Silvia Bakovic
SILVIA BAKOVIC-PhD DEFENSE
“Ex vivo expansion of hematopoietic stem cells for use in nonmyeloablative transplantation”
B. Sc. Universita’ degli Studi di Torino 1999
Monday, July 16, 2007, 9:00 am, Room 200, Graduate Student Centre
Silvia Bakovic- PhD Defense program-pdf
SUPERVISORY COMMITTEE
Dr. R. Keith Humphries, Research Supervisor (Medicine)Dr. Connie J. Eaves Research Co-Supervisor (Medical Genetics)
Dr. Dixie Mager (Medical Genetics)
Dr. Aly Karsan (Pathology and Laboratory Medicine)
EXAMINING COMMITTEE
Chair: Dr. James Berger (Zoology)Supervisory Committee: Dr. R. Keith Humphries, Research Supervisor (Medicine), Dr. Connie J. Eaves Research Co-Supervisor (Medical Genetics), Dr. Dixie Mager (Medical Genetics)
University Examiners: Dr. Lorne Clarke (Medical Genetics), Dr. Ross MacGillivray (Biochemistry and Molecular Biology)
External Examiner: Dr. James Ellis, Developmental and Stem Cell Biology Program. Hospital for Sick Children. University of Toronto. Toronto, ON
ABSTRACT
Hematopoietic stem cell transplantation (HSCT) is used to treat a wide range of hematologic and non-hematologic disorders. Recently, interest has grown in the potential of autologous HSCT coupled to gene therapy for the treatment of genetic blood disorders as a way of avoiding the severe immunologic reactions associated with allogeneic HSCT. However, the remaining risks in using myeloablative conditioning regimens to allow relatively small numbers of transplanted HSCs to be transplanted greatly limit the applicability of this approach. Nonmyeloablative regimens would be an appealing alternative but necessitate the generation of large numbers of genetically corrected HSCs to achieve therapeutic levels of chimerism. In this thesis I have explored the potential of forced overexpression of homeobox genes as a strategy to obtain the degree of HSC expansion required. In a first series of experiments, I found that HOXB4 and NUPHOX transduced and expanded HSCs maintain the ability of fresh HSCs to produce sustained, high level, polyclonal, lympho-myeloid chimerism when transplanted into mice given 200-250 cGy. I then tested the therapeutic efficacy of ex vivo expanded HSCs in nonmyeloablated mice with severe -thalassemia caused by the homozygous deletion of the -major globin gene (beta-MDD). The results of these experiments showed that this approach could produce a dramatic improvement in the hematocrit, hemoglobin and RBC morphology and ultimately the cure of the thalassemic phenotype that was not achievable in recipients of equivalent numbers of unmanipulated BM cells or of cells transplanted immediately after transduction. Again, the cured mice displayed a sustained, high level of polyclonal chimerism. Together these data provide “proof of principle” of the curative potential of ex vivo expanded HSCs in a preclinical model of beta-thalassemia treated with nonmyeloablative conditioning.PUBLICATIONS
Ohta H, Sekulovic S, Bakovic S, Eaves CJ, Pineault N, Gasparetto M, Smith C, Sauvageau G, and Humphries RK. Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions. Exp Hematol. 35: 817-830,2007Bakovic S, Ohta H, Imren S, Cavilla B, Eaves CJ and RK Humphries High level polyclonal reconstitution of nonmyeloablated mice with expanded HOXB4- transduced hematopoietic stem cells. In preparation.
Follenzi A, Ailles LE, Bakovic S, Geuna M & Naldini L. Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 25: 217-222, 2000.
Follenzi A, Bakovic S, Gual P, Stella MC, Longati P & Comoglio PM. Cross-talk between the proto-oncogenes Met and Ron. Oncogene 19: 3041-3049, 2000.
SELECTED PRESENTATIONS
Bakovic S, Ohta H, Sauvageau G, Eaves CJ & Humphries RK. Enhanced repopulation of sublethally conditioned mice using ex-vivo expanded HOXB4-transduced hematopoietic stem cells (HSC). Exp Hematol 32 (Suppl. 1): 36, 2004 Oral presentation at the 33rd Annual Meeting of the International Society for Experimental Hematology. July 17-20, 2004. New Orleans, Louisiana, USA.Bakovic S, Ohta H, Eaves C & Humphries K. High level polyclonal hematopoietic reconstitution of non-myeloablated mice with HOXB4-expanded stem cells. Exp Hematol 33 (Suppl. 1), 2005. Oral presentation at the 34th Annual Scientific Meeting of the International Society for Experimental Hematology. July 30-August 2, 2005. Glasgow, Scotland, UK.
Bakovic S, Rosten PM, Eaves CJ & Humphries RK. Ex-vivo expansion of hematopoietic stem cells (HSC) using HOXB4 to achieve polyclonal reconstitution and therapy of murine beta thalassemia with nonmyeloablative conditioning. Exp Hematol 34 (Suppl. 1) 2006. Oral presentation at the 35th Annual Scientific Meeting of the International Society for Experimental Hematology, September 27- 30, 2006. Minneapolis, Minnesota, USA.
Bakovic S, Rosten PM, Eaves CJ & Humphries RK. Therapeutic Effect of HOXB4-Expanded Stem Cells in Mice with Beta Thalassemia Given a Non-Myeloablative Conditioning Regimen. Oral presentation at the 48th Annual Meeting of the American Society of Hematology, Orlando, Florida, USA, December 9-12, 2006 Blood.
AWARDS
2001-2002 University Graduate Fellowship2002-2004 Cordula and Gunter Paetzold (UGF affiliated)
OTHER AWARDS
2004 International Society for Experimental Hematology; Travel Award2006 International Society for Experimental Hematology; Travel Award
2006 American Society of Hematology; Travel Award
GRADUATE STUDIES
Field of Study: Experimental HematologyCourses:
MEDG 520: Advanced Human Molecular Genetics, Dr. C. Brown
MEDG 521: Biology and Genetics of Neoplasia, Dr. F. Takei
MEDG 530: Advanced Human Genetics, Dr. B. Simpson
MEDG 540: Seminar, Dr. C. Brown and F. Dill
MEDG 548: Directed Studies, Dr. R.K. Humphries
BIOL 530: Biology of the Cell, Dr. D. Moerman
Silvia Bakovic- PhD Defense program-pdf