Despite significant efforts to develop gene therapy strategies for erythroid diseases, several roadblocks havelimited 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 acritical concern for gene therapy. Here we propose to overcome these problems by developing a HSC genetherapy for pyruvate kinase (PK) deficiency using foamy retrovirus vectors, and by developing means to targetintegration of foamy vectors using polydactyl zinc finger proteins. We have recently demonstrated efficientgene 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 thatfoamy vectors may be effective for HSC gene therapy for erythroid diseases, and that the Basenji PK-deficientcanine model should be an excellent preclinical model to evaluate their potential. We have incorporatederythroid-specific promoters into foamy vectors and will compare transgene expression levels in vitro towardsdeveloping effective erythroid-specific foamy vectors. We will also explore means to target the integration offoamy vectors using polydactyl zinc finger DNA binding proteins. Finally, we will attempt to cure the Basenjidog 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 likelybe required to observe a therapeutic effect. The Basenji canine PK model is thus ideal to evaluate in vivoselection 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 thelevel of marking post-transplantation to increase our chances of curing PK-deficiency. Unlike mouse modelsthe canine model has been predictive of HSC transplantation outcomes in the clinical setting and of genetransfer levels in humans. These data should thus be directly translatable to future clinical studies to treat PKdeficiency and for other erythroid diseases. Project Narrative The proposed research will evaluate the efficacy and safety of gene therapy for hematopoietic (blood)stem cells using a novel gene delivery vehicle, foamy retroviral vectors. These studies will be performed in ahighly relevant canine model of a human genetic disease of red blood cells, pyruvate kinase deficiency. Thisresearch may lead to more effective and safer therapies for pyruvate kinase deficiency and for other diseasesof red blood cells including thalassemias.
