Inclusion of the hepatic locus control region, an Intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro

Abstract

We systematically compared human factor IX gene expression from a variety of plasm ids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection into the livers of mice. Although there was a 1.5-to 2.0-fold variation in gene expression from cultured cells, a 65-fold variation was observed in the in vivo studies. We found that a plasm id containing the apolipoprotein E locus control region (HCR), human α1-antitrypsin (hAAT) promoter, hFIX minigene (hFlXmg) sequence including a portion of the first intron (intron A), 3′-untranslated region (3′-UTR), and a bovine growth hormone polyadenylation signal (bpA) produced the highest serum level of human factor IX, reaching 18 μg/ml (normal = 5 μg/ml) 1 day after injection. Although most of the plasm id DNAs resulted in transient gene expression, inclusion of an intron, a polyadenylation signal from either the 1.7-kb 3′UTR or the 0.3-kb bpA, and the HCR resulted in persistent and therapeutic levels of hRX gene expression, ranging from 0.5 to 2 μg/ml (10 to 40% of normal) for 225 days (length of experiment). These data underscore the importance of cis sequences for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.