Advancing brain health research through male germline editing in marmosets

项目概要/摘要 神经精神障碍是导致残疾的主要原因，影响近19%的美国人口。 进入临床试验的神经精神药物只有9%到达市场，这是成功率最低的药物之一 在所有治疗领域的应用。啮齿动物和人类神经生物学的根本差异 已经提出在开发有效的治疗策略中解释翻译失败， 减轻神经或神经变性疾病或病症。啮齿类动物行为测定也是非常重要的。 有效预测临床有效的神经精神药物。非人灵长类动物（NHP）被认为是 一个有价值的，临床相关的替代跨越差距之间的啮齿动物和人类的发展， 旨在促进大脑健康的疗法。在NHP中，普通的绒猴[Callithrix jacchus（cj）]提供 由于其体积小，寿命短，每年产生多个后代， 准确再现了人类神经解剖学然而，绒猴模型的最终效用仍然存在于 由于缺乏有效的工具来促进需要遗传修饰的研究，特别是那些 需要概括大脑健康的复杂方面。为了满足这一迫切需要，我们提出了一个创新的， 更有效的方法来实现基因编辑和转基因的基础上，新的使用高度 可操作的诱导多能干细胞（iPSC），其可以分化形成雄性生殖细胞， 最终用于生产转基因后代，这些后代携带与大脑相关的基因的精确编辑的等位基因。 健康和疾病。具体来说，我们将联合收割机1）紧密接近NIH指定的两种绒猴之一 繁殖群，维持在西南国家灵长类动物研究中心，2）NHP经验 多能干细胞、iPSC衍生和CRISPR/Cas9编辑，3）产生可移植的 4）将NHP生殖细胞移植到睾丸中以 生产精子，5）在使用尖端单细胞基因组学和多参数 综合表观基因组学评估任何细胞类型的正常性，以及6）大脑健康和疾病方面的领先专业知识 尤其是癫痫的神经遗传学在目标1中，我们将使用CRISPR编辑来生成 突变的ARX等位基因和报告转基因。在目标2中，我们将优化派生和移植 将雄性cjiPSC衍生的生殖细胞移植到受体睾丸中并移植以促进转基因精子的发育。在 目的3，我们将评估ARX突变对绒猴皮层神经元发育和迁移的影响。 总之，这些目标旨在推进基于以下方面的绒猴模型在大脑研究中的效用： cjiPSC的CRISPR/Cas9编辑，雄性生殖系介导的转基因，cjiPSC衍生的脑的发育 类器官，以及ARX突变对神经系统影响的具体知识。

PROJECT SUMMARY/ABSTRACT Neuropsychiatric disorders represent a leading cause of disability, affecting nearly 19% of the US population. Only 9% of neuropsychiatric drugs entering clinical trials reach the market, which is one of the lowest success rates across all therapeutic areas. Fundamental differences between the neurobiology of rodents and humans have been proposed to account for translational failures in development of effective therapeutic strategies to mitigate neurological or neurodegenerative diseases or disorders. Rodent behavioral assays are also variably effective in predicting clinically effective neuropsychiatric drugs. Nonhuman primates (NHPs) are recognized as a valuable, clinically relevant alternative to span the gap between rodents and humans in the development of therapies designed to advance brain health. Among NHPs, the common marmoset [Callithrix jacchus (cj)] affords a highly tractable option because of its small size, short lifespan, production of multiple offspring/year and accurate recapitulation of human neuroanatomy. However, the ultimate utility of the marmoset model remains in its infancy due to the paucity of efficient tools to facilitate studies requiring genetic modification, especially those needed to recapitulate complex aspects of brain health. To address this urgent need, we propose an innovative, more efficient approach to achieve gene editing and transgenesis in marmosets based on the novel use of highly manipulable induced pluripotent stem cells (iPSCs) that can be differentiated to form male germ cells that can ultimately be used to produce transgenic offspring carrying precisely edited alleles of genes relevant to brain health and disease. Specifically, we will combine 1) close proximity to one of two NIH-designated Marmoset Breeding Colonies, maintained at the Southwest National Primate Research Center, 2) experience with NHP pluripotent stem cells, iPSC derivation, and CRISPR/Cas9 editing, 3) a novel strategy to produce transplantable male germ cells from edited cjiPSCs, 4) documented expertise transplanting NHP germ cells into testes to produce sperm, 5) published experience in the use of cutting-edge single-cell genomics and multiparametric integrative epigenomics to assess normality of any cell type, and 6) leading expertise in brain health and disease in general and the neurogenetics of epilepsy in particular. In Aim 1, we will use CRISPR editing to generate mutant ARX alleles and reporter transgenes in cjiPSCs. In Aim 2, we will optimize derivation and transplantation of male cjiPSC-derived germ cells into recipient testes and grafts to foster development of transgenic sperm. In Aim 3, we will assess the impact of ARX mutations on marmoset cortical neuron development and migration. Together, these aims are designed to advance the utility of the marmoset model for brain research based on CRISPR/Cas9 editing of cjiPSCs, male germline-mediated transgenesis, development of cjiPSC-derived brain organoids, and specific knowledge of the neurological impact of ARX mutations.