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Sickle cell disease (SCD) afflicts an estimated 300,000 births per year globally (Rees et al., 2010; Piel et al., 2017), rendering it the most common monogenic disorder in the world (Hassel 2010). This genetic disorder is caused by a single nucleotide substitution which ultimately leads to the formation of sickle hemoglobin (HbS) in lieu of normal adult hemoglobin (HbA). Whereas, HbS polymerizes and deforms erythrocytes into their characteristic “sickled” shape, leading to a plethora of chronic symptoms, fetal hemoglobin (HbF) instead interrupts polymerization, thereby allowing erythrocytes to retain their standard shape and functionality. HbF usually only comprises approximately 1% of total hemoglobin in healthy adults, traditionally fluctuating on account of genetic differences no more than a few percent (Orkin and Bauer, 2019). In rare cases, HbF elevation may compose 10-30% of total hemoglobin, constituting a benign condition monikered hereditary persistence of fetal hemoglobin (HPFH) (Steinberg and Sebastiani, 2012). Because the significant induction of HbF is considered ameliorative of SCD symptoms, a gene-editing approach to inducing HPFH in patients with SCD presents a potential cure. This project’s goal is to design and validate an in vitro model of HPFH in an effort to induce HbF expression in an erythroblast cell line (HUDEP2). We used a cytidine-deaminase base editor to introduce an HPFH mutation into the HUDEP2 cell line, checked for the appropriate edits via restriction digest and deep sequencing, and ultimately ran a functional assay to assess HbF induction. This initial trial laid the groundwork for future modeling using iPSC-based platforms that take into account patients’ genetic background. In addition to summarizing and analyzing the modeling experiment, this four-part thesis preliminarily tackles the nuances of hemoglobin, clinical aspects of SCD, and the clinical basis for a gene-editing approach to SCD in order to provide the proper context and relevant background for the experimental investigation.



sickle cell disease, hemoglobin, fetal hemoglobin, gene editing, induced pluripotent stem cells