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

点击翻译按钮获取中文摘要

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.