Nucleic acids are an emerging class of therapeutics with the capacity to repair both DNA and RNA mutations in clinically relevant targets. We have used two approaches, mobile group II introns and Spliceosome Mediated RNA Trans-splicing (SMaRT), to correct β-globin mutations at the DNA and RNA levels respectively. We show that the group II intron inserts site-specifically into its DNA target, even when similar targets are available. Experiments transitioning this therapeutic into mammalian cell systems are then described. We also illustrate how SMaRT RNA repair can be used to correct β-globin mutations involved in sickle cell disease and some forms of β- thalassemia. We uncovered diverse repair efficiencies when targeting sickle cell versus β- thalassemia transcripts in mammalian cells. Possible reasons for this and how it might direct target choice for the SMaRT therapeutic approach are both discussed. The therapeutic molecule in SMaRT, a Pre-Trans-splicing Molecule or PTM, is also delivered via lentivirus to erythrocyte precursors cultured from the peripheral blood of sickle cell patients. Preliminary results from these experiments are discussed. / Dissertation

Sickle cell anemia is a hematological disorder that is caused by a single point mutation in the beta-globin chain of hemoglobin. It results in several complications related to the small and large vessels in patients with the disease. Large vessel complications include cerebral infarcts, which are observed in children under ten years old. The mechanism behind this complication is not completely understood. It is the goal of this project to begin to understand the role sickle erythrocytes may play in causing endothelial dysfunction as a precursor to sickle related complications. The hypothesis of this work is that exposure of large vessel endothelium to sickle erythrocytes causes an increase in endothelial permeability through loosening of adherens junctions. In the first goal of this work, bovine aortic endothelial cells (BAECs) are grown on coverslips and exposed to sickle erythrocytes for 5 minutes and either immediately fixed or incubated in 30 minutes and then fixed. Immunofluorescent studies labeling VE cadherin show changes in VE cadherin dynamics, suggesting sickle erythrocytes may be involved in this observation. Next, BAECs were grown on transwell inserts and exposed to sickle erythrocytes for 5 minutes. The erythrocytes are washed off and the BAEC are incubated with 10,000 MW dextran conjugated to lucifer yellow or FITC-BSA or to determine BAEC permeability. When dextran is used as the test molecule, endothelial permeability did not show a significant change from baseline. However, when BSA is used as the test molecule, increases in endothelial permeability are observed. Explanations into the differences between the transport mechanisms of the two molecules are discussed. These experiments show changes in VE cadherin localization due to sickle erythrocyte exposure. This may cause increases in endothelial permeability and an experimental model and preliminary studies are performed. This study provides potential mechanisms to explain the changes in VE cadherin localization and provide suggestions for further studies to test the effect of sickle erythrocytes on endothelial permeability. This work provides a strong foundation for continuing studies on the effects of sickle erythrocytes on endothelial dysfunction within the confines of sickle related complications.

Thesis (M. S. in Psychology)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

No description available.

Thesis (Ph.D)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on January 6, 2009). Research advisor: Ann K. Cashion, Ph.D. Document formatted into pages (x, 102 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 81-88).

Thesis (M.S.)--University of Tennessee Health Sciences Center, 2005. / Spine title: Effects of sickle cell disease on growth of the craniofacial complexes. Appendices: leaves 162-414 Bibliography: leaves 145-161.

Thesis (Ph. D.)--Florida State University, 2003. / Advisor: Dr. Marcia L. Rosal, Florida State University, College of Visual Arts and Dance, Dept. of Art Education. Title and description from dissertation home page (viewed Jan. 31, 2005). Includes bibliographical references.

Thesis (PH.D.)--Michigan State University. Chemistry, 2008. / Title from PDF t.p. (viewed on Aug. 11, 2009) Includes bibliographical references. Also issued in print.

Thesis (Ph. D.)--Alliant International University, California School of Professional Psychology, San Francisco, 2005. / Includes bibliographical references (61-64) and abstract.

The HbS gene had a limited number of origins during history, and these can be defined by the haplotype (a set of DNA polymorphisms inherited together) of the associated β-globin gene. Five major haplotypes have been identified, and associated with different ethnic groups. These are the Arab Indian haplotype, the Benin haplotype, the Cameroonian haplotype, the Central African Republic (CAR) or Bantu haplotype, and the Senegal haplotype. The polymorphisms defining these haplotypes are associated with fetal hemoglobin, the major modifier of sickle cell disease phenotype and severity. The Arab Indian haplotype, in particular, is associated with unusually high HbF levels (20%), and a significantly less severe clinical presentation. We found a novel (C>T) SNP -68 bp 5' to HBD in this region, expressed in patients with the Arab Indian haplotype, but not sickle cell disease patients expressing other β-globin cluster haplotypes. There is evidence that this -68 (C>T) polymorphism may play a functional role in the hemoglobin expression of these patients, and its effect on globin levels and disease severity is the long-term interest of this study. A previous reporter assay in K562 cells determined that the -68 SNP was associated with decreased δ-globin gene expression. This study aims to clone the HBD region of a patient positive for this SNP into a lentiviral firefly luciferase reporter vector, for use in more physiologically accurate CD34+ erythropoietic progenitor cells. If the mutations in these β-globin gene haplotypes, such as the HBD mutation described in this study, are responsible for the protective effects seen in patients, perhaps some of these genetic locations can serve as targets for therapeutics in sickle cell disease or other blood disorders.