Date of Award

8-1-2016

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Wilber, Andrew

Abstract

Sickle cell disease (SCD) is a severe hemoglobin disorder caused by co-inheritance of a single mutation in the β-globin gene of adult hemoglobin (HbA; α2β2). This alteration leads to the formation of sickle hemoglobin (HbS; α2βS2) and deformed, sickle-shaped red blood cells (RBCs). Sickle RBCs obstruct small blood vessels resulting in anemia, excruciating pain crises, organ damage, and stroke. For the millions of people affected by this disease, life expectancy is only 40-60 years of age. The only cure for SCD is hematopoietic stem cell (HSC, CD34+) transplantation, which requires a human leukocyte antigen (HLA)-matched donor. However, this option runs the risk of complications associated with graft versus host disease and infection. Before birth, individuals with SCD do well because their RBCs are filled with γ-globin containing fetal hemoglobin (HbF; α2γ2), which inhibits the formation of HbS. In fact, some SCD patients who co-inherit mutations that allow for high-level expression of HbF into adulthood are asymptomatic. This suggests that genetic modification of the patient’s own HSCs to permit HbF production would be a viable therapeutic alternative to HSC transplantation. Our work has focused on the use of lentiviral vectors to introduce an exogenous γ-globin gene or shRNA sequences designed to knockdown repressors of γ-globin, such as the zinc-finger transcription factor, BCL11A, to prevent silencing of the endogenous γ-globin genes allowing for persistent expression of HbF. Despite significant progress using both approaches, we have been unable to increase the level of HbF > 30%; a curative threshold for SCD patients who continue to produce HbF into adulthood. The goal of my project was to combine these approaches into a single lentiviral vector to achieve co-regulated, erythroid-specific expression and augmented levels of HbF. I successfully modified the insulated, erythroid-specific γ-globin vector (termed V5m3-400) to include microRNA (miR)-adapted shRNAs (or shmiRs) targeting BCL11A (based on miR-30 and miR-E architectures) in the first and second noncoding introns of the γ-globin genomic sequences. Inclusion of shmiRs had no appreciable effect on integrity of the integrated provirus or vector titer. Vector performance was initially tested using human K562 erythroleukemia cells expressing a flag-tagged version of BCL11A. In this cell line, BCL11A knockdown was significantly improved using miR-E-shRNAs due to a dramatic increase (up to 350-fold) in processing of mature shRNA sequences. The miR-E vectors also provided high-level expression of γ-globin. Erythroid-specific expression of the γ-globin transgene and BCL11A knockdown was confirmed in maturing erythroid cells derived from transduced CD34+ cells of a healthy donor resulting in a 50% increase in HbF levels compared with cells transduced with V5m3-400 as a control. While encouraging, I was unable to discriminate HbF derived from the vector-encoded versus endogenous γ-globin genes. To address this, I introduced a single base change in exon 2 of the γ-globin gene encoded by V5m3-400 such that threonine replaces isoleucine at amino acid 75 (I75T). This variant was successfully distinguished from endogenous γ-globin gene products by reverse phase high performance liquid chromatography (HPLC) in culture-differentiated erythroid cells. Based on these findings, I created compound γ-globin/shmiR-E vectors that include the I75T substitution (I75Tγ-globin/shmiR-E). Future studies will focus on testing this novel vector design in erythroid cells derived from transduced CD34+ cells of healthy donors and patients with SCD. I anticipate that this compound vector has the potential to maximize γ-globin expression and promote levels of HbF that are unlikely to be safely and effectively achieved by conventional globin gene addition or shRNA knockdown approaches alone.

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