Development and application of a real-time PCR method for pharmacokinetic and biodistribution studies of recombinant adenovirus.
A replication-deficient recombinant adenovirus (Ad5-LFA-3/IgG(1)) that encodes secreted LFA-3/IgG(1) was constructed for gene therapy treatment of psoriasis. The purpose of this study was to develop a real-time PCR method for pharmacokinetic and biodistribution studies of Ad5-LFA-3/IgG(1) within the circulation and organs. This method showed good specificity, sensitivity and reproducibility over a wide dynamic range of concentrations. Quantitative measurement of recombinant adenoviral DNA suggested that the level of Ad5-LFA-3/IgG(1) DNA in circulating blood peaked within 10 min following intravenous injection in rhesus macaques. Following this peak, the adenoviral DNA level dropped significantly to a very low level. Real-time PCR revealed that Ad5-LFA-3/IgG(1) DNA was enriched in the spleen, lung and liver after injection of the adenovirus into rats through the tail vein. The adenoviral DNA was barely detected in other tissues. These data provide important information for clinical trials of Ad5-LFA-3/IgG(1) and confirm the utility of the real-time PCR assay for monitoring gene therapy trials.
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, No. 20 Dongdajie Road, Fengtai District, Beijing, People's Republic of China. happyxuxian@sohu.com
Xian-Xing
XX
Xu
Xue
X
Shui
Zhi-Hang
ZH
Chen
Cheng-Qi
CQ
Shan
Yu-Nan
YN
Hou
Yuan-Guo
YG
Cheng
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism*; D000077944:Alefacept; D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000493:pharmacokinetics*; D008253:Macaca mulatta; D008297:Male; D008657:Metabolic Clearance Rate; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000493:pharmacokinetics*; D011994:Recombinant Proteins / Q000378:metabolism / Q000493:pharmacokinetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014018:Tissue Distribution
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000077944:Alefacept