Photoreceptors are subject to a greater number of Mendelian diseases than any other cell type in the human body. Unfortunately, the repertoire of cis-regulatory elements (CREs;i.e., promoters/enhancers) available for targeting gene therapy to photoreceptors is extremely limited. It is our aim to develop a quantitative understanding of photoreceptor CRE function that will facilitate the identification of novel photoreceptor-specific CREs for gene therapy and will inform the rational engineering of artificial gene circuits for therapeutic purposes in photoreceptors. Achieving these goals will require a detailed understanding of the cis-regulatory networks that control gene expression in photoreceptors. Accordingly, we have produced a comprehensive model of the photoreceptor transcriptional network controlled by the transcription factors (TFs), Crx and Nrl. In the course of this work, we developed a computational algorithm, Phastfind, to predict CREs around hundreds of genes in this network. We then created a high throughput validation pipeline to assay CRE activity in living retinas. This assay has so far led to the identification of 19 novel CREs around retinal disease gene loci, thus doubling the number currently available for gene therapy. The present proposal aims to extend this newly gained knowledge of photoreceptor cis-regulation by further elucidating the role of Crx and Nrl in controlling photoreceptor CRE activity and by exploiting these two key transcriptional regulators for therapeutic purposes. We hypothesize that the affinity, spacing and orientation of Crx and Nrl sites within a photoreceptor CRE quantitatively control its transcriptional activity in a predictable fashion. We will test this hypothesis in Specific Aim #1 by systematically elucidating the quantitative contributions of Crx and Nrl binding sites to transcriptional activity in both natural and synthetic CREs. Next, we will apply our knowledge of Crx and Nrl for therapeutic purposes in the retina.
In Specific Aim #2 we will use the results of the Phastfind algorithm to create a `minimalized'gene-specific CRE for Crx and use it in a gene therapy vector to treat a mouse model of congenital blindness.
This Aim will serve as test case for a general approach to CRE design that combines computational prediction with rapid in vivo validation. If this approach is successful, we believe it can be used to engineer compact, vector-ready gene-specific CREs for a wide range of human retinal disease genes.
In Specific Aim #3 we will exploit Nrl's role as a determinant of rod cell fate to engineer a synthetic drug-inducible cell fate switch which can be used to alter the fate of developing photoreceptors for therapeutic purposes. We hypothesize that this switch may permit treatment of a wide range of diseases caused by mutations in rod-specific genes by driving the transdifferentiation of diseased rods into cones. In addition, this switch could someday be used to regulate the differentiation of embryonic stem cells into photoreceptors for replacement therapy. Overall, the proposed studies promise to deliver tools that can be directly translated into clinical therapies for patients with blindness.
Photoreceptors in the retina are the main cell type affected in patients with blindness. Unfortunately, the repertoire of photoreceptor-specific promoters used to make gene therapy vectors to treat these patients is very limited. It is the aim of our research to significantly expand the repertoire of both natural promoters and synthetic gene circuits available for treating patients with blindness.
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