CRISPR/Cas9-Based Functional Characterization of ANK2 Mutations in ASD Neural Circuitry

基于 CRISPR/Cas9 的 ASD 神经回路中 ANK2 突变的功能表征

• 批准号: 9014157
• 负责人: Jacob C Garza
• 金额: $ 8.44万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2017-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9014157
• 关键词: Adipocytes; Affect; Amino Acids; Animal Behavior; Animal Model; Ankyrins; Antidepressive Agents; Architecture; Area; Autistic Disorder; Award; Axon; Basic Science; Behavior; Biological; Biological Models; Biological Neural Networks; Brain; Brain region; Candidate Disease Gene; Cell model; Cell physiology; Cells; Child; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cognition; Collaborations; Cytoskeleton; Development; Disease; Dissection; Doctor of Philosophy; Engineering; Ensure; Environment; Exhibits; Faculty; Fellowship; Funding; Gene Mutation; General Hospitals; Generations; Genes; Genetic; Genetic Research; Genomics; Germ-Line Mutation; Goals; Grant; Health Sciences; Hormones; Human; Human Development; Human Genetics; Immunohistochemistry; Impairment; Individual; Institutes; Institution; Knowledge; Laboratories; Lead; Leptin; Light; Link; Massachusetts; Mediating; Membrane; Mentors; Microscopy; Missense Mutation; Modeling; Molecular; Molecular Target; Morphology; Mus; Mutation; National Research Service Awards; Neurobiology; Neurologic; Neurons; Neurosciences; Nonsense Mutation; Organ; Outcome; Outcomes Research; Pathogenesis; Patients; Performance; Pharmacology; Phase; Phenotype; Play; Positioning Attribute; Prefrontal Cortex; Proteins; Psychiatry; Regulation; Research; Research Institute; Research Personnel; Research Project Grants; Research Technics; Research Training; Resolution; Role; Scaffolding Protein; Social Behavior; Structure; Survival Rate; Synaptic Transmission; System; Teaching Hospitals; Technology; Testing; Texas; Therapeutic Intervention; Training; Transgenic Mice; Translational Research; Universities; adeno-associated viral vector; autism spectrum disorder; axon growth; axon guidance; base; career; clinically relevant; effective intervention; effective therapy; executive function; exome sequencing; falls; genome editing; improved; in vitro Model; induced pluripotent stem cell; interest; loss of function; loss of function mutation; mammalian genome; medical schools; member; mouse model; nerve stem cell; nervous system disorder; neural circuit; neuromechanism; neuron development; neuropsychiatry; neurotransmission; new therapeutic target; novel; post-doctoral training; pre-clinical; pre-doctoral; programs; psychogenetics; public health relevance; relating to nervous system; research study; tool; vector

 DESCRIPTION (provided by applicant): The K22 applicant, Dr. Jacob Garza, obtained his Ph.D. in Pharmacology at the University of Texas Health Science Center at San Antonio (Supervisor: Dr. Xin-Yun Lu) supported by an F31 Individual Predoctoral NRSA fellowship. His thesis research focused on mechanisms underlying the antidepressant-like effects of the adipocyte-derived hormone leptin. He subsequently completed three years of postdoctoral training in neuroscience at the same institution studying neural stem cell regulation under the support of an NRSA T32 Institutional Research Training Grant. To diversify his research training, Dr. Garza accepted a new postdoctoral position in November 2013 in the Department of Psychiatry at the Massachusetts General Hospital (MGH) in the laboratory of his primary mentor on this application, Dr. Tracey Petryshen. This new position falls within his research interest in neurological and neuropsychiatric disorders, and expands his training to human psychiatric genetics and molecular neuroscience. Dr. Garza's immediate goals for this award are to obtain training in the following areas: 1) human genetics, 2) iPSC and human neural progenitor cell models, 3) genome editing technology and 4) clinical, neurobiological, and genetic aspects of autism spectrum disorder. In order to accomplish this, he has assembled a spectacular team of experts in each of the fields in which he will acquire training and gained support from world-class researchers. The mentoring committee is highly diverse and incorporates faculty members from a range of positions. The laboratories of the primary mentor (Dr. Tracey Petryshen) and the co-mentors (Dr. Mark Daly and Dr. Stephen Haggarty) are located within the MGH Center for Human Genetic Research (CHGR). These three investigators are also affiliated with the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard. These institutions are located in close proximity to Harvard University, Massachusetts Institute of Technology, Harvard Medical School and its teaching hospitals (of which the MGH is the flagship), and numerous other universities and research institutes. This incredibly rich scientific environment offers an outstanding training opportunity through interactions with leading researchers in autism research. Dr. Garza's long-term career goal is to establish himself as an independent investigator at an academic institution. He envisions maintaining a strong, well-funded research program focused at identifying mechanisms of neurological and neuropsychiatric disorders and would like to incorporate aspects of basic science and translational research. The environment at MGH is excellent for establishing collaborations between clinical and basic researchers and is extremely beneficial for Dr. Garza's long-term goals. The proposed research project focuses on autism spectrum disorder (ASD), a severe developmental neurological disorder that affects as many as 1 in 68 children. ASD is typically characterized by abnormal cognition, inhibition of social behaviors and onset of focused, isolated, or repetitive behaviors. While neuroanatomical studies have suggested abnormal brain connectivity and development of key structures involved in executive function, the underlying causes are not known. This lack of knowledge hinders the identification of new molecular targets for the development of effective treatments. Recent large-scale patient genomic studies have identified candidate genes that are highly associated with ASD, including the ankyrin-2 gene (ANK2), which has repeatedly been implicated by ASD exome sequencing studies. ANK2 encodes the ankyrin-B membrane associated scaffolding protein that plays an integral role in neuronal function, particularly axon guidance and growth, thus disruption to ankyrin-B may have critical consequences for brain function. This proposal will characterize the functional consequences of three loss-of-function mutations in ANK2 that were recently discovered in ASD patients. Aim 1 will utilize human neuronal model to study the mutation effects on neuronal development and morphology. Aim 2 will examine the impact of the most severe mutation on the function of neural circuits regulating behaviors that are abnormal in ASD. Both of these aims will be completed during the training phase of this award. During the independent phase of this award, Aim 3 will use the CRISPR/Cas9 system to generate mice with germline mutations in ANK2 that are present in ASD patients, and study the effects on brain development and ASD-like behaviors. The experiments utilized in this project will incorporate a wide array of research techniques including genomic engineering using the CRISPR/Cas9 system, adeno-associated viral vector delivery to the mouse brain, quantitative PCR, animal behavior analysis related to ASD, and development of a human neural progenitor cell model. The expected outcome of the research aims is dissection of the mechanisms of ANK2 in neural impairments underlying ASD, which is critical for improving our understanding of this disorder and developing effective interventions and treatments.

