Transgenic rescue of hemolytic anemia due to red blood cell pyruvate kinase deficiency

From the Department of Transfusion Medicine and Cell Processing, Tokyo Women’s Medical University, Tokyo, Japan (HK, TU, HO, HF); Institute of Medical Genetics, Tokyo Women’s Medical University, Tokyo, Japan (HK, TH); Division of Genomic Medicine, Department of Advanced Biomedical Engineering and Science, Graduate School of Medicine, Tokyo Women’s Medical University (HK); Department of Anatomy, Nihon University School of Medicine, Tokyo, Japan (SA); Division of Laboratory Animal Resources, Centers for Advanced Research Support, University of Fukui, Fukui, Japan (TK); Cellular & Molecular Toxicology Division, National Institute of Health Sciences, Tokyo, Japan (K-IA) Correspondence: Hitoshi Kanno, M.D. Ph.D., Department of Transfusion Medicine and Cell Processing, Tokyo Women’s Medical University, Tokyo 162-8666, Japan. E-mail: hikanno{at}clabo.twmu.ac.jp Background and Objectives: Red blood cell pyruvate kinase (R-PK) deficiency is the most common glycolytic enzyme defect associated with hereditary non-spherocytic hemolytic anemia. Cases with the most severe deficiency die in the peri- or neonatal period and no specific therapy exists at present. To test whether the targeted overexpression of the normal R-PK gene in erythroid cells could reduce hemolysis in R-PK mutant mice, we performed a genetic rescue study using human R-PK transgenic mice. Design and Methods: Human R-PK promoter driven with human µLCR of the human ß-globin locus was used for the erythroid-specific expression of human R-PK in murine erythrocytes. The transgenic lines were mated with homozygous R-PK mutant mice and subsequently back-crossed. Mutant homozygotes with the µLCR-R-PK transgene were examined for any therapeutic effects of transgene expression. Results: Two PK transgenic lines, hRPK_lo and hRPK_hi, were obtained. R-PK activity of the transgenic mice reached as high as three times that of the animals with the endogenous PK gene. Overexpression of human R-PK in the homozygous mutant mice successfully reduced hemolytic anemia. Improvements of hemolysis were evaluated by hemoglobin concentration, reticulocyte count, and spleen weight, which showed significant correlations with the levels of expression of the transgene. Recovery from metabolic disturbance in mutant red blood cells was shown as normalized concentrations of the glycolytic intermediates upstream of PK. In addition, there was a remarkable negative correlation between R-PK activity and the number of TUNEL-positive erythr