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

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
{"title":"Gene correction of HBB mutations in CD34<sup>+</sup> hematopoietic stem cells using Cas9 mRNA and ssODN donors.","authors":"Justin S Antony,&nbsp;Ngadhnjim Latifi,&nbsp;A K M Ashiqul Haque,&nbsp;Andrés Lamsfus-Calle,&nbsp;Alberto Daniel-Moreno,&nbsp;Sebastian Graeter,&nbsp;Praveen Baskaran,&nbsp;Petra Weinmann,&nbsp;Markus Mezger,&nbsp;Rupert Handgretinger,&nbsp;Michael S D Kormann","doi":"10.1186/s40348-018-0086-1","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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 HBB<sup>IVS1-110</sup> using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34<sup>+</sup> hematopoietic stem cells (HSCs).</p><p><strong>Results: </strong>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 HBB<sup>IVS1-110</sup> mutation showed indels in both K562 cells (up to 77%) and CD34<sup>+</sup> 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<sup>+</sup> HSCs.</p><p><strong>Conclusion: </strong>Our approach provides guidance on non-viral gene correction in CD34<sup>+</sup> 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.</p>","PeriodicalId":74215,"journal":{"name":"Molecular and cellular pediatrics","volume":null,"pages":null},"PeriodicalIF":2.4000,"publicationDate":"2018-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40348-018-0086-1","citationCount":"43","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular and cellular pediatrics","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1186/s40348-018-0086-1","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PEDIATRICS","Score":null,"Total":0}
引用次数: 43

Abstract

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.

Abstract Image

Abstract Image

使用Cas9 mRNA和ssODN供体对CD34+造血干细胞HBB突变进行基因校正。
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。
求助全文
约1分钟内获得全文 求助全文
来源期刊
CiteScore
2.20
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信