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. Erythroid cells (Phase 1 end) obtained from ZnF4-1 base-edited HSPC is the study sample. The AAVS1 edited (negative control), the Binding site (-115 cluster) disruption in HBG promoter and the BCL11A total knockout are the other controls used in the experiment to compare the Whole transcriptome analysis. Each sample is performed in duplicates.

BCL11A是一类核心的γ-珠蛋白阻遏因子，也是当前临床改善β-血红蛋白病（beta-hemoglobinopathies）的主流靶点。较长的BCL11A-XL同工型（isoform）在C端含有三个独特的锌指结构域（ZnF domains），该结构域已被证实可与HBG区域发生DNA互作。这些特异性锌指结构域存在于神经发育障碍患者的错义突变位点中，且有研究表明，该位点突变可在血液学参数正常的前提下提升胎儿血红蛋白（HbF）水平。然而，靶向BCL11A-XL特异性锌指结构域且不影响其他同工型的治疗潜力仍未被探索。因此，本研究利用CRISPR/Cas9系统敲除BCL11A-XL特异性锌指结构域，观察到显著的HbF诱导效果，但同时伴随造血干祖细胞（Hematopoietic Stem and Progenitor Cells, HSPC）植入缺陷与红细胞成熟障碍。后续研究发现，对参与DNA识别的锌指结构域关键残基进行碱基替换，可有效上调HbF水平，且仅伴随轻微缺陷。ZnF4突变仅会对红细胞基因表达产生极小影响，体外（in vitro）实验中未对红细胞成熟造成明显干扰，但会降低体内（in vivo）HSPC的植入能力。对ZnF6的修饰则意外地提升了HbF水平，尽管该区域并未直接与HBG启动子结合。本研究通过特异性改变BCL11A-XL的锌指结构域，有望筛选出对胎儿珠蛋白抑制至关重要、但对BCL11A-XL其他生物学功能影响较小的残基，以此为β-血红蛋白病的治疗提供潜在策略。本研究的实验样本为从经ZnF4-1碱基编辑的HSPC中分离得到的红细胞（第一阶段终末样本）。实验中设置了三类对照样本：AAVS1编辑样本（阴性对照）、HBG启动子结合位点（-115簇）敲除样本，以及BCL11A全敲除样本，用于对照全转录组分析（Whole transcriptome analysis）。所有样本均设置生物学重复。