Antony Justin S, Latifi Ngadhnjim, Haque A K M Ashiqul, Lamsfus-Calle Andrés, Daniel-Moreno Alberto, Graeter Sebastian, Baskaran Praveen, Weinmann Petra, Mezger Markus, Handgretinger Rupert, Kormann Michael S D
Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany.
University Children's Hospital, Department of Pediatrics I, University of Tuebingen, Tuebingen, Germany.
Mol Cell Pediatr. 2018 Nov 14;5(1):9. doi: 10.1186/s40348-018-0086-1.
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.
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 using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34 hematopoietic stem cells (HSCs).
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 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.
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.
β地中海贫血是一种遗传性血液疾病，由人类血红蛋白β（HBB）基因突变引起，该突变会降低或消除β珠蛋白的表达。尽管慢病毒介导的β珠蛋白表达和自体移植是一种有前景的治疗方法，但插入诱变或转基因低表达的风险很明显。然而，使用诸如CRISPR/Cas9、转录激活因子样效应物核酸酶（TALENs）和锌指核酸酶（ZFNs）等可编程核酸酶与非病毒修复模板对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。
我们比较了三种不同的基因编辑工具（CRISPR/Cas9、TALENs和ZFNs）对HBB基因座的靶向效率。作为概念验证，我们使用Cas9信使核糖核酸（mRNA）和几种优化设计的单链寡核苷酸（ssODN）供体，在K562和CD34造血干细胞（HSCs）中研究了针对常见β地中海贫血剪接变体HBB的个性化基因校正疗法。
我们的结果显示，CRISPR/Cas9的插入缺失频率优于TALENs和ZFNs（P<0.0001）。我们设计的靶向HBB突变位点的单向导RNA（sgRNA）在K562细胞（高达77%）和CD34造血干细胞-HSCs（高达87%）中均显示出插入缺失。通过下一代测序进行的绝对定量显示，在CD34 HSCs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。
我们的方法为使用Cas9 mRNA和化学修饰的ssODN在CD34 HSCs中进行非病毒基因校正提供了指导。然而，需要进一步优化以提高同源定向修复（HDR），从而为β地中海贫血带来真正的临床益处。
