QUANTITATIVE ANALYSIS AND ENGINEERING OF THE PHOTORECEPTOR TRANSCRIPTION NETWORK

描述（由申请人提供）：光感受器比人体中任何其他类型的细胞更容易受到孟德尔疾病的影响。不幸的是，可用于光感受器靶向基因治疗的顺式调控元件（cre，即启动子/增强子）非常有限。我们的目标是发展对光感受器CRE功能的定量理解，这将有助于识别用于基因治疗的新型光感受器特异性CRE，并将为光感受器治疗目的的人工基因回路的合理工程提供信息。实现这些目标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，我们建立了一个由转录因子（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，来预测这个网络中数百个基因的cre。然后，我们创建了一个高通量验证管道来测定活视网膜中的CRE活性。到目前为止，这项检测已经鉴定出19个新的视网膜疾病基因位点周围的cre，从而使目前可用于基因治疗的数量增加了一倍。本研究旨在进一步阐明Crx和Nrl在控制光受体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，从而扩展这一新获得的光受体顺式调节知识。我们假设光感受器CRE中Crx和Nrl位点的亲和力、间距和方向以可预测的方式定量控制其转录活性。我们将在Specific Aim #1中通过系统地阐明Crx和Nrl结合位点对天然和合成cre转录活性的定量贡献来验证这一假设。接下来，我们将把Crx和Nrl的知识应用于视网膜的治疗目的。在Specific Aim #2中，我们将使用Phastfind算法的结果为Crx创建一个“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，我们相信它可以用于设计紧凑的，载体就绪的基因特异性cre，用于广泛的人类视网膜疾病基因。在Specific Aim #3中，我们将利用Nrl作为杆状细胞命运决定因素的作用来设计一种合成药物诱导的细胞命运开关，该开关可用于改变发育中的光感受器的命运，以达到治疗目的。我们假设这种开关可以通过驱动患病的杆状细胞向锥体的转分化来治疗由杆状细胞特异性基因突变引起的广泛疾病。此外，这种开关有一天可以用来调节胚胎干细胞向光感受器的分化，用于替代疗法。总的来说，提出的研究有望提供可以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，用于制造基因治疗载体来治疗这些患者的光感受器特异性启动子的数量非常有限。我们的研究目的是显著扩大天然启动子和合成基因回路的曲目，可用于治疗失明患者。

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.