Modeling genetic anencephaly with human brain organoids

摘要 神经管缺陷(NTD)是妊娠期间常见的神经系统畸形。 摘要无脑畸形是最具戏剧性和破坏性的神经管畸形之一，其特征是脑部和颅骨功能衰竭。 在神经形成过程中关闭。它总是致命的。最近的三个外显子组测序研究已经确定了从头开始 无脑胎儿的基因突变(DNMs)，其中一些突变存在于基因中 与遗传性小鼠模型中的无脑有关。出于这些原因，DNM开始 在无脑儿和其他神经管畸形的病因中被认为是一个重要因素。但是，当前的模型 系统不容易直接验证这些变异是否会导致NTDS。拥有一种人类特有的 早期神经发育模型将允许进行这些类型的构象研究，并进一步 机械式的调查。利用人脑有机体技术达到这一目的的尝试受到限制，原因是 结构的异质性和有机体内的可变性。我们在可复制自组织方面的最新进展 来自人类诱导的多能干细胞的单玫瑰花结球体(SOSRS)使我们能够治疗SOSRS 与两种已知的神经致畸物质相一致，并观察到与NTDS一致的明显结构变化。中国的目标 目标1是通过敲除已知的导致无脑儿的基因来产生无脑儿的模型 (SHROOM3和CELSR1)，并表征指示NTDS的结构特征。在目标2中，我们 将生成特定于胎儿的模型。为此，我们将招募无脑胎儿的家庭进行采血 样本、脐带血和/或羊膜穿刺术。血液DNA将用于执行三个外显子组测序 识别DNM。脐带血细胞或无脑胎儿羊膜腔穿刺术中可能的致病原因 DNM将被重新编程为IPSC。潜在的致病突变也将通过以下方式纠正 CRISPR/Cas9产生等基因对照。最后，将生成来自这些模型的SOSR，并 测量在原则证明无脑模型中确定的表型。我们的研究很可能会放弃 阐明人类无脑的机制，并允许为NTD因果关系筛选新的DNM，因此， 极大地扩展了我们对人类NTD遗传学的理解。此外，我们与Basic的合作 拥有专门研究母婴医学遗传学的临床医生的研究人员将为 临床病例的快速建模，可能导致包括生育在内的临床实践的改变 建议和预防性治疗的效用，如叶酸补充。

Abstract Neural tube defects (NTDs) are common malformations of the nervous system that occur during pregnancy. Anencephaly is one of the most dramatic and devastating NTDs characterized by failure of the brain and skull to close during neurulation. It is always fatal. Recent trio exome sequencing studies have identified de novo genetic mutations (DNMs) in anencephalic fetuses, and some of these mutations have been in genes associated with anencephaly in genetic mouse models. For these reasons, DNMs are beginning to be considered an important factor in the etiology of anencephaly and other NTDs. However, current model systems do not easily allow for directly validating that these variants lead to NTDs. Having a human-specific model of early neurodevelopment would allow for these types of conformational studies and further mechanistic investigations. Attempts to utilize human brain organoid technology to this end are limited due to structural heterogeneity and intra-organoid variability. Our recent development of reproducible self-organizing single rosette spheroids (SOSRS) from human induced pluripotent stem cells has allowed us to treat SOSRS with two known neuroteratogens and observe distinct structural changes consistent with NTDs. The goal in Aim 1 is to generate a model of anencephaly by knocking out genes known to lead to anencephaly (SHROOM3 and CELSR1) in SOSRS and characterize structural signatures indicative of NTDs. In Aim 2, we will generate fetal-specific models. To this end, we will enroll families with anencephalic fetuses to obtain blood samples, cord blood, and/or amniocentesis. Blood DNA will be used to perform trio exome sequencing to identify DNMs. Cells from the cord blood or amniocentesis from anencephalic fetuses with likely causative DNMs will be reprogrammed into iPSCs. The potentially causative mutations will also be corrected by CRISPR/Cas9 to generate isogenic controls. Finally, SOSRS from these models will be generated and measured for phenotypes identified in the proof-of-principle anencephalic models. Our study will likely shed light on the mechanisms of human anencephaly and allow for screening novel DNMs for NTD causality, thus, greatly expanding our understanding of human NTD genetics. Furthermore, our collaboration of basic researchers with clinicians specializing in genetics of maternal and fetal medicine will provide a framework for rapid modeling of clinical cases, potentially resulting in altered clinical practices including fertility recommendations and the utility of prophylactic treatments, such as folate supplementation.