GENE THERAPY OF BLOOD DISEASES

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The long range goal of this project is to investigate the potential application and determine the safety of retroviral vector mediated gene transfer for treatment of blood diseases using a canine model of hemolytic anemia due to pyruvate kinase (PK) deficiency. The first major goal of this project is to develop a retroviral transduction/bone marrow culture protocol for efficient expression of exogenous genes in hematopoietic stem cells. A retroviral vector expressing the human multidrug resistance (MDR) gene will be used in these studies. To accomplish this goal bone marrow cells will be incubated with hematopoietic growth factors (IL-1, IL-3, IL- 6, IL-11, G-CSF, stem cell factor) to activate the hematopoietic stem cell from Go into the cell cycle. Cells will then be transduced using co- cultivation, supernatant and long-term bone marrow culture transduction protocols. In additional experiments animals will be treated with 5- fluorouracil (250 mg/m2) or sublethal irradiation (2.5 Gy) before obtaining bone marrow for retroviral transduction to activate the hematopoietic stem cell. The efficiency of retroviral transduction will be determined using an in vitro colony forming unit assay and by polymerase chain reaction analysis of DNA. Bone marrow reconstitution and expression of the MDR gene in vivo will also be used as the endpoint to determine the most efficient retroviral transduction protocol because that is presently the only reliable assay for the canine hematopoietic stem cell. The second major goal is to use a retroviral vector containing the canine R-type PK cDNA in the most efficient retroviral transduction protocol determined in the initial experiments to investigate gene therapy of PK deficiency. Standard hematologic parameters (CBC, RBC morphology, reticulocyte count, RBC t1/2) and PK enzyme levels will be used to evaluate PK deficient dogs treated with gene therapy. Individual animals will be closely monitored for spontaneous disease and periodically checked for helper virus to evaluate the safety of gene therapy in the treatment of blood diseases. The unique strengths of this proposal, not available elsewhere are the ability to investigate the efficacy and safety of gene therapy in a large animal red blood cell disease model using bone marrow culture/retroviral transduction protocols similar to that expected to be used in human clinical trials. The proposed studies will provide important new information on the applicability of gene therapy to treatment of blood diseases without human risk or suffering.