Differential regulation of multiple transgenes for treatment of eye disease

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DESCRIPTION (provided by applicant): Proper transgene expression is essential to achieve therapeutic effect and ensure safety in many gene therapy strategies. Current regulation systems for controlling transgene expression typically require use of a special promoter and co-delivery of a cassette expressing a trans-activator protein. While these systems have been proven effective, the requirements prohibit differential regulation of multiple transgenes. Additionally, the requirement of co-delivering a trans-activator adds significantly to the payload required of the chosen gene transfer vector. To develop a new type of regulation system without such drawbacks, we are exploring a novel regulation system based on alternative splicing. To date, we have developed minimal alternative splicing introns capable of regulating transgene expression. Furthermore, we have demonstrated the feasibility of using two different ASO to independently control the expression of two different transgenes. Using AAV vector for delivering our novel regulation system, we have successfully expressed a marker gene in mouse eye in a controlled manner. Therefore, we are proposing to further develop our novel regulation system and apply the resulting technology for gene therapy of ocular diseases. Our hypothesis is that our novel regulation system could be developed to independently regulate the expression of multiple transgenes, and that differential expression of multiple potent factors inhibiting angiogenesis via different pathways would have a synergistic effect in treating ocular neovascularization. Our long-term goal is to develop novel delivery systems that exploit the advantages of AAV mediated persistent gene transfer with the ability to control the expression of the therapeutic genes being delivered. The studies described in this proposal would allow us to further optimize our novel regulation system suitable for gene therapy of clinically relevant diseases. The Specific Aims of the proposal are as follows: 1. Study the kinetics of transgene expression for our inducible system in an eye model. We have already successfully demonstrated our inducible system both in vitro and in vivo. We 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. 2. Develop our inducible system to differentially regulate the expression of multiple transgenes. In our regulation system, transgene expression is controlled by using antisense oligonucleotides targeting alternative splice site to modulate the alternative splicing of transgene message. Since the alternative splice site and its flanking sequences can be varied, we will develop introns with different sequences to allow differential regulation of multiple transgenes. 3. Regulate the expression of multiple transgenes in eye models. We will first validate our 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 in an eye disease model in an effort to better validate this novel regulation system. PUBLIC HEALTH RELEVANCE: Regulating transgene expression is essential to achieve therapeutic effect and ensure safety in many gene therapy strategies. We are developing a novel regulation system to independently regulate the expression of multiple transgenes, such as those encoding potent factors inhibiting angiogenesis via different pathways and potentially having a synergistic effect in treating ocular neovascularization.