 描述（由申请人提供）：K22 申请人 Jacob Garza 博士在 F31 个人支持下在德克萨斯大学圣安东尼奥健康科学中心获得药理学博士学位（导师：Xin-Yun Lu 博士）他的论文研究重点是脂肪细胞源性激素瘦素的抗抑郁作用机制，随后他在同一机构完成了三年的神经科学博士后培训。为了使他的研究培训多样化，Garza 博士于 2013 年 11 月在马萨诸塞州综合医院 (MGH) 的实验室精神病学系接受了一个新的博士后职位。他在该应用程序上的主要导师 Tracey Petryshen 博士属于他对神经学和神经精神疾病的研究兴趣，并将他的培训扩展到人类精神遗传学和分子神经科学。 Garza 获得该奖项的直接目标是获得以下领域的培训：1) 人类遗传学，2) iPSC 和人类神经祖细胞模型，3) 基因组编辑技术和 4) 自闭症谱系障碍的临床、神经生物学和遗传方面。为了实现这一目标，他在各个领域组建了一支出色的专家团队，他将在这些领域接受培训，并获得世界一流研究人员的支持。指导委员会高度多元化，由来自不同职位的教员组成。这主要导师（Tracey Petryshen 博士）和共同导师（Mark Daly 博士和 Stephen Haggarty 博士）的实验室位于 MGH 人类基因研究中心 (CHGR)，这三位研究人员也隶属于斯坦利研究所。麻省理工学院和哈佛大学布罗德研究所的精神病学研究中心毗邻哈佛大学、麻省理工学院、哈佛医学院及其教学医院（麻省总医院是其中的旗舰医院），以及众多的医院。加尔萨博士的长期职业目标是在学术机构中成为一名独立研究者，这种丰富的科学环境为他提供了良好的培训机会。麻省总医院 (MGH) 是一个强大且资金充足的研究项目，专注于确定神经系统和神经精神疾病的机制，并希望将基础科学和转化研究的各个方面结合起来，MGH 正在为临床和基础研究人员之间的合作建立良好的环境，这对 Dr.加尔萨的拟议的研究项目重点关注自闭症谱系障碍 (ASD)，这是一种严重的发育性神经障碍，影响多达六分之一的儿童。自闭症谱系障碍的典型特征是认知异常、社交行为抑制和注意力不集中。虽然神经解剖学研究表明大脑连接和参与执行功能的关键结构发育异常，但这种知识的缺乏阻碍了新分子靶点的开发，以开发有效的治疗方法。大规模患者基因组研究已确定候选者与 ASD 高度相关的基因，包括锚蛋白 2 基因 (ANK2)，ASD 外显子组测序研究多次表明 ANK2 编码锚蛋白 B 相关膜支架蛋白，在神经元功能（尤其是轴突）中发挥着不可或缺的作用。指导和生长，因此锚蛋白-B 的破坏可能对大脑功能产生严重影响。该提案将描述最近在 ASD 患者中发现的 ANK2 中的三种功能丧失突变的功能后果。目标1将利用人类神经模型研究突变对神经发育和形态的影响，目标2将研究最严重的突变对自闭症谱系障碍中异常行为的神经回路功能的影响。在该奖项的独立阶段，Aim 3 将使用 CRISPR/Cas9 系统培育 ASD 患者中存在的 ANK2 种系突变小鼠，并研究其对大脑发育和类 ASD 的影响。该项目中使用的实验将结合广泛的研究技术，包括使用 CRISPR/Cas9 系统的基因组工程、腺相关病毒载体递送至小鼠大脑、定量 PCR、与 ASD 相关的动物行为分析以及开发该研究的预期结果是剖析 ANK2 在 ASD 神经损伤中的机制，这对于提高我们对这种疾病的理解并开发有效的干预措施和治疗方法至关重要。