Targeting KLF1 for the Treatment of Sickle Cell Disease Using Antisense Oligonucleotides

Sickle cell anemia (SCD) is a hereditary blood disorder in which red blood cells (RBC) become sickle-shaped and block blood vessels, leading to painful vaso-occlusive episodes. Sickling occurs because of a point-mutation in the β-globin gene of hemoglobin. Fetal hemoglobin (HbF, α2γ2) is the main oxygen transport protein in the fetus during the last months of embryonic development and the first few months of life after birth. HbF has a slightly greater oxygen binding affinity than adult hemoglobin (HbA, α2β2) and inhibits sickling by interfering with the polymerization of hemoglobin S. Higher HbF levels in SCD correlate with better survival and because HbF production can be reactivated pharmacologically in adults, it can be used for the treatment of SCD. Erythroid Kruppel-like factor (KLF1) is an erythroid-specific transcription factor that regulates β-globin expression through direct interaction with its promoter and indirectly regulates γ-globin expression through the regulation of BCL11A. By reducing the expression of KLF1, we can promote production of HbF through the upregulation of γ-globin expression. Since rodents don’t express γ-globin, we have employed both human and engineered mouse cell lines to demonstrate upregulation of γ-globin mRNA expression in vitro. We used MEL-h-b-BAC line#7 cells, a murine erythroleukemic cell line harboring the entire human beta globin locus and expressing mouse KLF1, and treated with antisense oligonucleotides (ASOs) targeting mouse KLF1. After 7 days of free uptake with the ASOs, we observed a 6-fold increase of human γ-globin mRNA expression after achieving 65% mRNA reduction of mouse KLF1 compared to the untreated control. We were also able to demonstrate significant upregulation of human γ-globin protein expression in these cells by western blot. We have shown similar results in a human erythroleukemia cell line, K562, using ASOs targeting human KLF1. K562 cells were electroporated with the KLF1 ASOs and 4 days later, we observed a 5-fold increase of human γ-globin mRNA expression after achieving 40% mRNA reduction of human KLF1 compared to the untreated control. These data indicate that targeting mouse or human KLF1 with ASO treatment can cause an increase in human γ-globin expression in vitrothat is necessary for the upregulation of fetal hemoglobin. We have also shown that we are able to target the bone marrow in both mice and rats through subcutaneous administration of our KLF1 ASOs. In wild type mice, at a dose of 100 mpk/wk for 4 weeks, we observed KLF1 target reduction of 88% and a β-globin reduction of 58% compared to the saline control in whole bone marrow. In Sprague-Dawley rats, at a dose of 50 mpk/wk for 4 weeks, we observed KLF1 target reduction of 83% and a β-globin reduction of 77% compared to the saline control in whole bone marrow. Therefore, we are able to achieve significant β-globin mRNA reduction in the bone marrow in both mice and rats after subcutaneous administration of KLF ASOs. These data indicate that reducing KLF1 with antisense oligonucleotides is a viable option for the treatment of sickle cell anemia. Disclosures Peralta: Isis Pharmaceuticals, Inc.: Employment. Low:Isis Pharmaceuticals, Inc.: Employment. Booten:Isis Pharmaceuticals, Inc.: Employment. Zhou:Univeristy of Alabama at Birmingham: Employment. Kim:Isis Pharmaceuticals, Inc.: Employment. Freier:Isis Pharmaceuticals, Inc.: Employment. Guo:Isis Pharmaceuticals, Inc.: Employment. Murray:Isis Pharmaceuticals, Inc.: Employment. Townes:University of Alabama at Birmingham: Employment. Hung:Isis Pharmaceuticals, Inc.: Employment.

Interference of globin genes with biomarker discovery for allograft rejection in peripheral blood samples

Microarray technology is a powerful tool in the discovery of new biomarkers for disease. After solid organ transplantation, where the detection of rejection is usually made on invasive biopsies, it could be hypothesized that noninvasive transcriptional profiling of peripheral blood will reveal rejection-specific expression patterns from circulating immune cells. However, in kidney transplant rejection, the analysis of gene expression data in whole blood has proven difficult for detecting significant genes specific for acute graft rejection. Previous studies have demonstrated that the abundance of globin genes in whole blood may mask the underlying biological differences between whole blood samples. In the present study, we compared the gene expression profiles of peripheral blood of nine stable renal allograft recipients with seven matched patients having an ongoing acute renal transplant rejection, using four different protocols of preparation, amplification, and synthesis of cRNA or cDNA and hybridization on the Affymetrix platform. We demonstrated that the globin reduction method is not sufficient to unmask clinically relevant rejection-specific transcriptome profiles in whole blood. Applying an additional mathematical depletion of the globin genes improves the efficacy of globin reduction but cannot remove the confounding influence of globin gene hybridization. Sampling of peripheral blood leukocytes alone, without the confounding influence of globin mRNA, provides sensitive and specific peripheral signatures for graft rejection, with many of these signals overlapping with rejection-driven tissue (kidney)-specific signatures from matched biopsies. Similar applications may exist for array-based biomarker discovery for other diseases associated with changes in leukocyte trafficking, activation, or function.