QUANTITATIVE ANALYSIS AND ENGINEERING OF THE PHOTORECEPTOR TRANSCRIPTION NETWORK

描述（由申请人提供）：光感受器比人体内任何其他细胞类型更易患孟德尔疾病。不幸的是，顺式调节元件（克雷斯;即，启动子/增强子）可用于将基因治疗靶向光感受器的方法极其有限。这是我们的目标，以发展感光细胞CRE功能的定量理解，这将有助于识别新的感光细胞特异性克雷斯的基因治疗，并将告知人工基因电路的合理工程，用于治疗目的的光感受器。实现这些目标将需要详细了解控制光感受器基因表达的顺式调控网络。因此，我们已经产生了由转录因子（TF）Crx和Nrl控制的光感受器转录网络的综合模型。在这项工作的过程中，我们开发了一种计算算法Phastfind，以预测该网络中数百个基因的克雷斯。然后，我们创建了一个高通量验证管道来测定活体视网膜中的CRE活性。到目前为止，该试验已经鉴定出19种围绕视网膜疾病基因位点的新型克雷斯，从而使目前可用于基因治疗的数量翻了一番。本提案旨在通过进一步阐明Crx和Nrl在控制光感受器CRE活性中的作用，并利用这两个关键的转录调节因子用于治疗目的，来扩展这种新获得的光感受器顺式调节知识。我们推测，在感光CRE内的Crx和Nrl位点的亲和力、间距和方向以可预测的方式定量地控制其转录活性。我们将在特定目标#1中通过系统地阐明Crx和Nrl结合位点对天然和合成克雷斯中的转录活性的定量贡献来测试该假设。接下来，我们将应用我们对Crx和Nrl的了解用于视网膜中的治疗目的。在具体目标#2中，我们将使用Phastfind算法的结果来创建Crx的“最小化”基因特异性CRE，并将其用于基因治疗载体中以治疗先天性失明的小鼠模型。该目标将作为CRE设计的一般方法的测试案例，该方法将计算预测与快速体内验证相结合。如果这种方法是成功的，我们相信它可以用来工程紧凑，载体准备基因特异性克雷斯的广泛的人类视网膜疾病基因。在具体目标#3中，我们将利用Nrl作为视杆细胞命运的决定因素的作用来工程化合成药物诱导的细胞命运开关，其可用于改变发育中的光感受器的命运以用于治疗目的。我们推测，这种开关可能允许治疗广泛的疾病所引起的杆特异性基因的突变驱动转分化的患病杆成锥。此外，这种开关有一天可以用来调节胚胎干细胞分化为替代疗法的光感受器。总体而言，拟议的研究有望提供可直接转化为失明患者临床治疗的工具。公共卫生相关性：视网膜中的光感受器是失明患者受影响的主要细胞类型。不幸的是，用于制备基因治疗载体以治疗这些患者的光感受器特异性启动子的库非常有限。我们研究的目的是显着扩大天然启动子和合成基因电路的库，用于治疗失明患者。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.