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

基本信息

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
项目类别：
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&apos 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

项目摘要

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）：法伯病是一种非常罕见且非常严重的溶酶体贮积症，最常见于出生后几个月。溶酶体酸性神经酰胺酶 (AC) 活性缺乏会导致神经酰胺在溶酶体中积聚，从而导致多种痛苦的临床表现，并严重影响生活质量。这种疾病是进行性的，通常会在最初几年内导致死亡。该项目将表征我们生成的小鼠模型，该模型含有人类 AC 基因突变。初步观察表明，我们当前模型中的疾病进展和临床表现与在人类中观察到的情况高度相关。此外，该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，和（2）LV改良的离体靶向HSC疗法。我们的法伯模型的基因治疗校正所获得的结果将为开发针对这种目前人类无法治愈的疾病的新疗法提供见解。该模型还将影响对神经酰胺代谢、信号传导、生物活性特性以及在癌症中的作用的理解的研究。具体目标： (1) 检验以下假设：携带人类保守 DNA 突变的新型“敲入”小鼠模型将重现法伯病的病理生理学。 (2) 探索骨髓移植和 LV 介导的人 AC 过表达在 Farber 小鼠造血干/祖细胞移植中作为治疗这种疾病的效果。研究设计和方法：（1）我们最近生成了一种新型法伯病小鼠模型。将分析活力和形态异常。将从正常、杂合和纯合小鼠中收集来自不同器官的 MEF 和组织，以测定 AC 酶活性和神经酰胺水平。将使用 EM 比较细胞水平的结构变化。沃基博士的小组还将评估神经结构和功能损伤。 (2)我们将设计一种新型LV，它可以指导人类AC的表达以及细胞命运控制盒，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 HSC 和 HPC 将被注入完全消融的 Farber 小鼠中。将随时间跟踪动物，并按照目标 1 分析法伯病的表现。