Sustained expression of human apolipoprotein A-I after adenoviral gene transfer in C57BL/6 mice: role of apolipoprotein A-I promoter, apolipoprotein A-I introns, and human apolipoprotein E enhancer.
Elevation of HDL cholesterol, after adenoviral apolipoprotein A-I (apo A-I) gene transfer, may delay or revert ischemic cardiovascular disease, provided transgene expression is persistent. Previously, we observed transient human apo A-I expression after adenoviral gene transfer with a cytomegalovirus (CMV)-driven construct containing the human apo A-I cDNA. Therefore, the effects of promoters (CMV or 256 base pairs of the human apo A-I promoter), introns of the human apo A-I gene, and the liver-specific human apolipoprotein E (apo E) enhancer on adenovirus-mediated human apo A-I expression were evaluated in C57BL/6 mice. In the presence of the CMV promoter, human apo A-I introns prolonged expression above 20 mg/dl from 14 to 35 days. Addition of one, two, or four copies of the human apo E enhancer in these constructs resulted in a copy-dependent but transient increase in expression for 14 days. The apo A-I promoter induced 3.2-fold lower peak levels of human apo A-I than did the CMV promoter, but insertion of four apo E enhancers in the apo A-I promoter-driven construct resulted in human apo A-I levels above 20 mg/dl for 6 months. The decline between day 6 and day 35 of human apo A-I expression driven by the CMV promoter was due to (1) a 2.5-fold decline in transgene DNA levels that is not observed with apo A-I promoter-driven constructs, and (2) CMV promoter attenuation as evidenced by a 7.6-fold decline in the human apo A-I mRNA/human apo A-I DNA copy number ratio between day 6 and day 35. Hepatotoxicity, as evidenced by up to 10-fold higher serum levels of transaminases on day 6 after gene transfer with CMV promoter-driven constructs than with apo A-I promoter-driven constructs, probably caused the accelerated decline of transgene DNA. In conclusion, gene transfer with an adenovirus comprising the 256-bp apo A-I promoter, the genomic apo A-I DNA, and four apo E enhancers, all of human origin, is associated with low hepatotoxicity and with the absence of promoter shutoff resulting in human apo A-I expression above 20 mg/dl for up to 6 months.
De Geest
B
B
Center for Molecular and Vascular Biology, Leuven, Belgium.
Van Linthout
S
S
Lox
M
M
Collen
D
D
Holvoet
P
P
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics*; D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000009:adverse effects; D006801:Humans; D007438:Introns; D008099:Liver / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D013601:T-Lymphocytes / Q000166:cytology
D016632:Apolipoprotein A-I; D001057:Apolipoproteins E; D017931:DNA Primers