Hematopoietic stem cell-based therapy for Friedrich Ataxia

基于造血干细胞的弗里德里希共济失调疗法

基本信息

批准号：
8807433
负责人：
Stephanie Cherqui
金额：
$ 19.38万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8807433
关键词：
15 year old Aconitate Hydratase Adult Affect Afferent Neurons Animal Model Animals Ataxia Backcrossings Biochemical Biological Assay Bone Marrow Bone Marrow Stem Cell Bone Tissue Breeding Cardiomyopathies Cell Separation Cell Therapy Cells Color Confocal Microscopy Cystine Cystinosis Cytoplasm Defect Diabetes Mellitus Disease Disease model DsRed Engraftment Exhibits Fibroblasts Flow Cytometry Friedreich Ataxia Genes Heart Hematopoietic Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Hereditary Disease Human Human Pathology In Vitro Integral Membrane Protein Introns Investigation Iron Laboratories Lead Life Lysosomes Manuscripts Measures Microtubules Mitochondria Mitochondrial Diseases Mitochondrial Proteins Modeling Motor Activity Multipotent Bone Marrow Stem Cell Mus Muscle Weakness Mutate Mutation Myocardium Nanotubes Nerve Degeneration Nervous system structure Neuraxis Organ Organelles Outcome Oxidative Stress Pathology Patients Phagocytes Phenotype Proteins Reporter Genes Sensory Skeletal Muscle Spinal Ganglia Stem cell transplant Stem cells Symptoms Systemic disease Testing Therapeutic Therapeutic Effect Tissue Preservation Tissues Transgenic Mice Transgenic Model Transplantation Vacuole Weight Wheelchairs Work base behavior test cellular transduction enhanced green fluorescent protein expectation frataxin gene delivery system hearing impairment in vivo insight iron metabolism macrophage migration mouse model neurobehavioral novel therapeutic intervention optic nerve disorder pre-clinical prevent public health relevance stem stem cell therapy therapeutic development treatment strategy

项目摘要

DESCRIPTION (provided by applicant): Friedreich's ataxia (FRDA) is a multi-systemic autosomal recessive disorder that is predominantly caused by a homozygous GAA repeat expansion mutation within intron 1 of the frataxin gene (FXN) leading to a decrease of its expression. FXN is a mitochondrial protein involved in iron metabolism. FRDA is characterized by ataxia, neurodegeneration, muscle weakness, and cardiomyopathy. There is no cure for this lethal disease. We are proposing to treat FRDA by hematopoietic stem and progenitor cell (HSPC) transplantation. This hypothesize is based on our previous work on cystinosis, which is a lysosomal storage disorder leading to multi-organ degeneration. Using the mouse model for cystinosis, we showed that transplantation of wild-type HSPC resulted in abundant bone marrow-derived tissue engraftment, robust tissue cystine reductions and long-term tissue preservation. One of the mechanisms underlying this surprising effect involves the differentiation of HSPCs into macrophages that provides "healthy lysosomes" carrying the functional protein cystinosin to the host disease cells via tunneling nanotubes (TNTs). Mitochondria can also be transferred via TNTs. Therefore, we believe that HSPC transplantation will allow the delivery of "healthy" mitochondria bearing fxn to the damaged tissues and will represent a life-long therapy that will prevent the long-term complications associated with FRDA. As a model for FRDA, we will use the YG8R mouse model, which is currently considered the best animal model of FRDA as it expresses only the human mutated frataxin containing GAA repeats, without endogenous murine frataxin, and develop symptoms similar to the human pathology. In Specific aim 1, we propose to test the therapeutic impact of HSPC transplantation in the YG8R mice. HSPCs will be isolated from eGFP-transgenic mice, so the cells can be tracked after transplantation, and will be transplanted in lethally irradiated YG8R mice at 2 month-old. The first objective will be t verify if bone marrow-derived cells engraft in the affected tissues, especially within the central nervous system, heart and skeletal muscle, and to characterize their phenotype. The second objective will be to evaluate the impact of HSPC transplantation on the mouse phenotype by behavioral testing, and histological and biochemical analyses. In Specific aim 2, we will investigate mitochondrial cross-correction in the context of FRDA in vitro and in vivo. We will generate the DsRed mtGFP-Tg transgenic mice expressing ubiquitously the DsRed reporter gene in the cytoplasm and eGFP in mitochondria by breeding available transgenic mice. HSPCs will be isolated from these mice and transplanted into YG8R mice. Tissues will be analyzed by confocal microscopy analysis to visualize if a transfer of eGFP-expressing mitochondria occurs from the DsRed bone marrow-derived cells to the host cells. Macrophages and fibroblasts will be isolated from the DsRed mtGFP-Tg mice and the YG8R, respectively, to study mitochondrial transfer in vitro and the resulting cellular and phenotypic outcomes. This work has the potential to lead to a new treatment for FRDA and be a proof of concept for other mitochondrial disorders.

