A Nonhuman Primate Model of Fragile X Associated Primary Ovarian Insufficiency

描述(申请人提供)：脆性X相关的原发卵巢功能不全的非人类灵长类动物模型摘要脆性X包括一系列遗传条件，所有这些都是由于FMR1基因内的变化和FMR1基因产物的异常产生和/或表达所致。正常等位基因(5-54个CGG重复)稳定传递给后代，FMR蛋白(FMRP)表达正常。在携带突变前序列(55-200个CGG重复)的个体中，等位基因保持未甲基化，并与FMR1转录本水平的增加和FMRP水平的降低相关。具有脆性X前突变(FXPM)的女性有患FXPOI和提前绝经的风险，这是唯一可归因于CGG重复长度前突变范围的非神经/心理疾病。为了全面研究导致FXPOI的人类前突变CGG重复序列扩张的分子机制和生物学后果，有必要设计和表征合适的体外和体内模型系统。胚胎干细胞(ESC)具有多能性、无限的自我更新能力、接受靶向基因操作和定向分化等特点，是研究发育调节细胞功能的理想工具。这项提议将寻求为第一个 FXPOI体内外NHP模型的建立。将解决三个具体目标：(1)生成基于NHP ESC的FXPOI模型。利用定点转基因技术，将人FXPM CGG重复序列导入现有的雌性NHP ESC系的内源性FMR1基因中。FXPM转基因胚胎干细胞系(ESCPOI)将用于FXPM原始生殖细胞的体外分化和鉴定。(2)一代女性ESCPOI嵌合NHP婴儿。使用来自AIM 1的ESCPOI系，结合体外生产ESC嵌合胚胎的优化技术，我们将产生在卵巢组织中显示FXPM表达的女性NHP婴儿(生殖系ESCPOI-嵌合体)。这一具体目标代表了一个“原则证明”步骤，这将是未来NHP FXPOI体内模型系统全面扩展的基础。(3)对于模型特征，将评估ESCPOI在活产婴儿的躯体组织和生殖系组织中的表达。我们将使用我们最先进的婴儿灵长类研究实验室的专门测试程序来监测6个月大婴儿的身体发育和神经行为评估，以确定婴儿是否表现出与FXPM相关的任何神经缺陷。我们的建议侧重于在NHP中使用基于ESC的技术，以努力向更广泛的脆性X研究社区提供可更新的、已定义的和临床相关的FXPOI体外和体内NHP模型系统。

DESCRIPTION (provided by applicant): A Nonhuman Primate Model of Fragile X Associated Primary Ovarian Insufficiency Abstract Fragile X encompasses a range of genetic conditions, all of which result as a function of changes within the FMR1 gene and abnormal production and/or expression of the FMR1 gene products. The normal allele (5-54 CGG repeats) is stably transmitted to offspring and FMR protein (FMRP) expression is normal. In individuals that carry the pre-mutation sequence (55-200 CGG repeat), the alleles remain unmethylated and are associated with increasing levels of FMR1 transcripts and decreasing levels of FMRP. Females with the Fragile X pre-mutation (FXPM) are at risk of developing FXPOI and early onset menopause, the only non- neurological/psychological condition attributed to the pre-mutation range of CGG repeat lengths. To fully investigate the molecular mechanisms and biological consequences of the human pre-mutation CGG repeat expansion responsible for FXPOI it is necessary to design and characterize appropriate in-vitro and in-vivo model systems. Embryonic stem cells (ESC) are an ideal tool with which to study developmentally regulated cell function due to their pluripotency, unlimited capacity for self-renewal, ability to accept targeted genetic manipulations and undergo directed differentiation. This proposal will seek to create for the first time in-vitro and in-vivo NHP models of FXPOI. Three specific aims will be addressed; (1) Generation of a NHP ESC-based model of FXPOI. Using site-specific transgenesis the human FXPM CGG repeat sequence is introduced into the endogenous FMR1 gene of a currently existing female NHP ESC line. The FXPM transgenic ESC line (ESCPOI) will then be used for the in-vitro differentiation and characterization of FXPM primordial germ cells. (2) Generation of female ESCPOI-chimeric NHP infants. Using the ESCPOI line from Aim 1, in conjunction with optimized techniques for the in-vitro production of ESC-chimeric embryos, we will generate female NHP infants that show expression of the FXPM within ovarian tissue (germline ESCPOI-chimeras). This specific aim represents a "proof-of-principle" step that will be fundamental to future full-scale expansion of an in-vivo NHP FXPOI model system. (3) For model characterization, ESCPOI expression in both somatic and germline tissues of liveborn infants will be assessed. Physical growth and neurobehavioral assessment in infants up to 6 months of age will be monitored using specialized testing procedures in our state-of-the-art Infant Primate Research Laboratory to determine if infants display any neurological deficits associated with the FXPM. Our proposal is focused on the use of ESC-based technologies in the NHP in an effort to provide renewable, defined and clinically relevant translational in-vitro and in-vivo NHP model systems of FXPOI to the broader Fragile X research community.