Stem Cell Gene Therapy for Pyruvate Kinase Deficiency Using Foamy Vectors

描述（由申请人提供）： 尽管为开发红系疾病的基因治疗策略做出了重大努力，但在该领域中的几个障碍取得了有限的成功，包括在大型动物和人类中重建造血干细胞（HSC）的基因转移效率低下，以及红细胞中转基因表达水平低。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关键问题。在这里，我们建议克服这些问题，通过开发一种HSC基因治疗丙酮酸激酶（PK）缺陷使用泡沫逆转录病毒载体，并通过开发手段，以目标泡沫载体的整合使用多指锌指蛋白。我们最近已经证明了使用基于非致病性泡沫病毒的载体将基因有效地转移到犬长期再生HSC中。在这些研究中，我们观察到相当多的GFP表达的红细胞。这些研究表明，泡沫载体可能是有效的HSC基因治疗红系疾病，Basenji PK缺陷犬模型应该是一个很好的临床前模型，以评估其潜力。我们已经将红系特异性启动子纳入泡沫载体，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。我们还将探索使用多指锌指DNA结合蛋白靶向泡沫载体整合的方法。最后，我们将尝试通过移植自体泡沫转导的CD 34+细胞来治愈Basenji犬PK缺陷症。在Basenji犬中，校正的细胞在体内不会具有显著的选择优势，并且可能需要高水平的基因标记来观察治疗效果。因此，Basenji犬PK模型是评价体内选择策略的理想模型。我们将使用除了PK转基因之外还含有P140 K突变体O 6-甲基鸟嘌呤-DNA-甲基转移酶（MGMT）选择盒的泡沫载体。这将使我们能够提高移植后标记的水平，以增加我们治愈PK缺陷的机会。与小鼠模型不同，犬模型已经预测了临床环境中的HSC移植结果和人类中的基因转移水平。因此，这些数据应可直接转化为未来治疗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.