Advancing brain health research through male germline editing in marmosets

通过狨猴雄性种系编辑推进大脑健康研究

基本信息

批准号：
10285904
负责人：
Brian Peter Hermann
金额：
$ 256.87万
依托单位：
UNIVERSITY OF TEXAS SAN ANTONIO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10285904
关键词：
3-Dimensional ATAC-seq Address Adult Affect Alleles Allogenic Anatomy Animals Area Behavioral Assay Biological Models Brain Brain Diseases Breeding CRISPR/Cas technology Callithrix Callithrix jacchus jacchus Cell Transplantation Cells ChIP-seq Chimerism Chromatin Clinic Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Communities Complex DNA Methylation Defect Derivation procedure Development Disease Embryo Enterobacteria phage P1 Cre recombinase Epigenetic Process Epilepsy Failure Fostering Gene Expression Gene Transfer Techniques Genes Genetic Genome Genomics Germ Germ Cells Germ Lines Goals Health Histology Homeobox Homeobox Genes Human In Vitro Injections Intellectual functioning disability Interneurons Knock-in Knowledge Laboratories Link Longevity Magnetic Resonance Imaging Mediating Methodology Methods Modeling Modification Mus Mutation Neuroanatomy Neurobiology Neurodegenerative Disorders Neurologic Neurons Newborn Infant Normalcy Operative Surgical Procedures Organoids Pathway interactions Pharmaceutical Preparations Pluripotent Stem Cells Population Pregnancy Primates Production Protocols documentation Publishing Reporter Research Research Personnel Rodent Rodent Model Seeds Source Spermatocytes Spermatogenesis Structure Study models System Testis Therapeutic Tissues Transgenes Transgenic Organisms Translating Translations Transplantation United States National Institutes of Health Validation Xenograft procedure base bisulfite sequencing brain health brain research cell type clinical predictors clinically relevant cognitive process design disability epigenomics experience fetal genome editing histone modification human model in vivo induced pluripotent stem cell infancy innovation innovative neurotechnologies interest lissencephaly male malformation migration mutant nervous system disorder neurogenetics neuron development neuropsychiatric disorder neuropsychiatry non-invasive imaging nonhuman primate novel novel strategies novel therapeutics offspring polyalanine postnatal reconstitution relating to nervous system single-cell RNA sequencing skills sperm cell success therapeutically effective therapy design/development tool transcriptome sequencing transmission process whole genome

项目摘要

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.

项目概要/摘要神经精神疾病是导致残疾的主要原因，影响着近 19% 的美国人口。进入临床试验的神经精神药物只有9%进入市场，这是成功率最低的药物之一所有治疗领域的比率。啮齿动物和人类神经生物学之间的根本差异已被提议解释开发有效治疗策略中的翻译失败减轻神经系统或神经退行性疾病或紊乱。啮齿动物行为测定也各不相同有效预测临床有效的神经精神药物。非人类灵长类动物（NHP）被认为是一种有价值的、临床相关的替代方案，可以跨越啮齿类动物和人类之间的发展差距旨在促进大脑健康的疗法。在 NHP 中，普通狨猴 [Callithrix jacchus (cj)] 提供由于其体型小、寿命短、每年可繁殖多个后代，因此是一种高度易于处理的选择准确再现人体神经解剖学。然而，狨猴模型的最终用途仍然在于由于缺乏有效的工具来促进需要基因改造的研究，尤其是那些需要概括大脑健康的复杂方面。为了解决这一紧迫需求，我们提出了一种创新的、基于高度的新用途，在狨猴中实现基因编辑和转基因的更有效方法可操作的诱导多能干细胞（iPSC），可以分化形成雄性生殖细胞，最终用于产生携带经过精确编辑的大脑相关基因等位基因的转基因后代健康和疾病。具体来说，我们将结合 1) 靠近 NIH 指定的两只狨猴之一繁殖群体，由西南国家灵长类研究中心维护，2) NHP 经验多能干细胞、iPSC 衍生和 CRISPR/Cas9 编辑，3) 生产可移植细胞的新策略来自经过编辑的 cjiPSC 的雄性生殖细胞，4) 记录了将 NHP 生殖细胞移植到睾丸中的专业知识产生精子，5）发表了使用尖端单细胞基因组学和多参数的经验用于评估任何细胞类型正常性的综合表观基因组学，以及 6) 大脑健康和疾病方面的领先专业知识一般而言，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 cjiPSC 中的突变 ARX 等位基因和报告基因转基因。在目标2中，我们将优化衍生和移植将雄性 cjiPSC 衍生的生殖细胞注入受体睾丸和移植物中，以促进转基因精子的发育。在目标 3，我们将评估 ARX 突变对狨猴皮质神经元发育和迁移的影响。总之，这些目标旨在促进狨猴模型在基于以下内容的大脑研究中的实用性： cjiPSC 的 CRISPR/Cas9 编辑、雄性种系介导的转基因、cjiPSC 衍生大脑的发育类器官，以及 ARX 突变对神经系统影响的具体知识。