Cellular therapies rely on the delivery of therapeutic cells into patients, but their safety can be compromised by the manipulation of cells ex vivo or their placement outside of their natural context in vivo. Cell Fate Control Gene Therapy (CFCGT) offers the possibility of establishing pharmacological controls over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. In its simplest form, 'suicide' gene therapy (SGT), stable introduction of a 'suicide' gene that can activate a non-toxic prodrug establishes control over the survival of GMCs. Current SGT modalities are sub-optimal in clinical setting.
To overcome the many limitation of current strategies, we have developed a next-generation CFCGT approach based on the active site-engineered variants of human deoxyCytidine Kinase (dCK), which enable robust activation of multiple Nucleoside Analogue (NA)-based prodrugs, act early in the pathway enabling rapid accumulation of activated NAs in target cells, and also provide the capabilities for the direct imaging of GMCs. Stable introduction of dCK variants into target cells by means of Lentiviral (LV) gene transfer significantly increases their sensitivity to multiple prodrugs. Our dCK variant with only two active site amino acid substitutions is expected to be non-immunogenic yet capable of specifically activating deoxythymidine- and deoxyuridine-based NAs that are not substrates for the wild-type enzyme, such as bromovinyldeoxyuridine (BVdU) and L-deoxythymidine (LdT). We show here that dCK can be used for controlling the survival of GMCs, in cell lines and primary cells in vitro and in a murine xenogeneic transplant models in vivo. To characterize dCK/prodrug-mediated killing mechanisms in GMCs, we have examined the levels of active metabolites in cells and the cellular pathways they antagonize.
We describe here the experimental basis for the application of this novel CFCGT in bone marrow transplantation for management of Graft-versus-Host Disease (GvHD) and in enhancing chemotherapy in direct treatment of tumors. In summary, we have developed a novel and robust strategy for effective CFCGT that addresses the many shortcomings of existing modalities. Future studies will validate this novel system in a variety of primary cells and animal disease models, including models of hematopoietic transplantation and ES/iPS-based cell therapies.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/31881 |
| Date | 11 January 2012 |
| Creators | Neschadim, Anton |
| Contributors | Medin, Jeffrey A. |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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