Disruption of SOX6 gene using CRISPR/Cas9 technology for gamma‐globin reactivation: An approach towards gene therapy of β‐thalassemia

Abstract

Elevation of Hemoglobin F ameliorates symptoms of β-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to β-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively. Relative quantification showed that the γ-globin mRNA level increased to 1.3-, 2.1-, and 1.1-fold in the cells treated with CRISPR/Cas9 a, b, and c, respectively, compared with untreated cells. Our results show that mutation induction in the binding site of the SOX6 gene leads to γ-globin reactivation. These findings support the idea that CRISPR interrupts the SOX6 binding site, and, as a result, SOX6 is incapable of binding the γ-globin promoter. In conclusion, SOX6 disruption could be considered as a therapeutic approach for β-thalassemia treatment. CRISPR/Cas9 was selected for this purpose as it is the most rapidly evolving technology.

Novel genetic therapeutic approaches for modulating the severity of β‑thalassemia (Review)

“…Various transcription factors are important in switching gene expression from γ-globin to β-globin. Shariati et al (111) described one such transcription factor, SOX6. A mutation was introduced in the SOX6 binding gene region with the γ-globin gene promoter using CRISPR/Cas9 preventing its binding, and this resulted in reactivation of γ-globin gene expression.…”

“…Increased levels of γ-globin mRNA expression was observed in K562 cells transfected with the CRISPR/Cas9 vectors. Thus, CRISPR/Cas9 can be used as a therapeutic approach for treating patients with β-thalassemia (111). It is hypothesized that the CRISPR/Cas9 system may be used to correct specifically harmful mutations of the HBB gene, and this could be confirmed by normal erythrocyte differentiation and their normal expression.…”

Emerging roles of Sox6 in the renal and cardiovascular system

“…The transcription factor Sox6 is a pivotal gene in switching the γ to β-globin gene. Shariati et al (2018), reported that disruption of Sox6 binding domain results in the reactivation of γ globin expression. This study concluded that silencing Sox6 may become therapeutic strategy for β-thalassemia treatment (Modares Sadeghi et al, 2018;Shariati et al, 2018).…”

“…(2018), reported that disruption of Sox6 binding domain results in the reactivation of γ globin expression. This study concluded that silencing Sox6 may become therapeutic strategy for β‐thalassemia treatment (Modares Sadeghi et al., 2018; Shariati et al., 2018). Another study showed the functional importance of a number of miRNAs and their target Sox6 in hereditary persistence of fetal hemoglobin deletion type‐2 (HPFH‐2) and Sicilian‐δβ‐thalassemia diseases (Fornari et al., 2017).…”

Development of α4 integrin DNA aptamer as a potential therapeutic tool for multiple sclerosis

“…There are some novel therapeutic tools that are capable of target therapy in different context . Aptamers as one of those therapeutic tools are single‐stranded DNA (ssDNA) or RNA molecules that are typically less than 100‐mer, which have the ability to bind to a wide range of targets, with high affinity and specificity .…”