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

Abstract

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.