Understanding and treating CRX-linked retinopathies

了解和治疗 CRX 相关视网膜病变

基本信息

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
项目类别：
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&apos 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

项目摘要

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。受以锥杆同源盒转录因子 CRX 突变为中心的遗传程序控制。人类 CRX 基因与多种表型的显性视网膜病相关对每个个体突变机制的了解不足使得开发变得困难。为了应对这些挑战，我们的实验室定义了四类致病的 CRX。到目前为止，我们和制作了携带每个类别的代表性突变的小鼠模型。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，证明了一致性这些研究已经提供了小鼠和人类因每种突变而产生的状况之间的深入关系。然而，其余类的突变的致病机制。（II 类）仍有待确定 II 类突变与早发性显性视网膜病 Leber 相关。我们已经产生了携带两种疾病的小鼠品系。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- 就像与光感受器基因表达失调相关的表型一样，因为这些突变是位于负责 DNA 结合的 CRX 同源域中，我们捕获到疾病蛋白通过改变 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。使用无偏见的高通量 DNA 结合和生物学、分子和功能基因组学方法。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，从而导致在目标 2 中，我们将解决光感受器基因表达失调和功能缺陷的问题。我们勇敢地引入适量的外源性CRX疾病的治疗策略。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。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。