Backgroundβ-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.MethodsWe 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).ResultsOur 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.ConclusionOur 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.Electronic supplementary materialThe online version of this article (10.1186/s40348-018-0086-1) contains supplementary material, which is available to authorized users.

β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies. Sickle cell disease (SCD) and β-thalassemia, commonly known as β-hemoglobinopathies, are inherited blood disorders caused by mutations in the human β-globin gene (HBB) 1-4. In healthy condition, adult human hemoglobin (HbA) consists of 2 α and 2 β chains, whereas fetal hemoglobin (HbF) expressed in early gestation comprises 2 α chains and 2 γ chains. Notably, HbF was observed to bind oxygen with greater affinity than HbA, being functional when reactivated in adults 3,5,6. Recent studies have generated substantial experimental evidence that HbF reactivation by gene disruption of specific transcription factors and regulators could provide a therapeutic benefit for β-hemoglobinopathies 7. It has long been appreciated that KLF1 and BCL11A are key regulators involved in the process of γto β-globin switching and the repression of these genes leads to HbF resurgence 6-11. Interestingly, healthy individuals with a benign genetic condition namely hereditary persistence of fetal hemoglobin (HPFH) were observed to exhibit persistent production of functional HbF 4,10,12,13. HPFH is caused by large deletions in the δand β-globin genes, or point mutations in the γ-globin promoter and γ-globin repressors, such as KLF1 and BCL11A 5. Importantly,

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