A cellular reporter system to evaluate endogenous fetal hemoglobin induction and screen for therapeutic compounds

IF 7.6 2区 医学 Q1 HEMATOLOGY HemaSphere Pub Date : 2024-08-06 DOI:10.1002/hem3.139
Thijs C. J. Verheul, Nynke Gillemans, Kerstin Putzker, Rezin Majied, Tingyue Li, Memnia Vasiliou, Bert Eussen, Annelies de Klein, Wilfred F. J. van IJcken, Emile van den Akker, Marieke von Lindern, Joe Lewis, Ulrike Uhrig, Yukio Nakamura, Thamar van Dijk, Sjaak Philipsen
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Abstract

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 HBG1 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.

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用于评估内源性胎儿血红蛋白诱导和筛选治疗化合物的细胞报告系统。
β-球蛋白病是一种严重的遗传性疾病,因其发病率和死亡率高而对全球健康产生重大影响。这些症状是在出生后从胎儿血红蛋白表达向成人血红蛋白表达过渡时出现的。大量研究集中于诱导胎儿γ-球蛋白亚基的表达,以逆转这种转换并改善这些症状。尽管进行了数十年的研究,但只有羟基脲一种化合物作为胎儿血红蛋白的诱导剂被应用于临床。遗憾的是,它的疗效因人而异,因此需要更有效的治疗方法。红细胞细胞系在研究逆转出生后血红蛋白转换的药物和遗传方法方面发挥了重要作用。在这里,我们描述了第一个基于成人红细胞祖细胞系 HUDEP2 的内源性标记胎儿血红蛋白报告细胞系。利用 CRISPR-Cas9 介导的基因敲入技术,在 HBG1 基因上整合了一个生物发光标签。随后进行的广泛表征证实,所得到的报告细胞系与 HUDEP2 的特征非常接近,而且细胞报告胎儿血红蛋白诱导的灵敏度和特异性都很高。因此,这种新型报告细胞系非常适合用于评估诱导胎儿血红蛋白的基因和药物策略。此外,它还提供了一种与高通量药物筛选兼容的检测方法,例如在一项试验研究中鉴定出了一组已知的胎儿血红蛋白诱导物。向研究界提供这一新工具的目的是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换。
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来源期刊
HemaSphere
HemaSphere Medicine-Hematology
CiteScore
6.10
自引率
4.50%
发文量
2776
审稿时长
7 weeks
期刊介绍: HemaSphere, as a publication, is dedicated to disseminating the outcomes of profoundly pertinent basic, translational, and clinical research endeavors within the field of hematology. The journal actively seeks robust studies that unveil novel discoveries with significant ramifications for hematology. In addition to original research, HemaSphere features review articles and guideline articles that furnish lucid synopses and discussions of emerging developments, along with recommendations for patient care. Positioned as the foremost resource in hematology, HemaSphere augments its offerings with specialized sections like HemaTopics and HemaPolicy. These segments engender insightful dialogues covering a spectrum of hematology-related topics, including digestible summaries of pivotal articles, updates on new therapies, deliberations on European policy matters, and other noteworthy news items within the field. Steering the course of HemaSphere are Editor in Chief Jan Cools and Deputy Editor in Chief Claire Harrison, alongside the guidance of an esteemed Editorial Board comprising international luminaries in both research and clinical realms, each representing diverse areas of hematologic expertise.
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