Hematopoietic Stem Cell Gene Therapy for Friedreich's ataxia

项目摘要 弗里德赖希共济失调（FRDA）是一种多系统常染色体隐性遗传疾病， 主要是由一个纯合的GAA重复扩增突变内， 第一内含子的共济失调蛋白（FXN）基因，导致其表达的减少。Frataxin是 一种与铁代谢有关的线粒体蛋白质。FRDA的特征是共济失调， 神经变性、肌无力和心肌病。没有治疗方法 这种致命的疾病。我们测试了一种治疗这种疾病的新疗法，包括野生型（WT） 造血干细胞和祖细胞（HSPC）在Y8 GR小鼠中的移植 FRDA模型该模型仅表达突变的人FXN转基因， 从而模拟了FRDA患者和临床上 表型。使用此策略的前提来自我们之前的数据， 胱氨酸病，一种多系统性溶酶体贮积症，经HSPC挽救 通过HSPC分化为组织内的巨噬细胞进行移植， 携带胱氨酸的溶酶体通过隧道纳米管（TNTs）转移到邻近的 病变细胞TNT也可以转移线粒体，因此我们假设这是一种 战略也可以处理FRDA。这种疗法的效果大大超出了我们的预期， FRDA作为神经、肌肉和心脏并发症得到完全纠正 移植后7个月（最后检测时间点），单次输注 致死性辐照的Y8 GR小鼠中的HSPC。鉴于发病率和死亡率的高风险 与异基因HSPC移植相关，我们的目标是开发一种 FRDA的自体HSPC基因治疗方法。因为过度表达 Frataxin是有毒的，我们将测试两种不同的方法来离体基因校正和恢复 基因在HSPC中的生理表达。一个策略是引入 HSPCs是在其短形式控制下的人FXN（hFXN）cDNA。 使用慢病毒载体在内源性启动子中表达。由于大多数患者携带GAA 第二种方法是使用CRISPR/Cas9介导的基因， 编辑方法来去除HSPC中的这种突变。人和鼠FRDA HSPC 将用于最后，我们还将确定HSPC移植逆转 既存并发症这项工作代表了第一个自体基因校正 FRDA的HSPC移植治疗策略，并为 这一策略的临床应用。

Project Summary Friedreich’s ataxia (FRDA) is a multi-systemic autosomal recessive disorder that is predominantly caused by an homozygous GAA repeat expansion mutation within the first intron of the frataxin (FXN) gene leading to a decrease of its expression. Frataxin is a mitochondrial protein involved in iron metabolism. FRDA is characterized by ataxia, neurodegeneration, muscle weakness, and cardiomyopathy. There is no treatment for this lethal disease. We tested a new therapy for this disease consisting in wildtype (WT) hematopoietic stem and progenitor cell (HSPC) transplantation in the Y8GR mouse model of FRDA. This model expresses exclusively the mutated human FXN transgene, thus mimicking the transcriptional deficiency seen in FRDA patients and the clinical phenotype. The premise for using this strategy came from our previous data on cystinosis, a multi-systemic lysosomal storage disorder, which was rescued by HSPC transplantation via differentiation of the HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes via tunneling nanotubes (TNTs) to the adjacent diseased cells. TNTs can also transfer mitochondria, thus we hypothesized that this strategy could also treat FRDA. This therapy worked quite beyond our expectation in FRDA as the neurologic, muscular and cardiac complications were completely corrected up to 7 months post-transplantation (latest time point tested) after a single infusion of HSPCs in lethally irradiated Y8GR mice. Given the high risk of morbidity and mortality associated with allogeneic HSPC transplantation, our objective is to develop an autologous HSPC gene therapy approach for FRDA. Because overexpression of the frataxin is toxic, we will test two different approaches to ex vivo gene-correct and restore a physiologic expression of the gene in the HSPCs. One strategy will be to introduce in HSPCs the human FXN (hFXN) cDNA under the control of a short form of its endogenous promoter using a lentivirus vector. As most of the patients carry a GAA repeat expansion, the second approach will be to use a CRISPR/Cas9-mediated gene editing approach to remove this mutation in HSPCs. Human and murine FRDA HSPCs will be used. Finally, we will also determine the ability of HSPC transplantation to reverse preexisting complications. This work represents the first autologous gene-corrected HSPC transplantation treatment strategy for FRDA and builds the foundation for a clinical application of this strategy.