Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia

Document Type: Original Article

Authors

1 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-communicable Disease, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

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 KLF1 binding sites to the K562 cell line.
Materials and Methods: A plasmid containing a 192 bp sequence with two repeats of KLF1 binding sites on β-globin and BCL11A promoters was constructed and used to transfect the K562 cell line. Positive selection was performed under treatment with 150 μg/ml hygromycin B. The remaining cells were expanded and harvested on day 28, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K562 cells. The cells were differentiated with 15 µg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD235a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells.
Results: A 1700 bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K562 cells was verified. Totally, 84% 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 KLF1-binding site on BCL11A and β-globin promoters can induce γ-globin expression in K562 cells. This novel strategy can be promising for the treatment of β-thalassemia and sickle cell disease.

Keywords


1. Sankaran VG, Nathan DG. Thalassemia: an overview of 50 years of clinical research. Hematol Oncol Clin North Am 2010; 24:1005-1020.

2. Sankaran VG, Orkin SH. The switch from fetal to adult hemoglobin. Cold Spring Harb Perspect Med 2013; 3:a011643.

3. Cao A, Galanello R. Beta-thalassemia. Genet Med; 12:61-76.

4. Fard AD, Hosseini SA, Shahjahani M, Salari F, Jaseb K. Evaluation of Novel Fetal Hemoglobin Inducer Drugs in Treatment of beta-Hemoglobinopathy Disorders. Int J Hematol Oncol Stem Cell Res 2013; 7:47-54.

5. Wilber A, Hargrove PW, Kim YS, Riberdy JM, Sankaran VG, Papanikolaou E, et al. Therapeutic levels of fetal hemoglobin in erythroid progeny of beta-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer. Blood 2011; 117:2817-2826.

6. Costa FC, Fedosyuk H, Chazelle AM, Neades RY, Peterson KR. Mi2β is required for γ-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in β-YAC transgenic mice. PLoS Genet 2012; 8:e1003155.

7. Sankaran VG. Targeted therapeutic strategies for fetal hemoglobin induction. Hematology Am Soc Hematol Educ Program 2011; 2011:459-465.

8. Wilber A, Tschulena U, Hargrove PW, Kim YS, Persons DA, Barbas CF, 3rd, et al. A zinc-finger transcriptional activator designed to interact with the gamma-globin gene promoters enhances fetal hemoglobin production in primary human adult erythroblasts. Blood 2010; 115:3033-3041.

9. Fathallah H, Atweh GF. Induction of fetal hemoglobin in the treatment of sickle cell disease. Hematology Am Soc Hematol Educ Program 2006:58-62.

10. Mai A, Jelicic K, Rotili D, Di Noia A, Alfani E, Valente S, et al. Identification of two new synthetic histone deacetylase inhibitors that modulate globin gene expression in erythroid cells from healthy donors and patients with thalassemia. Mol Pharmacol 2007; 72:1111-1123.

11. Fathallah H, Weinberg RS, Galperin Y, Sutton M, Atweh GF. Role of epigenetic modifications in normal globin gene regulation and butyrate-mediated induction of fetal hemoglobin. Blood 2007; 110:3391-3397.

12. Sankaran VG, Xu J, Byron R, Greisman HA, Fisher C, Weatherall DJ, et al. A functional element necessary for fetal hemoglobin silencing. N Engl J Med 2011; 365:807-814.

13. Wilber A, Nienhuis AW, Persons DA. Transcriptional regulation of fetal to adult hemoglobin switching: new therapeutic opportunities. Blood 2011; 117:3945-3953.

14. Xu J, Sankaran VG, Ni M, Menne TF, Puram RV, Kim W, et al. Transcriptional silencing of {gamma}-globin by BCL11A involves long-range interactions and cooperation with SOX6. Genes Dev 2010; 24:783-798.

15. Sankaran VG, Menne TF, Xu J, Akie TE, Lettre G, Van Handel B, et al. Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 2008; 19:1839-1842.

16. Xu J, Bauer DE, Kerenyi MA, Vo TD, Hou S, Hsu YJ, et al. Corepressor-dependent silencing of fetal hemoglobin expression by BCL11A. Proc Natl Acad Sci U S A 2013; 110:6518-6523.

17. Zhou D, Liu K, Sun CW, Pawlik KM, Townes TM. KLF1 regulates BCL11A expression and gamma- to beta-globin gene switching. Nat Genet 2010; 42:742-744.

18. Trakarnsanga K, Wilson MC, Lau W, Singleton BK, Parsons SF, Sakuntanaga P, et al. Induction of adult levels of beta-globin in human erythroid cells that intrinsically express embryonic or fetal globin by transduction with KLF1 and BCL11A-XL. Haematologica 2014; 99:1677-1685.

19. Vinjamur DS, Alhashem YN, Mohamad SF, Amin P, Williams DC Jr, Lloyd JA. Kruppel-Like Transcription Factor KLF1 Is Required for Optimal gamma- and beta-Globin Expression in Human Fetal Erythroblasts. PLoS One 2016; 11:e0146802.

20. Perrine SP, Mankidy R, Boosalis MS, Bieker JJ, Faller DV. Erythroid Kruppel-like factor (EKLF) is recruited to the gamma-globin gene promoter as a co-activator and is required for gamma-globin gene induction by short-chain fatty acid derivatives. Eur J Haematol 2009; 82:466-476.

21. Porcu S, Manchinu MF, Marongiu MF, Sogos V, Poddie D, Asunis I, et al. Klf1 affects DNase II-alpha expression in the central macrophage of a fetal liver erythroblastic island: a non-cell-autonomous role in definitive erythropoiesis. Mol Cell Biol 2011; 31:4144-4154.

22. Pandya K, Townes TM. Basic residues within the Kruppel zinc finger DNA binding domains are the critical nuclear localization determinants of EKLF/KLF-1. J Biol Chem 2002; 277:16304-16312.

23. Shariati L, Modaress M, Khanahmad H, Hejazi Z, Tabatabaiefar MA, Salehi M, et al. Comparison of different methods for erythroid differentiation in the K562 cell line. Biotechnol Lett 2016; 38:1243-1250.

24. Borg J, Papadopoulos P, Georgitsi M, Gutierrez L, Grech G, Fanis P, et al. Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet 2010; 42:801-805.

25. Romero Z, Campo-Fernandez B, Wherley J, Kaufman ML, Urbinati F, Cooper AR, et al. The human ankyrin 1 promoter insulator sustains gene expression in a beta-globin lentiviral vector in hematopoietic stem cells. Mol Ther Methods Clin Dev 2015; 2:15012.

26. Wang Y, Zheng CG, Jiang Y, Zhang J, Chen J, Yao C, et al. Genetic correction of beta-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice. Cell Res 2012; 22:637-648.

27. Macari ER, Lowrey CH. Induction of human fetal hemoglobin via the NRF2 antioxidant response signaling pathway. Blood 2011; 117:5987-5997.

28. Vikas U, Amit M, Mahavir Y, Archana T. Analyzing inhibition of BCL11A gene expression in K562 cells by RNAi. J Bio Sci Biotechnol 2013; 2:131-136.

29. Shariati l, Khanahmad H, Salehi M, Hejazi Z, Rahimmanesh I, Tabatabaiefar MA, Modarressi MH. Genetic disruption of the KLF1 gene to overexpress the γ-globin gene using the CRISPR/Cas9 system. J Gene Med 2016.