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, provid ed transgene expression is persistent. Previously, we observed transient hu man apo A-I expression after adenoviral gene transfer with a cytomegaloviru s (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 apolipopro tein E (apo E) enhancer on adenovirus-mediated human apo A-I expression wer e 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. Additi on of one, two, or four copies of the human apo E enhancer in these constru cts resulted in a copy-dependent but transient increase in expression for 1 4 days. The apo A-I promoter induced 3.2-fold lower peak levels of human ap o A-I than did the CMV promoter, but insertion of four apo E enhancers in t he 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 c onstructs, and (2) CMV promoter attenuation as evidenced by a 7.6-fold decl ine in the human apo A-I mRNA/hmman apo A-I DNA copy number ratio between d ay 6 and day 35. Hepatotoxicity, as evidenced by up to Ill-fold higher seru m levels of transaminases on day 6 after gene transfer with CMV promoter-dr iven constructs than with apo A-I promoter-driven constructs, probably caus ed the accelerated decline of transgene DNA. In conclusion, gene transfer w ith 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 l ow hepatotoxicity and with the absence of promoter shutoff resulting in hum an apo A-I expression above 20 mg/dl for up to 6 months.