228. Characterization of Hematopoietic Progenitors from Pyruvate Kinase Deficient (PKD) Patients and Transduction of PKD CD34+ Cells with a Therapeutic Lentiviral Vector

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene. PKD is the most common erythroid inherited enzymatic defect causing chronic non-spherocytic hemolytic anemia, high reticulocytosis, acute splenomegaly and intense iron overload in the liver, being life-threatening in severe patients. Up to date allogeneic bone marrow transplant represents the only curative treatment of patients affected by the severe form of the disease (5-10% of PKD patients aprox.). Preclinical gene therapy studies conducted in pyruvate kinase deficient mice have shown the safety and the efficacy of a new PGK-coRPK-Wpre therapeutic lentiviral vector that provided the designation by the European Medicinal Agency (EMA) of this PGK-coRPK-Wpre vector as a new orphan drug (EU/3/14/1130). To continue with the preclinical studies required to develop a clinical trial for PKD we have characterized the hematopoietic progenitor's content and the erythroid progenitors’ profile in peripheral blood (PB) from PKD patients, showing an increase in both types of cells. For transduction studies, CD34+ sorted cells from PKD PB were transduced with PGK-coRPK-Wpre viral vector supernatants. Hematopoietic progenitors’ content and final yield of cells and CFCs was evaluated to analyze the toxicity of the vector. Percentage of transduction ranged from 31% to 100%, showing the efficacy of the transduction protocol and of the PGK-coRPK therapeutic vector. Vector copy number was quantified in both, individual CFUs and CD34+ cells maintained in liquid culture for 14 days, ranging from 0.1 to 3.0 VCN/cells. Additional studies are being conducted to demonstrate PGK-coRPK functionality in human PKD cells.