Transgenic rescue of hemolytic anemia due to red blood cell pyruvate kinase deficiency
Transgenic rescue of hemolytic anemia due to red blood cell pyruvate kinase deficiency
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DOI：
10.3324/haematol.10945
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发表时间：
2007-06-01
期刊：
影响因子：
10.1
通讯作者：
Fujii, Hisaichi
中科院分区：
文献类型：
--
作者：
Kanno, Hitoshi;Utsugisawa, Taiju;Fujii, Hisaichi
背景与目的红细胞丙酮酸激酶（R-PK）缺陷是遗传性非球形红细胞溶血性贫血最常见的糖酵解酶缺陷。缺乏症最严重的病例在婴儿期或新生儿期死亡，目前没有具体的治疗方法。为了测试是否有针对性的过表达的正常R-PK基因在红系细胞中可以减少溶血R-PK突变小鼠，我们进行了遗传救援研究使用人R-PK转基因migrationsDesign和MethodsHuman R-PK启动子驱动的人μ LCR的人β-珠蛋白基因座用于红系特异性表达的人R-PK在小鼠红细胞。将转基因系与纯合R-PK突变小鼠交配，随后回交。突变纯合子与亩LCR-R-PK转基因进行了检查转基因expression.ResultsTwo PK转基因株系，hRPK_lo和hRPK_hi，获得任何治疗效果。转基因小鼠的R-PK活性高达内源性PK基因动物的3倍。在纯合突变小鼠中过表达人R-PK成功地减少了溶血性贫血。通过血红蛋白浓度、网织红细胞计数和脾重量来评价溶血的改善，这些指标与转基因的表达水平显著相关。突变红细胞中代谢紊乱的恢复显示为PK上游糖酵解中间体的标准化浓度。此外，有一个显着的负相关性R-PK活性和TUNEL阳性的红系祖细胞在spleen.Interpretation和ConclusionsThese结果表明，野生型PK基因突变红系细胞中的过表达改善红系细胞凋亡和PK突变小鼠中观察到的红细胞寿命缩短。达到明显治疗效果所需的转基因表达水平可能应等于或高于内源性PK基因的表达水平。如果在红系祖细胞/红细胞中存在高水平的R-PK转基因表达，则该基因添加策略可能适合于临床应用。
Background and ObjectivesRed 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 MethodsHuman R-PK promoter driven with human mu LCR of the human beta-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 mu LCR-R-PK transgene were examined for any therapeutic effects of transgene expression.ResultsTwo 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.Interpretation and ConclusionsThese 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.