Institutes of Health Central Plains, Xinxiang Medical University, Xinxiang 453003, China.
State Key Laboratory of Genetic Engineering and MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai, China.
Gene. 2022 Apr 30;820:146289. doi: 10.1016/j.gene.2022.146289. Epub 2022 Feb 7.
Reactivation of fetal hemoglobin by editing the B-cell lymphoma/leukemia 11A (BCL11A) erythroid enhancer is an effective gene therapy for β-thalassemia. Using the CRISPR/Cas9 system, fetal γ-globin expression can be robustly reactivated to mitigate the clinical course of β-thalassemia. In our study, we found that the transfection efficiencies of CD34 hematopoietic stem/progenitor cells (HSPCs) were significantly and negatively correlated with the length of plasmids and greatly affected by the linearization of plasmids. Furthermore, the transgene expression of minicircles (MC) without plasmid backbone sequences was better both in vitro and in vivo compared with conventional plasmids. Thus, MC DNA was used to deliver the cassette of Staphylococcus aureus Cas9 (SaCas9) into HSPCs, and a single-guide RNA targeting the erythroid enhancer region of BCL11A was selected. After electroporation with MC DNA, an evident efficiency of gene editing and reactivation of γ-globin expression in erythroblasts derived from unsorted HSPCs was acquired. No significant off-target effects were found by deep sequencing. Furthermore, fragments derived from lentiviral vectors, but not MC DNA, were highly enriched in promoter, exon, intron, distal-intergenic, and cancer-associated genes, indicating that MC DNA provided a relatively safe and efficient vector for delivering transgenes. The developed MC DNA vector provided a potential approach for the delivery of SaCas9 cassette and the reactivation of γ-globin expression for ameliorating syndromes of β-thalassemia.
通过编辑 B 细胞淋巴瘤/白血病 11A（BCL11A）红细胞增强子来重新激活胎儿血红蛋白是治疗β-地中海贫血的有效基因疗法。使用 CRISPR/Cas9 系统，可以强有力地重新激活胎儿γ-珠蛋白表达，从而减轻β-地中海贫血的临床病程。在我们的研究中，我们发现 CD34 造血干细胞/祖细胞（HSPC）的转染效率与质粒的长度呈显著负相关，并且受到质粒线性化的极大影响。此外，与具有质粒骨架序列的常规质粒相比，无质粒骨架序列的微环（MC）的转基因表达在体外和体内都更好。因此，MC DNA 被用于将金黄色葡萄球菌 Cas9（SaCas9）的盒式元件递送到 HSPC 中，并选择了靶向 BCL11A 红细胞增强子区域的单导向 RNA。在用 MC DNA 电穿孔后，从未分选的 HSPC 衍生的红细胞中获得了基因编辑和γ-珠蛋白表达重新激活的明显效率。通过深度测序未发现明显的脱靶效应。此外，来源于慢病毒载体的片段，而不是 MC DNA，在启动子、外显子、内含子、远端基因间和癌症相关基因中高度富集，表明 MC DNA 为递呈转基因提供了一种相对安全且高效的载体。所开发的 MC DNA 载体为递呈 SaCas9 盒式元件和重新激活 γ-珠蛋白表达以改善β-地中海贫血综合征提供了一种潜在的方法。
