Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression,Molecular Therapy

当前位置： X-MOL 学术 › Mol. Ther. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression
Molecular Therapy ( IF 12.1 ) Pub Date : 2024-01-24 , DOI: 10.1016/j.ymthe.2024.01.023
Vignesh Rajendiran ₁ , Nivedhitha Devaraju ₂ , Mahdi Haddad ₃ , Nithin Sam Ravi ₁ , Lokesh Panigrahi ₂ , Joshua Paul ₂ , Chandrasekar Gopalakrishnan ₄ , Stacia Wyman ₅ , Keerthiga Ariudainambi ₆ , Gokulnath Mahalingam ₇ , Yogapriya Periyasami ₇ , Kirti Prasad ₂ , Anila George ₁ , Dhiyaneshwaran Sukumaran ₄ , Sandhiya Gopinathan ₇ , Aswin Anand Pai ₈ , Yukio Nakamura ₉ , Poonkuzhali Balasubramanian ₈ , Rajasekaran Ramalingam ₄ , Saravanabhavan Thangavel ₇ , Shaji R Velayudhan ₁₀ , Jacon E Corn ₁₁ , Joel P Mackay ₁₂ , Srujan Marepally ₇ , Alok Srivastava ₁₀ , Merlin Crossley ₃ , Kumarasamypet M Mohankumar ₇

Affiliation

Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, Tamil Nadu 632002, India; Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695 011, India.
Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, Tamil Nadu 632002, India; Manipal Academy of Higher Education, Manipal, Karnataka 576104, India.
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.
Department of Integrative Biology, School of Bioscience and Technology, Vellore Institute of Technology (VIT, Deemed to be University), Vellore, Tamil Nadu 632014, India.
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94704, USA.
Department of Biochemistry, Auxilium College, Vellore, Tamil Nadu 632006, India.
Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, Tamil Nadu 632002, India.
Department of Haematology, Christian Medical College & Hospital, Vellore, Tamil Nadu 632 004, India.
Cell Engineering Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Bagayam, Vellore, Tamil Nadu 632002, India; Department of Haematology, Christian Medical College & Hospital, Vellore, Tamil Nadu 632 004, India.
Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94704, USA; Institute of Molecular Health Sciences, Department of Biology, Zurich, Switzerland.
School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia.

BCL11A-XL directly binds and represses the fetal globin gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the promoters. This first structure/function analysis of ZnF4–6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

中文翻译：

BCL11A-XL 特异性锌指结构域中关键残基的碱基编辑可去抑制胎儿珠蛋白表达

BCL11A-XL 使用 3 个锌指结构域（ZnF4、ZnF5 和 ZnF6）直接结合和抑制胎儿珠蛋白基因启动子，是 β-血红蛋白病治疗的潜在靶点。破坏 BCL11A-XL 会导致胎珠蛋白去抑制和高 HbF，但也会影响造血干细胞和祖细胞（HSPC）植入和红系成熟。有趣的是，具有 ZnF 结构域突变的神经发育患者 HbF 升高，血液学参数正常。受这种自然现象的启发，我们在特定 ZnF 结构域使用了 CRISPR-Cas9 和碱基编辑，并评估了对 HbF 产生和造血分化的影响。通过 CRISPR-Cas9 在各种 ZnF 结构域中产生插入缺失，阻止了 BCL11A-XL 与其在启动子中的位点的结合，并提高了 HbF 水平，但影响了正常的造血功能。碱基编辑观察到的副作用要少得多——例如，红细胞成熟接近正常。然而，我们观察到 HSPC 植入适度减少，B 细胞发育完全丧失，这可能是因为当前的碱基编辑无法精确概括在 BCL11A-XL 相关神经发育障碍患者中发现的突变。总体而言，我们的结果表明，破坏不同的 ZnF 结构域具有不同的效果。破坏 ZnF4 会显著提高 HbF 水平，而许多其他红细胞靶基因不受影响，有趣的是，破坏 ZnF6 也会升高 HbF 水平，这是出乎意料的，因为该区域不直接与启动子相互作用。 ZnF4-6 的首次结构/功能分析为抑制胎儿珠蛋白表达所需的 BCL11A-XL 结构域提供了重要见解，并为探索自然突变的引入提供了框架，这些突变可能使单个基因的去抑制，同时不影响其他功能。

更新日期：2024-01-24

点击分享查看原文

点击收藏

公开下载

阅读更多本刊新发论文本刊介绍/投稿指南