Understanding and treating CRX-linked retinopathies

了解和治疗 CRX 相关视网膜病变

• 批准号: 10468985
• 负责人: SHIMING CHEN
• 金额: $ 38.19万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10468985
• 关键词: Address; Aftercare; Age; Age of Onset; Alleles; Binding; Biochemical; Biological Assay; CRX protein; Cell Culture Techniques; Cellular biology; DNA Binding; DNA Binding Domain; Dependovirus; Development; Disease; Disease model; Dose; Doxycycline; Future; Gene Expression; Genes; Genetic; Genome; Genomic approach; Genomics; Goals; Homeobox; Human; Individual; Knowledge; Leber' s amaurosis; Link; Maintenance; Measures; Missense Mutation; Modeling; Molecular; Morphology; Mus; Mutation; Outcome; Outcomes Research; Pathogenesis; Pathogenicity; Patients; Pattern; Phenotype; Photoreceptors; Process; Property; Proteins; Publishing; Reporting; Research; Retina; Retinal Diseases; Site; Specificity; System; Testing; Tetracyclines; Therapeutic; Therapeutic Intervention; Transgenes; Transgenic Organisms; Vertebrate Photoreceptors; Virus; base; cone-rod dystrophy; deep sequencing; design; disease-causing mutation; early onset; efficacy testing; functional genomics; gain of function; homeodomain; improved; in vitro Assay; interdisciplinary approach; loss of function; mouse model; multidisciplinary; mutant; programs; therapy development; tool; transcription factor; treatment strategy; vector

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.

项目摘要 精确调控的基因表达对于光感受器的发育和维持是必不可少的。这个过程 是由一个以锥杆同源框转录因子CRX为中心的遗传程序控制的。突变 人CRX基因与具有广泛表型的显性视网膜病相关， 发病年龄由于对每一个突变的机制缺乏了解， 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，并制作携带每类代表性突变的小鼠模型。到目前为止，我们和 其他人已经描述并报道了这三类小鼠模型的发现，证明了一致性。 小鼠和人类之间的差异。这些研究已经提供了一个深刻的 了解疾病的发病机理。然而，其余类别的突变的致病机制 （第二类）有待确定。II类突变与早发性显性视网膜病变Leber相关 先天性黑蒙（adLCA）和视锥杆营养不良（adCoRD）。我们已经生成了携带两个 单独的II类突变，Crx-K88 N和Crx-E80 A，并发现每一个都产生了显性LCA或CoRD。 与光感受器基因表达失调相关的类表型。因为这些突变 位于CRX同源结构域负责DNA结合，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在靶点发生功能障碍 网站.在本提案的目标1中，我们将使用细胞培养和小鼠模型来测试我们的假设。 生物学、分子和功能基因组学方法。使用无偏的高通量DNA结合和 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，导致 光感受器中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。我们假设外源性引入适量的 在治疗窗期间正常的CRX可以改善患病视网膜中的光感受器表型。我们 设计了一种可调基因扩增方法，该方法结合了四环素（多西环素）开关， 由整合在基因组内或由腺病毒携带的转基因产生的开启或关闭治疗性CRX， 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促进我们对CRX疾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。