Sickle cell disease (SCD) is caused by a point mutation in the beta-globin gene and is characterized by recurrent, painful, vaso-occlusive crises, end-organ dysfunction, and premature death. Foetal haemoglobin (HbF) protects against SCD pathophysiology and pharmacological increases in HbF are an important, yet limited, component of SCD management. Gene therapy offers a long-term curative option for SCD by exploiting the molecular regulators of ‘haemoglobin switching’. Krüppel-like factor (KLF1) is an essential, erythroid-specific, transcription factor that binds the β-globin gene promoter to upregulate its expression, whilst regulating the expression of additional factors like BCL11A and LRF that directly repress γ-globin and HbF expression. Heterozygosity for loss of function mutations in KLF1 lead to significant increases in HbF. We aimed to modify KLF1 in HUDEP-2 cells by CRISPR-based gene editing and examine transcriptome changes, differentiation potential and HbF reactivation (1). HuDEP-2 cells were transfected with PX458, a plasmid containing a guide sequence directing Cas9 to cut at KLF1-exon 2, using the Neon nucleofection device. The on-target specificity was determined using Sanger sequencing and the web tools TIDE and CRISPR-ID. Globin gene expression was assessed by qRT-PCR and RNAseq was employed to examine other transcriptome changes. We examined differentiation potential using flow cytometry and HPLC. We have demonstrated that HUDEP-2 cells require at least one copy of KLF1 for survival. Heterozygous cells proliferate at a reduced rate but differentiate normally. Gamma-globin is upregulated 10-fold and HbF percentage increased. CD44, a cellular adhesion molecule, is downregulated suggesting additional benefit in treating SCD with KLF1 gene editing. In conclusion, KLF1 is an exciting target for future gene editing studies in SCD.
References