Hematopoietic Stem Cell Gene Therapy for Friedreich's ataxia

项目摘要 Friedreich‘s共济失调(FRDA)是一种多系统常染色体隐性遗传性疾病 主要由GAA重复序列内的纯合扩增突变引起 Frataxin(FXN)基因的第一个内含子导致其表达下降。Frataxin是 一种与铁代谢有关的线粒体蛋白质。FRDA的特点是共济失调， 神经变性、肌肉无力和心肌病。目前尚无治疗方法。 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) Y8GR小鼠的造血干/祖细胞移植 FRDA的模型。这个模型只表达突变的人FXN转基因， 从而模拟了FRDA患者和临床患者的转录缺陷 表型。使用这一策略的前提来自于我们之前关于 一种多系统溶酶体储存障碍，被HSPC抢救 通过将HSPC分化为组织内巨噬细胞进行移植 通过隧道纳米管(TNTs)将携带半胱氨酸氨基转移酶的溶酶体转移到相邻的 病态细胞。TNTs也可以转移线粒体，因此我们假设这 战略也可以治疗FRDA。这种疗法的效果完全出乎我们的预料。 FRDA作为神经、肌肉和心脏并发症被完全纠正 移植后长达7个月(最新时间点测试)：单次输注 致死剂量照射的Y8GR小鼠的HSPC。鉴于发病率和死亡率很高的风险 与同种异体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。因为过度表达了 Frataxin是有毒的，我们将测试两种不同的体外基因纠正和恢复方法 该基因在HSPC中的生理表达。一种战略将是引入 人FXN(HFXN)基因在ITS短型调控下的表达 使用慢病毒载体的内源性启动子。因为大多数患者都携带GAA 重复扩增，第二种方法将使用CRISPR/Cas9介导的基因 在HSPC中删除这种突变的编辑方法。人和小鼠FRDA HSPC 将会被使用。最后，我们还将测定HSPC移植逆转的能力 先前存在的并发症。这项工作是第一个自体基因矫正的工作。 HSPC移植治疗FRDA的策略和基础 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.