University of North Carolina At Chapel Hill
Grant: $810,937 - Department of Health and Human Services - Jun. 3, 2009
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Award Description: Differential regulation of multiple transgenes for treatment of eye disease - AR
Project Description: Our long term goal is to develop novel gene delivery systems that exploit the advantages of Adeno-Associated Virus mediated persistent gene transfer with the ability to control the expression of the therapeutic genes being delivered. We have developed a system in which the splicing of a transgene can be controlled by the application of an antisense oligonucleotide (ASO) to induce transgene expression. We hypothesize that our novel regulation system can be developed to independently regulate the expression of multiple transgenes, and that differential expression of multiple potent factors inhibiting angiogenesis via different pathways will have a synergistic effect in treating ocular neovascularization. To evaluate this, we proposed three specific aims: 1 To determine the kinetics of transgene expression for our inducible system in an eye model 2 To develop our inducible system to differentially regulate the expression of multiple transgenes 3 To regulate the expression of multiple transgenes in eye models The results from these will help to define the potential efficacy of combined gene therapy targeting different genes. In the eye model, expression of the potent survival factors will be controlled by using our novel regulation system, allowing evaluation of benefits and risks of this system. This could be adapted for future clinical trials for treatment of ocular neovascularization. Progress We have been generated five constructs that have sequences within the alternative 5? splice site that are targeted by different ASOs. We examined the differential expression of each construct by induced by its corresponding ASO in 293, a human embryonic kidney cell lines. We observed the induction of expression of the reporter gene by the corresponding ASO but not other ASOs. This confirms that the splicing of the transgene is controlled in a highly sequence specific manner by the ASO, allowing for the differential regulation of multiple transgenes.
Jobs Summary: 1 Principal Investigator- responsibility for the execution and completion of the proposed research relating to use of alternative splicing intron for regulating transgene expression. Design, oversee and participate in the construction and selection of mutated introns as well as the construction of transgene expression cassettes containing introns. Plan and schedule all in vitro experiments for characterizing the constructs. Analyze experimental data and apply the results obtained towards further studies. 2. 2 Post docs-will focus on using marker genes in an eye model to study the kinetics including the onset, duration and level of transgene expression under various conditions of induction. Test inducible system to differentially regulate the expression of multiple transgenes. She will validate proposed multiple-transgene regulation system using marker genes in normal eyes and then differentially regulate the expression of multiple potent blockers for survival factors supporting neovascularization (e.g. VEGFtrap, PDGFtrap, etc.) in an eye disease model. 3. 2 Research Techs-will be responsible for providing vector reagents to our collaborator Dr. Campochiaro to advance rate-limiting animal studies 4. 2 Graduate Students: will focus on using marker genes in an eye model to study the kinetics including the onset, duration and level of transgene expression under various conditions of induction. Test inducible system to differentially regulate the expression of multiple transgenes. will focus on using marker genes in an eye model to study the kinetics including the onset, duration and level of transgene expression under various conditions of induction. Test inducible system to differentially regulate the expression of multiple transgenes. Validate proposed multiple-transgene regulation system using marker genes in normal eyes and then differentially regulate the expression of multiple potent blockers for survival factors supporting neovascularization (e.g. VEGFtrap, PDGFtrap, etc.) in an eye disease model. (Total jobs reported: 7)
Project Status: Less Than 50% Completed
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