Abstract
Human erythrocyte R-type pyruvate kinase deficiency (PKD) is an autosomal recessive disorder produced by mutations in the PKLR gene. Haemolytic anaemia is the major symptom of the disease. A severe deficiency in PKD causes ATP depletion in the RBC metabolism, which ultimately leads to haemolysis that may require periodical blood transfusion, splenectomy, and in some cases bone marrow transplantation. These clinical features make this disease a good candidate for gene therapy. With this aim, we have developed different gammaretroviral and lentiviral vectors expressing the human RPK and have characterized the functionality of the erythroid specific expression regulatory sequences of the human RPK gene in a lentiviral backbone. The transduction of mouse hematopoietic stem cells from a mouse strain deficient in the pklr gene [AcB55: pklr269A/269A mice], which mainly resembles the human RPK deficiency, with a retroviral vector expressing the human RPK, followed by the transplantation into irradiated syngenic recipients completely recovered the red cell parameters in peripheral blood, spleen and bone marrow. Also, intracellular values of ATP, plasmatic iron and circulating erythropoietin levels were recovered to normal values. After 100 days of transplantation, treated mice did not show any clinical symptom of the disease. Secondary pklr269A/269A recipients were also transplanted and their hematological symptoms were also reverted, demonstrating the stable therapeutic efficacy of the vector. To specifically express the RPK in the erythroid lineage, a lentiviral vector expressing the EGFP marker gene under the above RPK promoter (LVpRPKEG) was constructed to test its specificity. EGFP expression was detected in erythroid, but not in non-erythroid cell lines, transduced with this vector. Human CD34+ cells were also transduced with the LVpRPKEG vector and transplanted into NOD/SCID mice. Forty days post-transplantation human bone marrow cells were obtained and seeded in semisolid media. Significantly, only human erythroid colonies expressed the EGFP protein, demonstrating the efficacy of the RPK promoter to specifically express proteins in the erythroid lineage. Experiments using a lentiviral vector expressing the human RPK gene under the control of the human RPK promoter are now in progress. Overall, the transfer of lentiviral vectors harbouring the hRPK cDNA driven by its own promoter in PKLR mutated hematopoietic stem cells could represent an efficient therapeutic treatment of severe clinical cases of human erythrocyte PKD.
345. Gene Therapy for Wiskott-Aldrich Syndrome Using Lentiviral Vectors: Evidence for Efficacy and Safety after Transduction of Human T Cells and Hematopoietic Stem Cells
