Engineered Zinc-Finger Nuclease to Generate Site-Directed Modification in the Klf1 Gene for Fetal Hemoglobin Induction Publisher Pubmed



Shariati L^{1, 2} ; Modarressi MH³ ; Tabatabaiefar MA^{4, 5} ; Kouhpayeh S⁶ ; Hejazi Z⁴ ; Shahbazi M⁴ ; Sabzehei F⁴ ; Salehi M⁴ ; Khanahmad H^{4, 5} Show Affiliations
Source: Journal of Cellular Biochemistry Published:2019

Abstract

Elevation of hemoglobin F (HbF) ameliorates symptoms of β-thalassemia, as a common autosomal recessive disorder. In this study, the ability of an engineered zinc-finger nuclease (ZFN) system was assesed to disrupt the KLF1 gene to inhibit the γ to β hemoglobin switching in K562 cells. This study was performed using a second generation integration-deficient lentiviral vector assigned to transient gene targeting. The sequences coding for zinc finger protein arrays were designed and subcloned in TDH plus as a transfer vector. Transduction of K562 cells was performed with the integrase minus lentivirus containing ZFN. The indel percentage of the transducted cells with lentivirus containing ZFN was about 29%. Differentiation of K562 cell line into erythroid cell lineage was induced with cisplatin concentration of 15 µg/mL. After differentiation, γ-globin and HbF expression were evaluated using real-time reverse-transcription polymerase chain reaction and hemoglobin electrophoresis methods. The levels of γ-globin messenger RNA were nine-fold higher in the ZFN treated cells compared with untreated cells 5 days after differentiation. Hemoglobin electrophoresis method showed the same results for HbF level measurement. Application of the ZFN tool to induce KLF1 gene mutation in adult erythroid progenitors might be a candidate to stimulate HbF expression in β-thalassemia patients. © 2018 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.