Liver-Directed Gene Therapy: A Retroviral Vector with a Complete LTR and the ApoE Enhancer-α1-Antitrypsin Promoter Dramatically Increases Expression of Humanα1-AntitrypsinIn Vivo

Abstract

Hepatic gene therapy could improve the treatment of many inherited disorders. Although retroviral vectors result in long-term expression in hepatocytes in vivo, their low level of expression currently precludes most clinical applications. Four copies of the liver-specific apolipoprotein E (ApoE) enhancer were placed upstream of the human α₁-antitrypsin (hAAT) promoter in either orientation into a retroviral vector with a complete long terminal repeat (LTR) and the hAAT cDNA to generate ApoE(+)hAAT-LTR and ApoE(–)hAAT-LTR. In addition, the ApoAI promoter was placed upstream of the hAAT cDNA in a similar retroviral vector backbone. Amphotropic retroviral vectors were transferred into regenerating rat liver cells in vivo by intraportal injection. ApoE(–)hAAT-LTR and ApoE(+)hAAT-LTR led to average hAAT levels of 5 μg/ml (0.5% of normal levels of a very abundant protein), and 2.5 μg/ml, respectively, which was stable for at least 10 months after transduction. This level of serum hAAT was >25-fold higher than what was observed from the ApoAI promoter used in this study. Serum levels of hAAT were >15-fold higher than what was observed from retroviral vectors containing the hAAT cDNA that were analyzed previously by this lab. In some cases, improved expression was due to the promoter chosen. In other cases, the increase in expression was primarily due to the higher titers obtained by using a retroviral backbone with an intact LTR as opposed to a vector with a deletion in the LTR. The increased expression levels observed from this enhancer/promoter combination in an intact retroviral backbone may enable one to achieve therapeutic levels of clinically important genes from a retroviral vector in liver cells of animals. Low levels of expression from retroviral vectors in vivo are a limiting factor in the successful application of gene therapy to the treatment of genetic disorders. Three retroviral vectors containing liver-specific transcriptional elements were tested for their ability to direct expression of the serum protein human α₁-antitrypsin (hAAT) gene from rat liver cells. The apolipoprotein E enhancer-hAAT promoter combination resulted in high-level expression of the hAAT reporter gene in vivo when placed into a retroviral vector with an intact LTR. The level of serum hAAT observed was > 15-fold higher than what was observed with all previously tested retroviral vectors. This retroviral vector may significantly improve the overall levels of expression of other clinically important genes from retroviral vectors that have been transferred into liver cells by using ex vivo or in vivo techniques.