Gene Editing of KLF1 to Cure Sickle Cell Disease

Abstract

Sickle cell disease (SCD) affects millions of people worldwide and represents the most common monogenic disease of mankind (1). It is due to a homozygous T to A transversion in the β-globin gene that results in an amino acid variant - G6V - and production of HbS, which polymerises in red blood cells (RBCs) under hypoxic conditions. This generates irreversibly sickled cells that fail to traverse the microcirculation, resulting in micro-infarcts, hypoxia and pain, or 'sickle cell crises'. During gestation RBCs utilise different sets of globin genes to produce embryonic and fetal hemoglobins (HbF), so it is not until after birth when adult hemoglobin (HbA) is first produced that the first signs of SCD become apparent. This process termed 'hemoglobin switching' has been the focus of research efforts for decades because it offers an opportunity to reactivate HbF in adult cells of patients with hemoglobinopathies. A number of transcription factors, including Krüppel-like factor 1 (KLF1), play critical roles in hemoglobin switching. KLF1 is an essential erythroid transcription factor that co-ordinates the expression of more than a thousand genes critical to the formation of adult RBCs. KLF1 directly binds the β-globin gene promoter to up regulate its expression, whilst regulating the expression of additional factors like BCL11A and LRF that directly repress γ-globin expression (HbF). Heterozygosity for loss of function mutations in KLF1 leads to a significant increase in HbF that is beneficial to patients with β-thalassemia. We propose this can be recreated by advanced gene editing techniques to provide an effective therapy for SCD. We have employed CRISPR-based gene editing to knockout the expression of KLF1 in human cells. We designed two separate sgRNAs with corresponding HDR templates to target the second exon of KLF1 and ablate its function. We optimised transfection protocols and tested the on-target specificity of our sgRNAs achieving >90% efficacy in all cell types assayed. Using HUDEP-2 cells (2), a conditionally immortalised erythroid cell line which harbors three copies of KLF1 (3), we have demonstrated that these cells require at least one copy (>1/3) for survival; heterozygous cells (+/-/- or +/+/-) proliferate at a reduced rate, but are able to differentiate normally. Using RNA-seq, we identified some genes, including ICAM-4 and BCAM, which are down-regulated accordingly in a KLF1 gene dosage-dependent manner. ICAM-4 and BCAM are cellular adhesion molecules implicated in triggering vaso-occlusive episodes (4; 5), so it is anticipated their reduced expression may provide additional benefit in treating SCD. Gamma-globin is upregulated 10-fold, BCL11A down-regulated 3-fold, and HbF+ RBCs generated at ~20% of total RBCs in KLF1 +/-/- HUDEP-2 cell lines. We also engineered the ablation of KLF1 in CD34+ cells harvested from the peripheral blood of SCD patients undergoing exchange transfusions. Following transfection of the two guides, we performed directed differentiation using an erythroid differentiation medium and analysed the levels of HbF. We observed HbF at levels of between 40-60% of total Hb by HPLC, and HbF+ cells of ~50% by FACS. There was no measurable block in erythroid differentiation by FACS. We documented the types of gene editing using a high throughout NGS assay (6). We compared efficiencies of CRISPR repair of the HbS mutation with CRIPSR damage of the KLF1 gene. Lastly, we transplanted gene-edited CD34 cells into NSGW41 mice (where human erythropoiesis is established) to determine the efficiency and safety of editing long term HSCs from SCD patients. We will report on the results of these xenotransplantation assays. Taken together these results reveal the potential utility in targeting KLF1 to cure SCD. References: Wastnedge, E. et al..J Glob Health 8, 021103 (2018). Kurita, R. et al.PLoS One 8, e59890 (2013). Vinjamur, D. S. & Bauer, D. E. Methods Mol Biol 1698, 275-284 (2018). Bartolucci, P. et al..Blood 116, 2152-9 (2010). Zhang, J., et al. PLoS One 14, e0216467 (2019). Bell, C. C., et al. BMC Genomics 15, 1002 (2014). Perkins, A. et al..Blood 127, 1856-62 (2016). Disclosures Kaplan: Celgene: Honoraria; Novartis: Honoraria. Perkins:Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees.