Gamma reactivation using the spongy effect of KLF۱-binding site sequence: an approach in gene therapy for beta-thalassemia

Objective(s): β-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF۱ binding sites to the K۵۶۲ cell line. Materials and Methods: A plasmid containing a ۱۹۲ bp sequence with two repeats of KLF۱ binding sites on β-globin and BCL۱۱A promoters was constructed and used to transfect the K۵۶۲ cell line. Positive selection was performed under treatment with ۱۵۰ μg/ml hygromycin B. The remaining cells were expanded and harvested on day ۲۸, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K۵۶۲ cells. The cells were differentiated with ۱۵ µg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD۲۳۵a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells. Results: A ۱۷۰۰ bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K۵۶۲ cells was verified. Totally, ۸۴% of cells were differentiated. The transfected cells significantly increased γ-globin expression after differentiation compared to untransfected ones. Conclusion: The findings demonstrate that the spongy effect of KLF۱-binding site on BCL۱۱A and β-globin promoters can induce γ-globin expression in K۵۶۲ cells. This novel strategy can be promising for the treatment of β-thalassemia and sickle cell disease.

Keywords:

Hb F induction , KLF۱ gene , β-switching , β-thalassemia , γ-globin