Genetic Correction of a Novel "Knock-in" Mouse Model for Farber Disease

法伯病新型“敲入”小鼠模型的基因校正

• 批准号: 8598114
• 负责人: JEFFREY A MEDIN
• 金额: $ 15.4万
• 依托单位: UNIVERSITY HEALTH NETWORK
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598114
• 关键词: Age-Months; Amino Acids; Animal Model; Animals; Autologous Transplantation; Biological Assay; Bone Marrow; Bone Marrow Cell Transplantation; Bone Marrow Transplantation; Cell Death; Cell Fate Control; Cell Proliferation; Cell Transplants; Cell membrane; Cells; Ceramides; Cessation of life; Clinical; Clinical Data; Complex; Critiques; Defect; Deterioration; Development; Diffusion; Disease; Disease Progression; Embryo; Engineering; Ensure; Exhibits; Exploratory/Developmental Grant for Diagnostic Cancer Imaging; Farber' s lipogranulomatosis; Functional disorder; Funding; Galactosylceramides; Gangliosides; Gene Mutation; Generations; Genes; Genetic; Glycolipids; Goals; Granuloma; Hematopoietic; Homeostasis; Homozygote; Human; Hydrolase; Hydrolysis; Impairment; In Vitro; Individual; Joints; Knock-in Mouse; Knockout Mice; Lead; Learning; Lentivirus Vector; Lipid-Laden Macrophage; Liver; Lysosomes; Macaca mulatta; Malignant Neoplasms; Mediating; Membrane; Metabolic; Metabolism; Modeling; Modification; Mus; Mutation; Myelin; Neurologic; Organ; Outcome; Pain; Patients; Pattern; Peripheral Blood Mononuclear Cell; Peripheral Nervous System; Positioning Attribute; Production; Property; Quality of life; Recombinants; Recording of previous events; Reporting; Research; Research Design; Research Methodology; Role; Safety; Signal Transduction; Site; Sphingolipids; Sphingomyelins; Spleen; Stem cells; Structure; Subcutaneous Tissue; Sulfoglycosphingolipids; Testing; Therapeutic; Time; Tissues; Transplantation; United States National Institutes of Health; Vertebral column; Work; base; clinically relevant; enzyme activity; enzyme replacement therapy; functional disability; galactosylgalactosylglucosylceramidase; gene therapy; human DNA; in vivo; insight; mouse model; nonhuman primate; novel; novel therapeutic intervention; overexpression; palliative; postnatal; pre-clinical; public health relevance; research study; sphingosine 1-phosphate; stem; vector

DESCRIPTION (provided by applicant): Farber disease is a very rare and very severe lysosomal storage disorder that most commonly presents in the first few months of age. A deficiency of lysosomal acid ceramidase (AC) activity results in the accumulation of ceramide in lysosomes causing multiple clinical manifestations that are painful and severely impact quality of life. The illness is progressive, and often leads to death within the first few years. The propose project will characterize a murine model we generated that harbors a human mutation in the AC gene. Preliminary observations show that disease progression and clinical manifestations in our current model highly correlates with what is observed in humans. Furthermore, this project will carry out correction of the mouse model using two different transplantation platforms: (1) bone marrow transplantation, and (2) LV- modified ex vivo-targeted HSC therapy. Results acquired from gene therapy correction of our Farber model will provide insight for the development of novel therapies for this currently incurable disease in humans. This model will also impact research on the understanding of ceramide metabolism, signaling, bioactive properties, and role in cancer. Specific Aims: (1) To test the hypothesis that a novel 'knock-in' mouse model harboring a conserved human DNA mutation will recapitulate Farber disease pathophysiology. (2) Explore the effects of bone-marrow transplantation and LV-mediated overexpression of human AC in hematopoietic stem/progenitor cell transplants in Farber mice as treatments for this disorder. Research Design and Methods: (1) We have very recently generated a novel murine model for Farber disease. Viability and morphological abnormalities will be analyzed. MEFs and tissues from various organs will be collected from normal, heterozygous, and homozygous mice to assay for AC enzyme activity and ceramide levels. Structural changes on the cellular level will be compared using EM. Dr. Walkey's group will also assess neurological structure and functional impairment. (2) We will engineer a novel LV that directs the expression of human AC along with a cell fate control cassette to ensure safety of the vector. We will attempt bone marrow transplantation and cell-directed gene therapy of Farber mice using a clinically relevant treatment schema. Syngeneic HSCs and HPCs modified using the novel LV will be infused into fully ablated Farber mice. Animals will be followed over time and manifestations of Farber disease will be analyzed as in Aim 1.

描述（由申请人提供）：Farber疾病是一种非常罕见且非常严重的溶酶体储存障碍，最常见的是在头几个月大时出现。溶酶体酸神经酶（AC）活性的缺乏导致神经酰胺在溶酶体中的积累，导致多种临床表现，这些临床表现痛苦且严重影响生活质量。这种疾病是进步的，通常会在头几年导致死亡。 建议的项目将表征我们生成的鼠模型，该模型携带了AC基因中的人类突变。初步观察表明，我们当前模型中的疾病进展和临床表现与人类观察到的高度相关。此外，该项目将使用两个不同的移植平台对小鼠模型进行校正：（1）骨髓移植和（2）（2）LV-修饰的Ex Vivo-TargeT靶向HSC治疗。从我们的Farber模型的基因疗法校正获得的结果将为人类目前无法治愈的疾病开发新疗法提供见解。该模型还将影响了解神经酰胺代谢，信号传导，生物活性特性和在癌症中的作用的研究。具体目的：（1）检验一个假说，即具有保守人类DNA突变的新型“敲入”小鼠模型将概括Farber疾病病理生理学。 （2）探索骨髓移植和LV介导的人类AC在造血茎/祖细胞移植物中人类AC的过表达作为这种疾病的治疗方法。研究设计和方法：（1）我们最近已经生成了一种用于法伯疾病的新型鼠模型。将分析生存力和形态异常。来自各种器官的MEF和组织将从正常，杂合和纯合小鼠中收集，以测定AC酶活性和神经酰胺水平。使用EM将比较细胞水平的结构变化。 Walkey博士的小组还将评估神经系统结构和功能障碍。 （2）我们将设计一种新型的LV，该LV和细胞命运控制盒一起指导人类AC的表达，以确保矢量的安全性。我们将尝试使用临床相关的治疗模式对Farber小鼠的骨髓移植和细胞指导的基因治疗。使用新型LV修饰的合成性HSC和HPC将被注入完全消融的Farber小鼠中。随着时间的流逝，动物将被遵循，而AIM 1中将分析Farber疾病的表现。

项目成果

Chronic lung injury and impaired pulmonary function in a mouse model of acid ceramidase deficiency.

• DOI: 10.1152/ajplung.00223.2017
• 发表时间: 2018-03
• 期刊: American journal of physiology. Lung cellular and molecular physiology
• 作者: Fabian P. S. Yu;D. Islam;J. Sikora;Shaalee Dworski;Jiri Gurka;Lucía López-Vásquez;Mingyao Liu;W. Kuebler;T. Levade;Haibo Zhang;J. Medin