GYRATE ATROPHY-MODEL FOR MOLECULAR STUDY OF EYE DISEASES

描述：(申请人摘要)。其广泛的、长期的目标是 工作是了解遗传性视网膜的分子基础 退行性疾病，这涉及到疾病基因的识别， 说明基因缺陷的具体性质，并加以说明 所涉及的病理生理机制，所有这些都可能使 未来的基因替代疗法。在继承的各种 导致人类失明的视网膜退行性疾病，回旋萎缩 (GA)作为一种独特的模型，因为了解潜在的 生化缺陷，即线粒体的全身性缺陷 鸟氨酸动物转移酶(OAT)，使我们能够 从基因上解决这种疾病的相对简单的方法 水平。 OAT基因的特异性AIMS 1和2分析及其在GA中的表达 病例：基因缺陷与疾病表型的相关性。GA是一种 在临床水平上表现出相当大的异质性的疾病。 我们和其他人对GA病例的分子遗传分析揭示了一种 非常复杂的画面，其中不是一个，而是各种不同的 燕麦蛋白基因失活导致疾病的机制。几乎每一个案例 提供了有价值的新信息，不仅是关于 疾病，还包括基因表达的基本机制和 监管。为此，将继续对GA案例进行分析。 患者组织将在DNA、RNA和蛋白质水平上进行分析。这个 每个缺陷的特定性质，无论是基因缺失、重排、点 突变或其他变化将被识别。微妙的基因变化，如 将对点突变进行功能测试，以确定它们对OAT的影响 为了确认(我们和其他人)发现的突变类型， 将确定其在我们所有案件中发生的频率，并 与临床结果对照，确定是否能得出结论 关于突变的类型和引起的疾病表型 它。 具体目的3.探讨心绞痛的病理生理机制 佐治亚州燕麦片缺陷导致的精确机制 脉络膜视网膜变性，甚至主要是哪种细胞类型 受影响的，目前仍不清楚，主要是由于无法获得眼睛组织 从病人和缺乏动物模型。正在进行的项目将是 继续试图重现燕麦片缺陷 体内外内源性OAT失活对GA状态的影响。 体外方法涉及反义表达OAT基因的转移 转化成各种类型的培养细胞，包括视网膜色素上皮， 以及燕麦失活效果的生化分析。活体燕麦 灭活，通过反义和转基因的组合实现 方法，是一种尝试构建GA的小鼠模型，该模型将被 从多个层面进行分析。阐明其病理生理学基础 不仅有助于了解这种疾病，而且鉴于 GA与其他视网膜退行性疾病如视网膜炎的相似性 着色剂(RP)，也可能为可能的机制提供一些线索 在RP中。最后，对其分子和病理生理机制进行了阐述。 GA的机制将使这种疾病成为最好的 遗传性眼病考虑基因替代治疗。

DESCRIPTION: (Applicant's abstract). The broad, long-term goal of this work is to understand the molecular basis of hereditary retinal degenerative diseases, which involves identification of the disease genes, demonstration of the specific nature of the gene defects, and elucidation of the pathophysiologlogical mechanisms involved, all of which may enable gene replacement therapy in the future. Among the various inherited retinal degenerative diseases that lead to blindness in man, gyrate atrophy (GA) serves as a unique model because knowledge of the underlying biochemical defect, i.e. a generalized deficiency of the mitochondrial enzyme ornithine animotransferase (OAT), has enabled us to take a relatively straightforward approach to tackling this disease at the gene level. Specific Aims 1 and 2 Analysis of the OAT gene and its expression in GA cases: correlation of the gene defects to disease phenotypes. GA is a disease that exhibits considerable heterogeneity at the clinical level. Molecular genetic analysis of GA cases by us and others are revealing a remarkably complex picture in which not one, but a variety of different mechanisms of OAT gene inactivation results in disease. Almost every case analyzed so far has provided valuable new information not only about the disease but also on fundamental mechanisms of gene expression and regulation. For this reason, analysis of GA cases will be pursued. Patient tissues will be analyzed at the DNA, RNA and protein levels. The specific nature of each defect, be it gene deletion, rearrangement, point mutation or other changes will be identified. Subtle gene changes such as point mutations will be tested functionally for their effects on OAT in order to confirm type of mutation revealed (by us and others), the frequency of its occurrence in all of our cases will be established and compared with clinical findings, to determine if conclusions can be drawn concerning the type of mutation and the disease phenotype that results from it. Specific Aim 3. Investigation into the pathophysiological mechanisms of GA. The precise mechanism by which the OAT defects lead to the chorioretinal degeneration, or even which cell types are primarily affected, are still not known, largely due to unavailability of eye tissue from patients and lack of an animal model. Ongoing projects will be continued in which attempts are being made to reproduce the OAT deficiency state of GA through inactivation of endogenous OAT in vitro and in vivo. The in vitro approach involves transfer of antisense expression OAT gene into cultured cells of various types including retinal pigment epithelium, and biochemical analysis of the effects of OAT inactivation. in vivo OAT inactivation, achieved by a combination of the antisense and transgenic approaches, is an attempt to construct a mouse model of GA which will be analyzed at multiple levels. Elucidation of the pathophysiological basis of GA will not only help in understanding this disease but, in view of the similarity of GA to other retinal degenerative diseases such as retinitis pigmentosa (RP), may also shed some light on possible mechanisms involved in RP. Finally, elucidaiton of the molecular and pathophysiological mechanisms of GA will render this disease one of the best models of hereditary ocular diseases for consideration of gene replacement therapy.