Chromosome 21 Elimination In A New Mouse Model of Down Syndrome

新唐氏综合症小鼠模型中 21 号染色体的消除

• 批准号: 9926296
• 负责人: Roger H Reeves
• 金额: $ 20.47万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-06 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926296
• 关键词: Ablation; Adult; Affect; Animal Model; Appearance; Artificial Chromosomes; Behavior; Breeding; Cell Differentiation process; Cell physiology; Cells; Centromere; Cephalic; Cerebellum; Characteristics; Chromosome 21; Chromosomes; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Conceptus; Congenital cerebellar hypoplasia; Development; Down Syndrome; Electrophysiology (science); Equus caballus; Expression Profiling; Face; First Pharyngeal Arch; Functional disorder; Future; Generations; Genes; Genetic; Genetic Diseases; Genetic Models; Genome; Heart Abnormalities; Hippocampus (Brain); Human; Human Chromosomes; Individual; Karyotype; Knowledge; Learning; Meckel' s cartilage; Memory; Modeling; Molecular; Molecular Genetics; Mosaicism; Mus; Mutate; Nature; Neural Crest Cell; Neural tube; Orthologous Gene; Outcome; Perinatal; Persons; Pharmaceutical Preparations; Pharmacological Treatment; Phenotype; Pilot Projects; Recording of previous events; Research; Role; Skeleton; Spontaneous abortion; Structure; Sum; System; Tc1 mouse; Testing; Tissues; Transgenic Mice; Trisomy; Universities; Work; base; craniofacial development; dosage; gene product; granule cell; improved; migration; mouse Ts65Dn; mouse development; mouse genome; mouse model; offspring; overexpression; pre-clinical; prenatal therapy; skeletal; therapy design; therapy development; transcriptome sequencing; zygote

Project Summary Down syndrome (DS), caused by trisomy for human chromosome 21 (Hsa21), is among the most complex genetic perturbations compatible with human survival past term; 80% of conceptuses with trisomy 21 miscarry. The interactions of ca. 600 transcribed sequences on Hsa21 affect cells directly and, by altering the actions of individual cells during development, via secondary effects on neighboring cells. The resulting outcomes comprise 80+ clinical features affecting every system in the body, although only a subset of these features is observed in any individual with trisomy 21. The advent of molecular genetics in DS research brought with it single gene transgenic mice made with newly discovered Hsa21 genes; phenotypes were interpreted from the reductionist perspective that the DS phenotype is the sum of independently acting single gene effects. Davisson’s Ts65Dn mouse fundamentally changed the paradigm for DS research, providing a model trisomic for a large subset of Hsa21 orthologs. Analysis of these mice showed that they manifest trisomy in ways that are comparable to some outcomes of trisomy 21 in people. Ts65Dn has been the work horse for more than two decades, however, advances such as the genome project show weaknesses in that model. Given the critical role for the mouse in developing therapies to ameliorate features of DS, better genetic models are needed. We will characterize a newly established model, “MAC21”, carrying Hsa21 as a mouse artificial chromosome. It contains a better genetic representation of Hsa21 than any extant model and unlike other models with Hsa21, these mice are not mosaic. Thus MAC21 avoids many limitations of existing DS models (Specific Aim 1). While all effects of trisomy are ultimately a product of misexpression in specific cells, we can usefully differentiate trisomic phenotypes that are a product of maldevelopment from those which represent an ongoing impact of trisomy on cell function. For example, midface skeletal retrusion that is substantially responsible for the characteristic facial appearance of people with DS and Ts65Dn mice arises due to a reduction of cranial neural crest cells at all stages of delamination from the neural tube, migration and proliferation that forms the first pharyngeal arch, thus there are too few cells to properly form Meckel’s cartilage. Hence the “flat” skeletal aspect is due to hypocellularity as a consequence of a developmental anomaly due to trisomy, while the cells composing these structures appear to be function normally. Similarly, hypoplastic cerebellum in DS and Ts65Dn is a product of inadequate proliferation of granule cell precursors, but function of adult cells appears largely normal. Using CRISPR-based ablation of Hsa21 in specific cells at specific stages, we will seek to differentiate these effects. The distinction may seem academic, but in fact, specific knowledge of the timing as well as the nature of DS phenotypes is critical to the successful application of animal models in the development of prenatal therapies for complex genetic disease (Specific Aim 2).

项目概要 唐氏综合症 (DS) 由人类 21 号染色体 (Hsa21) 三体性引起，是最复杂的疾病之一 与人类过去的生存相容的遗传扰动； 80% 的 21 三体受孕者会流产。 约的相互作用。 Hsa21 上的 600 个转录序列直接影响细胞，并通过改变 发育过程中的单个细胞通过对邻近细胞的二次影响。由此产生的结果 包括影响身体每个系统的 80 多个临床特征，尽管这些特征中只有一部分是 在任何患有 21 三体性的个体中都可以观察到。分子遗传学在 DS 研究中的出现带来了 用新发现的Hsa21基因制成的单基因转基因小鼠；表型是从 还原论者认为 DS 表型是独立作用的单基因效应的总和。 Davisson 的 Ts65Dn 小鼠从根本上改变了 DS 研究的范式，提供了三体模型 对于 Hsa21 直向同源物的一个大子集。对这些小鼠的分析表明，它们表现出三体性的方式如下： 与人类 21 三体性的一些结果相当。 Ts65Dn 已成为超过两年的主力 然而，几十年来基因组计划等进步显示出该模型的弱点。鉴于关键 为了了解小鼠在开发改善 DS 特征的疗法中的作用，需要更好的遗传模型。我们 将表征新建立的模型“MAC21”，携带 Hsa21 作为小鼠人工染色体。它 与任何现有模型相比，包含更好的 Hsa21 遗传表征，并且与其他具有 Hsa21 的模型不同， 这些老鼠不是马赛克。因此，MAC21 避免了现有 DS 模型的许多限制（具体目标 1）。 虽然三体性的所有影响最终都是特定细胞中错误表达的产物，但我们可以有效地 区分发育不良产物的三体表型与代表持续发育的三体表型 三体性对细胞功能的影响。例如，中面部骨骼后缩主要是造成 DS 和 Ts65Dn 小鼠的特征性面部外观是由于颅骨减少而出现的 神经嵴细胞处于从神经管分层、迁移和增殖的各个阶段，形成神经管 第一咽弓，因此细胞太少，无法正确形成梅克尔软骨。因此，“扁平”骨骼 方面是由于三体性发育异常造成的细胞减少，而细胞 组成这些结构似乎功能正常。同样，DS 和 DS 中的小脑发育不全 Ts65Dn是颗粒细胞前体增殖不足的产物，但成体细胞的功能出现 基本上是正常的。在特定阶段的特定细胞中使用基于 CRISPR 的 Hsa21 消融，我们将寻求 区分这些影响。这种区别可能看起来很学术，但事实上，具体的时间知识 DS 表型的性质对于动物模型在动物模型中的成功应用至关重要 开发复杂遗传病的产前治疗（具体目标 2）。