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Background: In humans, the β-like globin genes are encoded from a single locus comprising five globin genes (ε-, Gγ-, Aγ-, δ-, and β-globin in sequence) and their expression is under developmental control. The γ-globin genes (Gγ- and Aγ-) are expressed during fetal life and replaced by adult β-globin after birth. Mutations in the β-globin gene cause β-hemoglobinopathies such as sickle cell disease (SCD) and β-thalassemia. The clinical severity of SCD and β-thalassemia can be mitigated by elevated fetal hemoglobin (HbF) levels, which have been found in individuals with the benign hereditary persistence of fetal hemoglobin (HPFH) syndrome. Thus, reactivating the expression of γ-globin genes is an attractive treatment strategy for β-hemoglobinopathies. Reactivation of γ-globin expression by disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter, provides a novel approach for inducing fetal hemoglobin. Methodology: The BCL11A binds a 5′-TGACCA-3′ element (spanning nucleotides −118 to −113) of globin genes using its three C-terminal Zinc fingers (Znf). The binding efficiency of Znf4, Znf5, and Znf6 was predicted by using in silico tools (SIFT, SNAP, PolyPhen-2, PANTHER, I-Mutant, PROVEAN, SNPs&GO, mCSM, and PhD-SNP), molecular dynamic simulation and homology modelling. K562 cells were electroporated with CRISPR-Cas9 targeting the BCL11A Znf4. Results: The binding energy scores illustrate that deleting Znf5 and Znf6 decreased the binding affinity by ~3.7-fold, whereas deleting Znf4 decreased the affinity by > 70-fold. Using CRISPR-Cas9 genome-editing strategy, we deleted Znf4 (260bp genomic region) within the BCL11A in K562 cell lines. Znf4 deletion resulted in a readily detectable γ-globin increase with a preferential increase in G-gamma. Conclusion: Altogether, our findings highlight the valuable insights for improving gene editing therapy strategies by either deleting Znf4 of BCL11A or the TTGACCA motif to disrupt the interaction between BCL11A and other interacting partners and the γ-globin gene promoter. Complete failure in the protein-protein interactions with functional partners and to the γ-globin gene promoter revealed that Znf4 is a suitable target for disrupting BCL11A-mediated hemoglobin switching. Citation Format: Irfan Hussain, Kainaat Mumtaz, Hammad Hassan, Afsar Ali Mian. Amelioration of hemoglobinopathies by targeted deletion of zinc finger domain within BCL11A gene using CRISPR-Cas9 technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4746. |
