Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes.
Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes.
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DOI：
10.1182/blood.v84.10.3394.bloodjournal84103394
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发表时间：
1994-11
期刊：
影响因子：
20.3
通讯作者：
D. Hafenrichter;X. Wu;S. Rettinger;S. Kennedy;M. Flye;K. Ponder
中科院分区：
文献类型：
--
作者：
D. Hafenrichter;X. Wu;S. Rettinger;S. Kennedy;M. Flye;K. Ponder
肝脏基因治疗可用于治疗许多遗传性血液病，如血友病或血栓形成。尽管肝脏定向逆转录病毒转导可以导致体内基因的长期表达，但蛋白质产量低限制了其临床应用。我们推断，在逆转录病毒载体中插入肝脏特异性启动子将增加体内基因表达。将347-bp的人α 1-抗胰蛋白酶（hAAT）、810-bp的鼠白蛋白（mAIb）、490-bp的大鼠磷酸烯醇式丙酮酸羧激酶（rPECK）和596-bp的大鼠肝脂肪酸结合蛋白启动子插入到含有hAAT报告基因的莫洛尼鼠白血病逆转录病毒骨架中。通过转导再生的大鼠肝脏在体内测试在体外产生适当大小的RNA和hAAT蛋白的载体。将hAAT报告基因的长期血清表达标准化为逆转录病毒转导效率，如通过使用来自转导的大鼠肝脏的基因组DNA的基于聚合酶链反应的测定所确定的。的hAAT，mAIb，和rPEPCK启动子，分别为35-，8-和0.02倍，如先前研究的组成型Pol-II启动子强。我们得出结论，hAAT启动子导致从逆转录病毒载体中的最高表达，并可能导致其他临床显著的血液蛋白的治疗显著的表达。
Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347-bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596-bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35-, 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.