Targeted disruption of BCL11A-XL specific zinc finger motif for de-repression of fetal globin expression

BCL11A is a major gamma globin repressor widely used target for the clinical amelioration of beta-hemoglobinopathies. The longer BCL11A-XL isoform has three distinct ZnF domains at the C-terminal which has been implicated in DNA interactions with the HBG region. These unique ZnF domains harbor missense mutations in neurodevelopmental disorder patients and have been shown to elevate HbF levels with normal hematological parameters. However, the therapeutic potential of targeting the BCL11A-XL-specific ZnF domains without affecting other isoforms remains unexplored. Hence, wedisrupted the BCL11A-XL specific ZnF domains usingCRISPR/Cas9 and observed high HbF induction albeit with defects in Hematopoietic Stem and Progenitor Cells(HSPC) engraftment and erythroid maturation. Subsequently, we observed that base substitutions at the key residues of ZnF-domainsinvolved in DNA recognition effectively upregulated HbF with minor defects. A ZnF4 mutation resulted in minimal changes to erythroid gene expression, no impact on erythroid maturation in vitro, but a reduction in HSPC engraftment in vivo. A modification of ZnF6showeda potential elevation of HbF levels, which was unexpected as this region does not directly interact with DNA at the HBG promoter.Our strategy of specifically altering ZnF domains of BCL11A-XL will potentially identify residues critical for fetal globin repression but less important in the other biological functions of BCL11A-XL for the treatment of beta-hemoglobinopathies.