Verheul Thijs C J, Gillemans Nynke, Putzker Kerstin, Majied Rezin, Li Tingyue, Vasiliou Memnia, Eussen Bert, de Klein Annelies, van IJcken Wilfred F J, van den Akker Emile, von Lindern Marieke, Lewis Joe, Uhrig Ulrike, Nakamura Yukio, van Dijk Thamar, Philipsen Sjaak
Department of Cell Biology Erasmus MC Rotterdam The Netherlands.
EMBL Chemical Biology Core facility Heidelberg Germany.
Hemasphere. 2024 Aug 6;8(8):e139. doi: 10.1002/hem3.139. eCollection 2024 Aug.
Reactivation of fetal hemoglobin expression alleviates the symptoms associated with β-globinopathies, severe hereditary diseases with significant global health implications due to their high morbidity and mortality rates. The symptoms emerge following the postnatal transition from fetal-to-adult hemoglobin expression. Extensive research has focused on inducing the expression of the fetal γ-globin subunit to reverse this switch and ameliorate these symptoms. Despite decades of research, only one compound, hydroxyurea, found its way to the clinic as an inducer of fetal hemoglobin. Unfortunately, its efficacy varies among patients, highlighting the need for more effective treatments. Erythroid cell lines have been instrumental in the pursuit of both pharmacological and genetic ways to reverse the postnatal hemoglobin switch. Here, we describe the first endogenously tagged fetal hemoglobin reporter cell line based on the adult erythroid progenitor cell line HUDEP2. Utilizing CRISPR-Cas9-mediated knock-in, a bioluminescent tag was integrated at the gene. Subsequent extensive characterization confirmed that the resulting reporter cell line closely mirrors the HUDEP2 characteristics and that the cells report fetal hemoglobin induction with high sensitivity and specificity. This novel reporter cell line is therefore highly suitable for evaluating genetic and pharmacologic strategies to induce fetal hemoglobin. Furthermore, it provides an assay compatible with high-throughput drug screening, exemplified by the identification of a cluster of known fetal hemoglobin inducers in a pilot study. This new tool is made available to the research community, with the aspiration that it will accelerate the search for safer and more effective strategies to reverse the hemoglobin switch.
胎儿血红蛋白表达的重新激活可缓解与β-珠蛋白病相关的症状，β-珠蛋白病是严重的遗传性疾病，因其高发病率和死亡率而对全球健康具有重大影响。这些症状在出生后从胎儿血红蛋白表达向成人血红蛋白表达的转变后出现。广泛的研究集中在诱导胎儿γ-珠蛋白亚基的表达以逆转这种转变并改善这些症状。尽管经过了数十年的研究，但只有一种化合物羟基脲作为胎儿血红蛋白的诱导剂进入了临床。不幸的是，其疗效在患者中存在差异，这凸显了对更有效治疗方法的需求。红系细胞系在寻求逆转出生后血红蛋白转变的药理学和遗传学方法方面发挥了重要作用。在此，我们描述了首个基于成人红系祖细胞系HUDEP2的内源性标记胎儿血红蛋白报告细胞系。利用CRISPR-Cas9介导的敲入技术，在基因处整合了一个生物发光标签。随后的广泛表征证实，所得的报告细胞系紧密反映了HUDEP2的特征，并且这些细胞能够以高灵敏度和特异性报告胎儿血红蛋白的诱导情况。因此，这种新型报告细胞系非常适合评估诱导胎儿血红蛋白的遗传和药理学策略。此外，它提供了一种与高通量药物筛选兼容的检测方法，在一项初步研究中鉴定出一组已知的胎儿血红蛋白诱导剂就是例证。这个新工具已提供给研究界，希望它能加速寻找更安全、更有效的策略来逆转血红蛋白转变。
