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 plasmids 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 plasmid containing the apolipoprotein E locus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX minigene (hFIXmg) 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 microg/ml (normal = 5 microg/ml) 1 day after injection. Although most of the plasmid 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 hFIX gene expression, ranging from 0.5 to 2 microg/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.

Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer

“…[1][2][3] In addition, we have previously shown that the combination of the AT promoter and four copies of the human apo E enhancer results in superior expression levels compared to the ubiquitously active CMV promoter. 1,8,23 The most frequently used hepatocyte-specific promoters, with or without fusion of one or two heterologous enhancer elements, are the human AT promoter, 4,12,26,27 the albumin promoter 28,29 and the LSP promoter. 3,7,9 In this direct comparative study using hydrodynamic and adenoviral gene transfer, we show that the DC172 promoter, consisting of a 890 bp human AT promoter and two copies of the 160 bp a 1 -microglobulin enhancer, combined with two or four copies of the HCR-1 3 0 of the transgene, constitutes the most potent expression cassette.…”

“…A 251 bp sequence of the genomic human gA-I containing the complete 5 0 -UTR including the first apo A-I intron was subcloned to generate pTG14681-AT.A-I intron.cDNA.E 4 . Multiple sublconing steps using a truncated human FIX intron 12,53,54 isolated from pGEM-FIX-intron by ClaI and NotI digestion led to the formation of pTG14681-AT.FIX intron.cDNA.E 4 .…”

“…Transcriptional targeting by use of hepatocyte-specific expression cassettes may lead to immunological ignorance or tolerance for the transgene product. [1][2][3] Expression cassettes for hepatocyte-directed transfer have been improved by using new promoterenhancer combinations, [3][4][5][6][7][8][9] inclusion of introns 5,[10][11][12][13] and inclusion of additional transcriptional sequences like scaffold matrix-attached regions (SMAR) and hepatic control regions (HCR). 12,[14][15][16][17][18] However, because different gene transfer vectors, different doses and different transgenes were used in these studies, direct comparative data for different expression cassettes are often lacking and hamper the interpretation of existing literature.…”

Gene Therapy Progress and Prospects: Nonviral vectors

“…Thus, Kay's group constructed a plasmid DNA containing the apolipoprotein E locus control region, a1-antitrypsin promoter, human factor IX minigene sequence including a portion of the first intron, 3 0 -untranslated region, and the bovine growth hormone polyadenylation signal. 59 When the plasmid DNA was delivered to mouse liver by hydrodynamic injection, it produced not only increased gene expression of factor IX (in the therapeutic range), but also maintained these levels for at least 10 months. Furthermore, a linear DNA expression cassette originating from this plasmid showed 10-to 100-fold higher expression than the closed circular DNA for a period of 9 months.…”

Development of an efficient endothelial cell specific vector using promoter and 5' untranslated sequences from the human preproendothelin-1 gene

“…Simari et al showed that addition of 5' intronic sequences to cytomegalovirus immediate early promoter resulted in about 6 fold increase in gene expression (Simari et al, 1998). Kay's group constructed a vector with hepatic factor IX intronic sequences expressing enhanced levels of proteins (Miao et al, 2000). In this report, we show that our newly constructed vector based on ppET-1 gene promoter and 5' untranslated region drives high level of gene expression in endothelial cells and the specificity is even further enhanced under hypoxiamimic conditions due to a natural hypoxia responsive element within the promoter region.…”