Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle.
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.
Brooks
Alan R
AR
Department of Gene Therapy, Berlex Biosciences, Richmond, CA 94804, USA. alan_brooks@berlex.com
Harkins
Richard N
RN
Wang
Peiyin
P
Qian
Hu Sheng
HS
Liu
Pengxuan
P
Rubanyi
Gabor M
GM
D000818:Animals; D018899:CpG Islands; D003587:Cytomegalovirus / Q000235:genetics*; D019175:DNA Methylation; D004742:Enhancer Elements, Genetic; D051519:Fibroblast Growth Factor 4; D005346:Fibroblast Growth Factors / Q000235:genetics; D005786:Gene Expression Regulation; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D006801:Humans; D007273:Injections, Intramuscular; D008297:Male; D018482:Muscle, Skeletal / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic
C496445:FGF4 protein, human; C496447:Fgf4 protein, rat; D051519:Fibroblast Growth Factor 4; D011518:Proto-Oncogene Proteins; D005346:Fibroblast Growth Factors