Genetically Reprogramming Immune Modulatory Activity in Cancer Cells using TALE and CRISPR/Cas9 Technologies
| dc.contributor.advisor | Zhang, Xiaoliu Shaun | |
| dc.contributor.committeeMember | Briggs, James M. | |
| dc.contributor.committeeMember | Widger, William R. | |
| dc.contributor.committeeMember | Otteson, Deborah C. | |
| dc.creator | Anthony-Gonda, Kim 1980- | |
| dc.date.accessioned | 2017-07-18T20:16:19Z | |
| dc.date.available | 2017-07-18T20:16:19Z | |
| dc.date.created | August 2014 | |
| dc.date.issued | 2014-08 | |
| dc.date.submitted | August 2014 | |
| dc.date.updated | 2017-07-18T20:16:19Z | |
| dc.description.abstract | The advent of TALE and CRISPR/Cas9 technologies has ushered in an era of new genome-engineering tools to precisely manipulate the human genome. These tools hold great promise to treat and cure complex human diseases, such as cancer. In the past few decades, cancer immunotherapy has emerged as an attractive cancer therapy that harnesses the natural ability of the immune system to treat malignancy. Major approaches in cancer immunotherapy include exogenously introducing an immune stimulatory gene into tumor cells for use as a tumor vaccine and manipulation of immunosuppressive mechanisms. However, current strategies to do so lack advanced techniques to manipulate the immune system in a precise way; and thus, compromises therapeutic efficacy or has unforeseen side effects. In this dissertation, we provide strong evidence for the ability of TALE and CRISPR/Cas9 technologies to reprogram endogenous expression of immune modulatory genes implicated in cancer immunotherapy. Overall, TALE-based genetic activators potently transactivated two immune stimulatory genes, IL-2 and GM-CSF, in which these genes are otherwise completely silenced in epithelial cancers. Furthermore, robust gene activation was linked to chromatin remodeling of silent cytokine gene promoters. To further enhance TALE potency, we engineered a novel multimerized TALE activator and demonstrated that it was highly efficient in transactivation of the IL-2 gene in a context-specific manner. Moreover comparative studies exploring CRISPR/Cas9 as genetic activators clearly identified TALE technology as a more efficacious strategy. We also present evidence that TALE and CRISPR/Cas9 technologies can be adapted as genetic repressors to regulate immune modulatory genes. For this purpose, we developed a collection of TALE and CRISPR/Cas9 genetic repressors and interrogated their functions on the IL-2 and CMV gene promoters as proof of principle. We clearly demonstrated that TALE and CRISPR/Cas9 repressors potently disrupted IL-2 gene activity when targeted to strong cis-regulatory elements. Moreover, we demonstrated that TALE-based repressors regulated genes downstream of their primary target. In conclusion, we have developed a novel collection of TALE and CRISPR/Cas9 genetic tools to translate our conceptual model in vivo and explore its implication for cancer immunotherapy. | |
| dc.description.department | Biology and Biochemistry, Department of | |
| dc.format.digitalOrigin | born digital | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.citation | Portions of this document appear in: K. Anthony, A. More, X. Zhang. "Activation of Silenced Cytokine Gene Promoters by the Synergistic Effect of TBP-TALE and VP64-TALE Activators." PLoS ONE 9(4) (2014): e95790. doi:10.1371/journal.pone.0095790 | |
| dc.identifier.uri | http://hdl.handle.net/10657/1904 | |
| dc.language.iso | eng | |
| dc.rights | The author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s). | |
| dc.subject | Genome-engineering | |
| dc.subject | TALE technology | |
| dc.subject | CRISPR/Cas9 technology | |
| dc.title | Genetically Reprogramming Immune Modulatory Activity in Cancer Cells using TALE and CRISPR/Cas9 Technologies | |
| dc.type.dcmi | text | |
| dc.type.genre | Thesis | |
| thesis.degree.college | College of Natural Sciences and Mathematics | |
| thesis.degree.department | Biology and Biochemistry, Department of | |
| thesis.degree.discipline | Biochemistry | |
| thesis.degree.grantor | University of Houston | |
| thesis.degree.level | Doctoral | |
| thesis.degree.name | Doctor of Philosophy |