Liver-directed gene therapy: quantitative evaluation of promoter elements by using in vivo retroviral transduction.

Liver-directed gene therapy will be applicable to many inherited diseases. Although various protocols have been devised for in vivo delivery of retrovirus, comparison of hepatocyte transduction frequencies has been difficult due to variations in retroviral titer and a paucity of DNA data. We have previously reported an in vivo rat hepatocyte transduction technique which involves 70% hepatectomy followed 24 hr later by portal vein injection of retrovirus during hepatic in-flow occlusion. In this study, we employed this method and concentrated retroviral preparations to achieve transduction of up to 15% of hepatocytes as determined by a quantitative PCR assay. As an initial step toward identifying promoters which lead to high-level long-term expression of retroviral transduced genes, we used our in vivo delivery system to compare the Moloney murine leukemia virus long terminal repeat (LTR) promoter with the promoter for the large subunit of murine RNA polymerase II (Pol-II). Human alpha 1-antitrypsin (hAAT) was used as the reporter gene to facilitate long-term analysis of expression. Serum hAAT levels were higher for the Pol-II promoter (143 ng/ml) than for the LTR promoter (50 ng/ml). This difference was consistent with the higher transduction frequency observed for the Pol-II-hAAT vector. Although serum hAAT expression was sustained for up to 1 year in six of eight Pol-II-hAAT-transduced rats and three of five LTR-hAAT-transduced rats and was proportional to hAAT mRNA level and proviral DNA frequency, in vivo expression was significantly lower than in transduced tissue culture cells. We conclude that a high frequency of in vivo transduction can be achieved by using retroviral vectors and our rapid transduction protocol, but transduced gene expression remains a serious problem. The quantitative assays described herein will facilitate in vivo comparisons of gene regulatory elements.