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Cas9-AAV6 Gene Correction of Beta-Globin in Autologous HSCs Improves Sickle Cell Disease Erythropoiesis in Mice
bioRxiv - Developmental Biology Pub Date : 2020-10-13 , DOI: 10.1101/2020.10.13.338319
Adam C. Wilkinson , Daniel P. Dever , Ron Baik , Joab Camarena , Ian Hsu , Carsten T. Charlesworth , Chika Morita , Hiromitsu Nakauchi , Matthew H. Porteus

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of Sickle Cell Disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a novel paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated. Here, we used a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observed a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

中文翻译:

自体HSC中β-球蛋白的Cas9-AAV6基因校正可改善小鼠镰状细胞病的红细胞生成

CRISPR / Cas9介导的镰状细胞病(SCD)患者来源的造血干细胞(HSC)与自体移植相结合的β-珠蛋白(HBB)基因校正代表了基因治疗的新范式。尽管已提出了几种基于Cas9的HBB校正方法,但尚未研究体内红细胞生成的功能校正。在这里,我们使用人源化的球蛋白簇SCD小鼠模型来研究自体移植范围内功能性HSC中Cas9-AAV6介导的HBB校正。我们发现,自体移植后,长期多能HSC可以通过离体基因校正,并且可以在体内从HBB校正的HSC体内获得稳定的血红蛋白-A生产。我们观察到HBB校正的骨髓嵌合体增多与体内RBC正常化之间存在直接关系,但即使是低水平的嵌合,也能导致强大的血红蛋白A水平。此外,该研究为基础和翻译研究的小鼠HSC基因编辑提供了一个平台。
更新日期:2020-10-15
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