Media Optimization Results in the Retention of Long and Short-Term Hematopoietic Stem Cells in Zinc Finger Modified Healthy Donor CD34+ Cells

Abstract

Sickle Cell Disease (SCD) is a genetic disease effecting millions of people around the world. SCD is the result of a point mutation in the beta-globin chain of hemoglobin which causes glutamic acid to be replaced with valine at the sixth position. This mutation causes the beta hemoglobin to sickle and become a crescent shape when there is a lack of oxygen, and this shape change can lead to many physiological problems, such as organ damage, anemia, jaundice, pulmonary hypertension, and painful acute crises where the sickled red blood cells cause blockages in small capillaries. Many times, patients end up in the emergency room during these painful vaso-occlusive crises (VOC) and their only option for treatment is a blood transfusion. There is currently no cure for SCD, and it generally leads to a premature death. A potential cure for SCD lies in the form of genetic modification. Raising the levels of fetal hemoglobin (HbF) combats the symptoms of SCD and therefore creates a palliative effect in these patients. BCL11A is a repressor of gamma globin and HbF production in adult red blood cells. By silencing this gene, levels of HbF in the blood will increase, and the symptoms of SCD will be attenuated. Using zinc finger nucleases (ZFNs) we can achieve this genetic modification and improve the lives of SCD patients everywhere. One caveat of this therapy is ensuring that enough of the modified cells engraft into the patient after modification. Therefore, there needs to be a significant amount of long-term hematopoietic stem cells (LT-HSCs) that will engraft in the bone marrow and produce these modified healthy red blood cells (RBCs). This work focuses on retaining these essential omnipotent cells by optimizing the media that the CD34+ cells are cultured in. By changing the media that the edited CD34+ cells in are cultured in, it is possible to retain the basal level of LT-HSCs in the genomic editing manufacturing process. Currently there is significant loss of the long-term and short-term hematopoietic stem cells (ST-HSCs) because these naïve cells are extremely sensitive, and the shock of electroporation causes them to either die or differentiate. The work in this thesis will support changing the media from XVIVO-10 to CellGenix SCGM to show the retention of LT and ST-HSC’s when cultured in CellGenix SCGM media versus XVIV-10. This work will also focus on setting-up and performing subsequent assays to ensure that there is no other detrimental effect on the cells, such as lower levels of editing or lower engraftment potential. The editing will be verified with MiSeq and the colony forming ability of the cells will be confirmed by performing a CFU assay. The LT and ST-HSC’s will be evaluated via flow cytometry.