使用Cas9 mRNA和ssODN供体对CD34+造血干细胞HBB突变进行基因校正。

IF 2.4 Q1 PEDIATRICS
Justin S Antony, Ngadhnjim Latifi, A K M Ashiqul Haque, Andrés Lamsfus-Calle, Alberto Daniel-Moreno, Sebastian Graeter, Praveen Baskaran, Petra Weinmann, Markus Mezger, Rupert Handgretinger, Michael S D Kormann
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引用次数: 43

摘要

背景:β-地中海贫血是一种遗传性血液学疾病,由人血红蛋白β (HBB)基因突变导致β-珠蛋白表达减少或消除引起。虽然慢病毒介导的β-珠蛋白表达和自体移植是一种很有前景的治疗方法,但插入突变或低转基因表达的风险是显而易见的。然而,使用可编程核酸酶(如CRISPR/Cas9、TALENs和ZFNs)和非病毒修复模板对HBB突变进行靶向基因校正可确保更高的安全性和内源性表达控制。方法:我们比较了三种不同的基因编辑工具(CRISPR/Cas9、TALENs和ZFNs)对HBB基因位点的靶向效率。为了证明这一概念,我们研究了在K562和CD34+造血干细胞(hsc)中使用Cas9 mRNA和几种优化设计的单链寡核苷酸(ssODN)供体对常见的β-地中海贫血剪接变体HBBIVS1-110的个性化基因校正治疗。结果:我们的研究结果显示,CRISPR/Cas9的indel频率优于TALENs和ZFNs (P IVS1-110突变在K562细胞(高达77%)和CD34+造血干细胞- hsc(高达87%)中均显示indel。下一代测序的绝对定量显示,在CD34+ hsc中,使用Cas9 mRNA和化学修饰的ssODN可实现高达8%的NheI标签位点特异性插入。结论:我们的方法为利用Cas9 mRNA和化学修饰的ssODN对CD34+造血干细胞进行非病毒基因校正提供了指导。然而,需要进一步优化以增加同源定向修复(HDR)以获得β-地中海贫血的真正临床益处。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Gene correction of HBB mutations in CD34<sup>+</sup> hematopoietic stem cells using Cas9 mRNA and ssODN donors.

Gene correction of HBB mutations in CD34<sup>+</sup> hematopoietic stem cells using Cas9 mRNA and ssODN donors.

Gene correction of HBB mutations in CD34+ hematopoietic stem cells using Cas9 mRNA and ssODN donors.

Background: β-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Although lentiviral-mediated expression of β-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control.

Methods: We have compared three different gene-editing tools (CRISPR/Cas9, TALENs, and ZFNs) for their targeting efficiency of the HBB gene locus. As a proof of concept, we studied the personalized gene-correction therapy for a common β-thalassemia splicing variant HBBIVS1-110 using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34+ hematopoietic stem cells (HSCs).

Results: Our results exhibited that indel frequency of CRISPR/Cas9 was superior to TALENs and ZFNs (P < 0.0001). Our designed sgRNA targeting the site of HBBIVS1-110 mutation showed indels in both K562 cells (up to 77%) and CD34+ hematopoietic stem cells-HSCs (up to 87%). The absolute quantification by next-generation sequencing showed that up to 8% site-specific insertion of the NheI tag was achieved using Cas9 mRNA and a chemically modified ssODN in CD34+ HSCs.

Conclusion: Our approach provides guidance on non-viral gene correction in CD34+ HSCs using Cas9 mRNA and chemically modified ssODN. However, further optimization is needed to increase the homology directed repair (HDR) to attain a real clinical benefit for β-thalassemia.

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