@article {6585,
	title = {Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.},
	journal = {Mol Ther},
	volume = {32},
	year = {2024},
	month = {2024 Mar 06},
	pages = {663-677},
	abstract = {<p>BCL11A-XL directly binds and represses the fetal globin (HBG1/2) 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 HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in\&nbsp;vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in\&nbsp;vivo, 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 HBG1/2 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.</p>
},
	keywords = {Fetal Hemoglobin, gamma-Globins, Gene Editing, Hematopoietic Stem Cells, Humans, Repressor Proteins, Zinc Fingers},
	issn = {1525-0024},
	doi = {10.1016/j.ymthe.2024.01.023},
	author = {Rajendiran, Vignesh and Devaraju, Nivedhitha and Haddad, Mahdi and Ravi, Nithin Sam and Panigrahi, Lokesh and Paul, Joshua and Gopalakrishnan, Chandrasekar and Wyman, Stacia and Ariudainambi, Keerthiga and Mahalingam, Gokulnath and Periyasami, Yogapriya and Prasad, Kirti and George, Anila and Sukumaran, Dhiyaneshwaran and Gopinathan, Sandhiya and Pai, Aswin Anand and Nakamura, Yukio and Balasubramanian, Poonkuzhali and Ramalingam, Rajasekaran and Thangavel, Saravanabhavan and Velayudhan, Shaji R and Corn, Jacon E and Mackay, Joel P and Marepally, Srujan and Srivastava, Alok and Crossley, Merlin and Mohankumar, Kumarasamypet M}
}
