the investigators demonstrated successful CRISPR-Cas9-mediated disruption of the LRF binding site within the transcriptional promoters of the 2 γ-globin genes, HBG1 and HBG2, via homology-directed repair, which generates double-stranded breaks in the DNA to edit a small gene region.² Hereditary persistence of fetal hemoglobin (HPFH) mutations in promoter regions of HBG1 and HBG2 disrupt the binding sites for LRD and other transcriptional repressors.  

“By disrupting binding sites [for transcriptional repressors] using the CRISPR-Cas9 nuclease system, we generated small insertions or deletions that basically mimic the effect of the HPFH mutation,” said Dr Miccio.

The team first conducted studies in liquid cell culture, editing hematopoietic stem/progenitor cells (HSPCs) obtained from patients with sickle cell disease (SCD). Editing the binding sites yielded robust reactivation of the HbF genes accompanied by decreased β^S-globin levels that corrected the SCD cell phenotype in red blood cells derived from the edited HSPCs. Similar results were demonstrated in patient-derived β⁰-thalassemic cells, which could potentially correct the α/β-like globin imbalance.

Next, xenotransplantation of edited human HSPCs demonstrated robust engraftment of the cells, which were able to differentiate into multiple lineages. Ex vivo-differentiated erythroid progenitors derived from the engrafted human cells demonstrated relevant γ-globin expression in approximately 40% of the total β-like chain, and ex vivo-differentiated mature red cells achieved therapeutically relevant HbF levels, with HbF representing up to 47% of the total Hb tetramers.

Because CRISPR-Cas9 editing resulted in some off-target editing events, the investigators also explored the use of base editing, another Cas9-based editing system that can directly modify individual base pairs in DNA without generating double-stranded breaks. Following a similar strategy as before, HSPCs in liquid culture were successfully edited for cytosine to thymine base conversions, and erythroid differentiation demonstrated high percentages of nonsickled cells similar to those seen with the CRISPR-Cas9 strategy (>60% in both).

Dr Miccio noted that the “[D]isruption of the LRF binding site in the γ-globin promoter by using the Cas-9 nuclease ameliorates both the [SCD] and β-thalassemic cell phenotype” in cells derived from patients with SCD. “Using the Cas-9 nuclease is probably [safer] using the base editing strategy and represents a promising therapeutic approach for the treatment of β-hemoglobinopathies.”

Disclosure: Some authors have declared affiliations with or received funding from the pharmaceutical industry. Please refer to the original study for a full list of disclosures.

Read more of Hematology Advisor’s coverage of the ASH 2020 meeting by visiting the conference page.

References

1. Frati G, Antoniou P, Hardouin G, et al. Editing the LRF repressor binding site in the γ-globin promoters induces therapeutically relevant fetal hemoglobin levels for the treatment of β-hemoglobinopathies. Presented at: American Society of Hematology (ASH) 62nd Annual Meeting and Exposition; December 5-8, 2020. Abstract 91. 
2. Weber L, Frati G, Felix T, et al. Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype. Sci Adv. 2020;6(7):eaay9392. doi:10.1126/sciadv.aay9392