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


METHODS
Human R-PK promoter driven with human mLCR of the human b-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 backcrossed. Mutant homozygotes with the mLCR-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 erythroid progenitors in the spleen.


CONCLUSIONS
These results indicate that overexpression of the wild-type PK gene in mutant erythroid cells ameliorates both erythroid apoptosis and the shortened red blood cell lifespan observed in PK mutant mice. It is likely that the level of transgene expression required to achieve evident therapeutic effects should be equivalent to or more than that of the endogenous PK gene. This gene-addition strategy may be suitable for clinical application if there is a high level of transgene expression of R-PK in erythroid progenitors/red blood cells.
Kanno
Hitoshi
H
Department of Transfusion Medicine and Cell Processing, Tokyo Women's Medical University, Tokyo, Japan. hikanno@clabo.twmu.ac.jp
Utsugisawa
Taiju
T
Aizawa
Shin
S
Koizumi
Tsutomu
T
Aisaki
Ken-Ichi
K
Hamada
Takako
T
Ogura
Hiromi
H
Fujii
Hisaichi
H
D000743:Anemia, Hemolytic / Q000209:etiology / Q000628:therapy*; D000746:Anemia, Hemolytic, Congenital Nonspherocytic / Q000209:etiology / Q000628:therapy; D000818:Animals; D017209:Apoptosis; D004905:Erythrocyte Aging; D004912:Erythrocytes / Q000201:enzymology / Q000473:pathology; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D011770:Pyruvate Kinase / Q000172:deficiency* / Q000235:genetics
D011770:Pyruvate Kinase