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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Exploratory/Developmental Grants (R21)
Project #
7R21DK077806-03
Application #
8201617
Study Section
Special Emphasis Panel (ZRG1-GTIE-A (01))
Program Officer
Wright, Daniel G
Project Start
2008-05-01
Project End
2012-04-30
Budget Start
2010-09-01
Budget End
2012-04-30
Support Year
3
Fiscal Year
2009
Total Cost
$162,788
Indirect Cost
Name
Washington State University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Jackson, Melissa F; Hoversten, Knut E; Powers, John M et al. (2013) Genetic manipulation of myoblasts and a novel primary myosatellite cell culture system: comparing and optimizing approaches. FEBS J 280:827-39
Trobridge, Grant D; Horn, Peter A; Beard, Brian C et al. (2012) Large animal models for foamy virus vector gene therapy. Viruses 4:3572-88
Trobridge, Grant D (2011) Genotoxicity of retroviral hematopoietic stem cell gene therapy. Expert Opin Biol Ther 11:581-93
Trobridge, Grant D; Wu, Robert A; Hansen, Michael et al. (2010) Cocal-pseudotyped lentiviral vectors resist inactivation by human serum and efficiently transduce primate hematopoietic repopulating cells. Mol Ther 18:725-33
Beard, Brian C; Trobridge, Grant D; Ironside, Christina et al. (2010) Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates. J Clin Invest 120:2345-54
Trobridge, G D; Kiem, H-P (2010) Large animal models of hematopoietic stem cell gene therapy. Gene Ther 17:939-48
Kiem, H-P; Wu, R A; Sun, G et al. (2010) Foamy combinatorial anti-HIV vectors with MGMTP140K potently inhibit HIV-1 and SHIV replication and mediate selection in vivo. Gene Ther 17:37-49
Kiem, Hans-Peter; Ironside, Christina; Beard, Brian C et al. (2010) A retroviral vector common integration site between leupaxin and zinc finger protein 91 (ZFP91) observed in baboon hematopoietic repopulating cells. Exp Hematol 38:819-22, 822.e1-3
Trobridge, Grant D (2009) Foamy virus vectors for gene transfer. Expert Opin Biol Ther 9:1427-36
Trobridge, Grant D; Allen, James; Peterson, Laura et al. (2009) Foamy and lentiviral vectors transduce canine long-term repopulating cells at similar efficiency. Hum Gene Ther 20:519-23