Hematopoietic stem cell-based therapy for Friedrich Ataxia

描述（由申请人提供）：弗里德赖希共济失调症（FRDA）是一种多系统常染色体隐性遗传病，主要由frataxin基因（FXN）内含子1内纯合子GAA重复扩增突变导致其表达减少引起。FXN是一种参与铁代谢的线粒体蛋白。FRDA的特点是共济失调、神经退行性变、肌肉无力和心肌病。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之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贮积紊乱。通过小鼠胱氨酸病模型，我们发现野生型HSPC移植导致大量骨髓来源的组织植入，组织胱氨酸大量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HSPCs向巨噬细胞的分化，巨噬细胞通过隧道纳米管（TNTs）向宿主疾病细胞提供“健康溶酶体”，携带功能性蛋白质胱氨酸蛋白。线粒体也可以通过tnt转移。因此，我们相信HSPC移植将允许携带fxn的“健康”线粒体输送到受损组织，并将代表一种终身治疗，将预防与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，该模型目前被认为是最好的FRDA动物模型，因为它只表达含有GAA重复序列的人类突变frataxin，不含内源性小鼠frataxin，并且出现类似于人类病理的症状。在Specific aim 1中，我们提议测试HSPC移植对YG8R小鼠的治疗作用。HSPCs将从egfp转基因小鼠中分离出来，以便在移植后可以追踪细胞，并将在2月龄时移植到致命辐照的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，特别是在中枢神经系统、心脏和骨骼肌中，并表征它们的表型。第二个目标将是通过行为测试、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。我们将通过培育现有的转基因小鼠，产生细胞质中普遍表达DsRed报告基因和线粒体中普遍表达eGFP的DsRed mtGFP-Tg转基因小鼠。从这些小鼠中分离HSPCs并移植到YG8R小鼠中。组织将通过共聚焦显微镜分析来观察表达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YG8R中分离巨噬细胞和成纤维细胞，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带来一种新的治疗方法，并为其他线粒体疾病的概念提供证据。

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.