Understanding and treating CRX-linked retinopathies

项目概要 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。这个过程 受以锥杆同源盒转录因子 CRX 为中心的遗传程序控制。突变在 人类 CRX 基因与多种表型的显性视网膜病相关 发病年龄。对每个个体突变机制的了解不足导致开发变得困难 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 突变并制作了携带每类代表性突变的小鼠模型。到现在为止，我们和 其他人已经对三个此类类别的小鼠模型进行了表征并报告了结果，证明了一致性 由于每种突变而导致的小鼠和人类状况之间的差异。这些研究已经提供了深刻的 疾病发病机制的知识。然而，其余类别突变的致病机制 （II 类）仍有待确定。 II 类突变与早发显性视网膜病 Leber 相关 先天性黑蒙（adLCA）和锥体营养不良（adCoRD）。我们生成了携带两个的鼠标线 单独的 II 类突变 Crx-K88N 和 Crx-E80A，并发现每个突变都发展出显性的 LCA 或 CoRD- 与光感受器基因表达失调相关的类似表型。因为这些突变是 位于负责 DNA 结合的 CRX 同源域，我们假设疾病蛋白 通过改变 CRX 的 DNA 结合特异性来错误调节基因表达，导致 CRX 在靶标上出现故障 网站。在本提案的目标 1 中，我们将使用细胞在细胞培养物和小鼠模型中检验我们的假设 生物学、分子和功能基因组学方法。使用无偏倚的高通量 DNA 结合和 通过调节功能测定，我们将确定这些突变如何改变 CRX 的调节活性，从而导致 基因表达失调和光感受器功能缺陷。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 治疗窗口期间正常的 CRX 可以改善患病视网膜的光感受器表型。我们 设计了一种可调节的基因增强方法，其中结合了四环素（强力霉素）开关 打开或关闭治疗性 CRX，由整合在基因组内的转基因产生或由腺苷携带 相关病毒（AAV）。我们将使用已建立的多学科评估表型改善 方法并期望通过 CRX 在不同的小鼠模型中看到不同程度的表型拯救 增强。这项研究的结果将增进我们对 CRX 疾病和光感受器的理解 的发展，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.