Stem Cell Gene Therapy for Pyruvate Kinase Deficiency Using Foamy Vectors

描述(由申请人提供)： 尽管在开发红系疾病的基因治疗策略方面做出了重大努力，但有几个障碍在这一领域取得的成功有限，包括在大型动物和人类中对重新繁殖的造血干细胞(HSCs)的基因转移效率低下，以及转基因在红细胞中的低水平表达。最近，在法国X连锁SCID试验中观察到的导致白血病的插入突变也成为基因治疗的一个关键问题。在这里，我们建议通过开发一种使用泡沫逆转录病毒载体的丙酮酸激酶(PK)缺乏症的HSC基因疗法来克服这些问题，并通过开发使用多指锌指蛋白靶向整合泡沫载体的方法来克服这些问题。我们最近使用基于非致病性泡沫病毒的载体证明了有效的基因转移到犬的长期重新繁殖的HSCs。在这些研究中，我们观察到红细胞中有相当多的绿色荧光蛋白表达。这些研究表明，泡沫载体对红系疾病的HSC基因治疗可能是有效的，Basenji PK缺陷的犬模型应该是评估其潜力的良好的临床前模型。我们已经将红系特异性启动子整合到泡沫载体中，并将在体外比较转基因表达水平，以开发有效的红系特异性泡沫载体。我们还将探索以多指锌指DNA结合蛋白为靶点整合泡沫载体的方法。最后，我们将尝试通过移植自体泡沫转导的CD34+细胞来治愈Basenji犬PK缺乏症。在巴森吉犬身上，校正后的细胞在体内不会有明显的选择优势，很可能需要高水平的基因标记来观察治疗效果。因此，Basenji犬PK模型是评估活体选择策略的理想模型。除了PK转基因外，我们还将使用含有P140K突变体O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)选择盒的泡沫载体。这将允许我们提高移植后的标记水平，以增加我们治愈PK缺乏症的机会。与小鼠模型不同，犬类模型可以在临床环境中预测造血干细胞移植的结果，并预测人类的基因转移水平。因此，这些数据应该可以直接用于未来治疗PK缺乏症和其他红系疾病的临床研究。

DESCRIPTION (provided by applicant): Despite significant efforts to develop gene therapy strategies for erythroid diseases, several roadblocks have limited success in this field including inefficient gene transfer to repopulating hematopoietic stem cells (HSCs) in large animals and humans, and low levels of transgene expression in red blood cells. More recently, insertional mutagenesis leading to leukemia, as observed in the French X-linked SCID trial, has also become a critical concern for gene therapy. Here we propose to overcome these problems by developing a HSC gene therapy for pyruvate kinase (PK) deficiency using foamy retrovirus vectors, and by developing means to target integration of foamy vectors using polydactyl zinc finger proteins. We have recently demonstrated efficient gene transfer to canine long term repopulating HSCs using vectors based on the non-pathogenic foamy virus. In these studies we observed considerable GFP expression in red blood cells. These studies suggest that foamy vectors may be effective for HSC gene therapy for erythroid diseases, and that the Basenji PK-deficient canine model should be an excellent preclinical model to evaluate their potential. We have incorporated erythroid-specific promoters into foamy vectors and will compare transgene expression levels in vitro towards developing effective erythroid-specific foamy vectors. We will also explore means to target the integration of foamy vectors using polydactyl zinc finger DNA binding proteins. Finally, we will attempt to cure the Basenji dog PK-deficiency by transplantation with autologous foamy-transduced CD34+ cells. In the Basenji dog, corrected cells will not have a significant selective advantage in vivo, and high levels of gene marking will likely be required to observe a therapeutic effect. The Basenji canine PK model is thus ideal to evaluate in vivo selection strategies. We will use foamy vectors that contain a P140K mutant O6-methylguanine-DNA- methyltransferase (MGMT) selection cassette in addition to the PK transgene. This will allow us to increase the level of marking post-transplantation to increase our chances of curing PK-deficiency. Unlike mouse models the canine model has been predictive of HSC transplantation outcomes in the clinical setting and of gene transfer levels in humans. These data should thus be directly translatable to future clinical studies to treat PK deficiency and for other erythroid diseases.