Hematopoietic stem cell-based therapy for Friedrich Ataxia

描述（由申请方提供）：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疾病，主要由共济失调蛋白基因（FXN）内含子1内的纯合GAA重复扩增突变引起，导致其表达降低。FXN是参与铁代谢的线粒体蛋白。FRDA的特征在于共济失调、神经变性、肌无力和心肌病。这种致命的疾病无法治愈。我们建议通过造血干细胞和祖细胞（HSPC）移植治疗FRDA。这一假设是基于我们以前对胱氨酸病的研究，胱氨酸病是一种导致多器官变性的溶酶体储存疾病。使用胱氨酸病的小鼠模型，我们表明野生型HSPC的移植导致了丰富的骨髓来源的组织植入、稳健的组织胱氨酸减少和长期的组织保存。这种令人惊讶的作用的机制之一涉及HSPC分化成巨噬细胞，巨噬细胞通过隧道纳米管（TNT）向宿主疾病细胞提供携带功能性蛋白质半胱氨酸蛋白的“健康溶酶体”。线粒体也可以通过TNT转移。因此，我们认为HSPC移植将允许将携带fxn的“健康”线粒体递送至受损组织，并且将代表预防与FRDA相关的长期并发症的终身治疗。作为FRDA的模型，我们将使用YG 8 R小鼠模型，其目前被认为是FRDA的最佳动物模型，因为其仅表达含有GAA重复序列的人突变共济失调蛋白，而不表达内源性鼠共济失调蛋白，并产生与人病理学相似的症状。在具体目标1中，我们提出在YG 8 R小鼠中测试HSPC移植的治疗影响。HSPC将从eGFP转基因小鼠中分离，因此可以在移植后跟踪细胞，并将在2月龄时移植到致死性辐照的YG 8 R小鼠中。第一个目标是验证骨髓来源的细胞是否能移植到受影响的组织中，特别是在中枢神经系统、心脏和骨骼肌中，并表征其表型。第二个目的是通过行为测试以及组织学和生化分析来评价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。本研究通过培育转基因小鼠，获得胞质中广泛表达DsRed报告基因，线粒体中广泛表达eGFP的DsRed mtGFP-Tg转基因小鼠。将从这些小鼠中分离HSPC并移植到YG 8 R小鼠中。将通过共聚焦显微镜分析来分析组织，以观察表达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YG 8 R中分离巨噬细胞和成纤维细胞，以研究体外线粒体转移和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。

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.