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

We systematically compared human factor IX gene expression from a variety o f plasmids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection in to the livers of mice. Although there was a 1.5- to 2.0-fold variation in g ene expression from cultured cells, a 65-fold variation was observed in the In vivo studies. We found that a plasmid containing the apolipoprotein E l ocus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX m inigene (hFIXmg) sequence including a portion of the first intron (intron A ), 3'-untranslated region (3'-UTR), and a bovine growth hormone polyadenyla tion signal (bpA) produced the highest serum level of human factor IX, reac hing 18 mug/ml (normal = 5 mug/ml) 1 day after injection. Although most of the plasmid DNAs resulted in transient gene expression, inclusion of an int ron, a polyadenylation signal from either the 1.7-kb 3'-UTR or the 0.3-kb b pA, and the HCR resulted in persistent and therapeutic levels of hFIX gene expression, ranging from 0.5 to 2 mug/ml (10 to 40% of normal) for 225 days (length of experiment), These data underscore the importance of cis sequen ces for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.