Abstract
Nuclear factor (erythroid derived-2)-like 2 (Nfe2l2; NRF2) is a transcription factor that orchestrates activation of antioxidant genes in response to oxidative stress. In this thesis dissertation, NRF2 was studied for its potential beneficial role in red blood cells and for its therapeutic relevance in hemoglobinopathies including sickle cell disease (SCD). The two main roles of NRF2 activation that are highlighted in this study include regulation of fetal hemoglobin (HbF), a protein that is known to provide therapeutic benefits in SCD and regulation of antioxidant response, that can improve the red blood cell health and prevent its propensity to hemolyze.
NRF2 activation was studied genetically by knocking out its binding partner KEAP1, using CRISPR Cas9 in erythroid cellular models including HUDEP-2 cell line and primary CD34+ cells. KEAP1 KO in HUDEP-2 and CD34+ cells showed robust induction in HbF at both transcriptional and protein levels. Epigenetic studies revealed increase in NRF2 occupancy in γ-globin promoter region.
Pharmacological activation of NRF2 in SCD trangenic mouse model showed reduction in total ROS levels in erythroid populations in bone marrow and whole blood. Activation of antioxidant enzymes including G6PD and total GSH levels in whole blood indicated towards an improvement in the glutathione response, a pathway that is significantly impaired in SCD. Chronic treatment with C1 resulted in improvement in overall disease pathophysiology including hemolysis parameters and improvement in endothelial inflammation marked by reduction in soluble p-selectin in the plasma and reduction in p-selectin mRNA in liver and lungs.
Overall, these studies focus on evaluating the functional effects of NRF2 activation in RBCs in the context of hemoglobinopathies with a focus on SCD that is characterized by hemolysis and chronic inflammation. Activation of NRF2 resulted in increase in HbF and improvement in markers of oxidative stress thereby strongly emphasizing its potential therapeutic relevance in SCD.