描述（由申请人提供）：Friedreich的共济失调（FRDA）是一种多系统性常染色体隐性疾病，主要是由Frataxin基因（FXN）内含子1中的纯合GAA重复扩张突变引起的，导致其表达降低。 FXN是参与铁代谢的线粒体蛋白。 FRDA的特征是共济失调，神经变性，肌肉无力和心肌病。这种致命疾病无法治愈。我们提议通过造血茎和祖细胞（HSPC）移植来治疗FRDA。该假设是基于我们先前关于囊肿性的工作，这是一种导致多器官变性的溶酶体储存障碍。使用小鼠模型进行囊肿性，我们表明野生型HSPC的移植导致丰富的骨髓衍生的组织植入，可靠的组织囊蛋白减少和长期的组织保存。这种令人惊讶的效果的基础机制之一是将HSPC分化为巨噬细胞，该巨噬细胞通过隧穿纳米管（TNT）将功能性蛋白质塞甲撒素的“健康溶酶体”分化为宿主病细胞。线粒体也可以通过TNT转移。因此，我们认为HSPC的移植将允许将轴承FXN的“健康”线粒体递送到受损的组织中，并代表一种终身治疗，可防止与FRDA相关的长期并发症。作为FRDA的模型，我们将使用YG8R小鼠模型，该模型目前被认为是FRDA的最佳动物模型，因为它仅表达了含有GAA重复序列的人类突变的Frataxin，而无需内源性鼠Frataxin，并且会产生与人类病理类似的症状。在特定目标1中，我们建议测试HSPC移植在YG8R小鼠中的治疗影响。 HSPC将从EGFP-转基因小鼠中分离出来，因此可以在移植后跟踪细胞，并将在2个月大的儿童辐照的YG8R小鼠中移植。第一个目标是验证骨髓衍生的细胞是否植入受影响的组织中，尤其是在中枢神经系统，心脏和骨骼肌中，并表征其表型。第二个目标是通过行为测试以及组织学和生化分析来评估HSPC移植对小鼠表型的影响。在特定的目标2中，我们将在FRDA的体外和体内研究线粒体交叉校正。我们将通过繁殖可用的转基因小鼠，在细胞质中生成DSRED MTGFP-TG转基因小鼠在细胞质中无处不在的DSRED报告基因和线粒体EGFP。 HSPC将从这些小鼠中分离出来，并将其移植到YG8R小鼠中。将通过共聚焦显微镜分析来分析组织，以形象化表达EGFP的线粒体是否从DSRED骨髓衍生的细胞转移到宿主细胞。巨噬细胞和成纤维细胞将分别从DSRED MTGFP-TG小鼠和YG8R中分离出来，以在体外研究线粒体转移以及所得的细胞和表型结果。这项工作有可能导致FRDA的新治疗方法，并成为其他线粒体疾病的概念证明。