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. 2004 Apr;6(4):395-404.
doi: 10.1002/jgm.516.

Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle

Alan R Brooks  1 , Richard N Harkins, Peiyin Wang, Hu Sheng Qian, Pengxuan Liu, Gabor M Rubanyi
Affiliations

Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle

Alan R Brooks et al. J Gene Med. 2004 Apr.

Abstract

Background: Although the transient nature of transgene expression using first-generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain.

Methods: Rats were given intramuscular (i.m.) injections of a first-generation Ad containing the human fibroblast growth factor 4 (hFGF-4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV-PE). The copy number of hFGF-4 mRNA and viral DNA was measured in the same muscles by quantitative RT-PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF-4 transcription unit DNA was also performed, and the methylation status of the CMV-PE DNA in the muscle was determined using bisulfite sequencing.

Results: The copy number of hFGF-4 mRNA peaked at 6 h then decreased 56-fold by 24 h, and a further 240-fold between days 3 and 28. Although the viral DNA copy number also decreased 23-fold between 6 h and 28 days, the ratio of copies of hFGF-4 mRNA per copy of viral DNA decreased 385-fold over this period. Methylation of the CMV-PE DNA in the muscle at both CpG and non-CpG sites was observed 24 h after virus administration and had increased at day 7.

Conclusions: Decreased transcription associated with extensive methylation of the CMV-PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors.

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