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: 8426987
• 负责人: JEFFREY A MEDIN
• 金额: $ 14.04万
• 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/8426987
• 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）：法伯氏病是一种非常罕见和非常严重的溶酶体贮积疾病，最常见于出生后的头几个月。溶酶体酸性神经酰胺酶（AC）活性缺乏导致神经酰胺在溶酶体中的积累，导致多种临床表现，这些临床表现是痛苦的，并严重影响生活质量。这种疾病是进行性的，通常在最初几年内导致死亡。该项目将描述我们生成的含有人类AC基因突变的小鼠模型。初步观察表明，我们目前模型中的疾病进展和临床表现与在人类中观察到的高度相关。此外，本项目将使用两种不同的移植平台对小鼠模型进行校正：(1)骨髓移植，(2)LV修饰的体外靶向HSC治疗。从我们的法伯模型的基因治疗校正中获得的结果将为这种目前无法治愈的人类疾病的新疗法的发展提供见解。该模型还将影响神经酰胺代谢、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)验证含有保守人类DNA突变的新型敲入小鼠模型将重现法伯病病理生理的假设。(2)探讨骨髓移植和lv介导的人AC在Farber小鼠造血干细胞/祖细胞移植中的过表达对该疾病的治疗作用。研究设计和方法：(1)我们最近建立了一种新的法伯氏病小鼠模型。将分析存活能力和形态异常。将从正常、杂合子和纯合子小鼠身上收集mef和各种器官组织，以测定AC酶活性和神经酰胺水平。细胞水平上的结构变化将通过电子显微镜进行比较。Walkey博士的团队还将评估神经结构和功能损伤。(2)我们将设计一种新的LV，它可以指导人类AC的表达，并带有细胞命运控制盒，以确保载体的安全性。我们将尝试使用临床相关的治疗方案对法伯小鼠进行骨髓移植和细胞定向基因治疗。使用新型LV修饰的同基因造血干细胞和造血干细胞将被注入完全消融的法伯小鼠体内。随着时间的推移，动物将被跟踪，法伯病的表现将被分析，如第1章所述。