Understanding and treating CRX-linked retinopathies

项目总结 精确调控的基因表达对于光感受器的发育和维持至关重要。这一过程 是由以锥杆同源框转录因子CRX为中心的遗传程序控制的。基因突变 人类CRX基因与显性视网膜病变有广泛的表型和 发病年龄。对每一种个体突变的机制缺乏了解，这使得开发变得困难 治疗策略。为了应对这些挑战，我们的实验室定义了四类致病CRX 并制作了每类携带有代表性突变(S)的小鼠模型。到目前为止，我们和 其他人则描述并报告了三个这样的类别的老鼠模型的发现，证明了一致性 由于每一种突变，老鼠和人类的条件之间存在差异。这些研究已经提供了一种深入的 疾病发病机制方面的知识。然而，致病机制的突变在剩下的一类 (II类)仍有待确定。II类突变与早发性显性视网膜病变Leber有关 先天性黑色素症(AdLCA)和视锥视杆细胞营养不良(AdCoRD)。我们已经产生了携带两个 个体II类突变，CRX-K88N和CRX-E80A，发现每一个都会发展成一个显性的LCA或脐带- 与光感受器基因表达失调相关的表型相似。因为这些突变是 位于负责DNA结合的CRX同源结构域中，我们假设疾病蛋白 通过改变CRX的DNA结合特异性而错误调节基因表达，导致CRX靶向故障 网站。在本提案的目标1中，我们将使用细胞在细胞培养和小鼠模型中验证我们的假设 生物学、分子和功能基因组学方法。使用无偏见的高通量DNA绑定和 调节功能分析，我们将确定这些突变如何改变CRX的调节活性，导致 光感受器基因表达的失调和功能缺陷。在目标2中，我们将解决缺乏 CRX疾病的治疗策略。我们假设外源引入适量的 在一个治疗窗口内正常的CRX可以改善病变视网膜的光感受器表型。我们 我设计了一种可调的基因增强方法，它结合了四环素(多西环素)开关来 打开或关闭由整合到基因组中的转基因或由腺病毒携带的治疗性CRX 相关病毒(AAV)。我们将使用已建立的多学科来评估表型改进 方法并期望看到CRX在不同小鼠模型中不同程度的表型挽救 增强功能。这项研究的结果将促进我们对CRX病和光感受器的理解 发展，并为未来治疗CRX疾病患者的努力提供信息。

PROJECT SUMMARY Precisely regulated gene expression is essential for photoreceptor development and maintenance. This process is governed by a genetic program centered on the cone-rod homeobox transcription factor CRX. Mutations in the human CRX gene have been associated with dominant retinopathies with a wide-range of phenotypes and ages of onset. A poor understanding of the mechanism of each individual mutation has made it difficult to develop treatment strategies. To address these challenges, our lab has defined four classes of disease-causing CRX mutations and made mouse models carrying a representative mutation(s) of each class. Up to now, we and others have characterized and reported findings on mouse models for three such classes, proving concordance between the mouse and human conditions due to each mutation. These studies have already provided a deep knowledge of disease pathogenesis. However, the pathogenic mechanism of mutations in the remaining class (Class II) remains to be determined. Class II mutations are linked to the early-onset dominant retinopathies Leber congenital amaurosis (adLCA) and cone rod dystrophy (adCoRD). We have generated mouse lines carrying two individual Class II mutations, Crx-K88N and Crx-E80A, and find that each develops a dominant LCA or CoRD- like phenotype associated with misregulation of photoreceptor gene expression. Because these mutations are located in the CRX homeodomain responsible for DNA binding, we hypothesize that the disease proteins misregulate gene expression by altering CRX’s DNA binding specificity, leading to CRX malfunction at target sites. In Aim 1 of this proposal, we will test our hypothesis in both cell culture and mouse models using cell biology, molecular and functional genomics approaches. Using unbiased high-throughput DNA binding and regulatory function assays, we will determine how these mutations alter CRX’s regulatory activity, leading to misregulation of gene expression and functional deficits in photoreceptors. In Aim 2, we will address the lack of treatment strategies for CRX diseases. We hypothesize that exogenous introduction of the proper amount of normal CRX during a therapeutic window can improve the photoreceptor phenotype in diseased retinae. We have designed a tunable gene augmentation approach that incorporates a tetracycline (doxycycline) switch to turn-on or turn-off therapeutic CRX produced by a transgene integrated within the genome or carried by an adeno associated virus (AAV). We will evaluate phenotypic improvement using established multidisciplinary approaches and expect to see varying degrees of phenotype rescue in different mouse models by CRX augmentation. The outcome of this research will advance our understanding of CRX disease and photoreceptor development, and inform future efforts to treat patients with CRX disease.