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

• 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
• 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

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）：弗里德赖希共济失调（FRDA）是一种多系统性常染色体隐性遗传病，主要是由 frataxin 基因（FXN）内含子 1 内的纯合 GAA 重复扩增突变导致其表达降低引起的。 FXN 是一种参与铁代谢的线粒体蛋白。 FRDA 的特点是共济失调、神经变性、肌肉无力和心肌病。这种致命疾病无法治愈。我们建议通过造血干细胞和祖细胞（HSPC）移植来治疗 FRDA。这一假设基于我们之前对胱氨酸沉积症的研究，胱氨酸沉积症是一种导致多器官变性的溶酶体贮积症。使用胱氨酸中毒小鼠模型，我们发现野生型 HSPC 的移植可导致丰富的骨髓源性组织植入、组织胱氨酸的强烈减少和长期组织保存。这种令人惊讶的作用背后的机制之一涉及 HSPC 分化为巨噬细胞，巨噬细胞提供“健康的溶酶体”，通过隧道纳米管 (TNT) 将功能蛋白胱氨酸蛋白携带至宿主疾病细胞。线粒体也可以通过 TNT 转移。因此，我们相信 HSPC 移植将允许将携带 fxn 的“健康”线粒体输送到受损组织，并将代表一种终生治疗，可预防与 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，该模型被认为是目前最好的 FRDA 动物模型，因为它仅表达含有 GAA 重复序列的人类突变 frataxin，而没有内源性小鼠 frataxin，并且出现与人类病理学相似的症状。在具体目标 1 中，我们建议测试 HSPC 移植对 YG8R 小鼠的治疗效果。 HSPC将从eGFP转基因小鼠中分离出来，因此可以在移植后追踪细胞，并将在2个月大时移植到经过致死照射的YG8R小鼠中。第一个目标是验证骨髓来源的细胞是否移植到受影响的组织中，特别是中枢神经系统、心脏和骨骼肌中，并表征它们的表型。第二个目标是通过行为测试、组织学和生化分析来评估 HSPC 移植对小鼠表型的影响。在具体目标 2 中，我们将在体外和体内研究 FRDA 背景下的线粒体交叉校正。我们将通过培育可用的转基因小鼠，产生在细胞质中普遍表达 DsRed 报告基因和在线粒体中表达 eGFP 的 DsRed mtGFP-Tg 转基因小鼠。 HSPC将从这些小鼠中分离出来并移植到YG8R小鼠体内。将通过共聚焦显微镜分析来分析组织，以可视化表达 eGFP 的线粒体是否从 DsRed 骨髓来源的细胞转移到宿主细胞。将分别从 DsRed mtGFP-Tg 小鼠和 YG8R 中分离巨噬细胞和成纤维细胞，以研究体外线粒体转移以及由此产生的细胞和表型结果。这项工作有可能为 FRDA 带来新的治疗方法，并成为其他线粒体疾病的概念证明。