title: Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes.
abstract: 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.
authors.affiliation: Department of Surgery, Washington University School of Medicine, St Louis, MO.
authors.forename: D G
authors.initials: DG
authors.lastname: Hafenrichter
authors.forename: X
authors.initials: X
authors.lastname: Wu
authors.forename: S D
authors.initials: SD
authors.lastname: Rettinger
authors.forename: S C
authors.initials: SC
authors.lastname: Kennedy
authors.forename: M W
authors.initials: MW
authors.lastname: Flye
authors.forename: K P
authors.initials: KP
authors.lastname: Ponder
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D012709:Serum Albumin / Q000096:biosynthesis / Q000235:genetics; D014161:Transduction, Genetic; D014162:Transfection / Q000379:methods*; D025521:Tumor Suppressor Proteins; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C107608:FABP7 protein, human; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D012709:Serum Albumin; D025521:Tumor Suppressor Proteins; D000515:alpha 1-Antitrypsin; D010729:Phosphoenolpyruvate Carboxykinase (GTP)