Discordance between expression and genome transfer titering of HSV amplicon vectors: recommendation for standardized enumeration.
Herpes simplex virus-derived amplicon vectors are well suited to the development of gene-based therapy for neurodegenerative diseases. The plasmid-based amplicon vector system allows for facile introduction of transcription units, possesses the potential for carrying gene inserts up to approximately 130 kb in length, and can be packaged into infectious virus devoid of contaminating cytotoxic helper virus. For accurate assessments to be made regarding vector comparison and improvements in vector design, a standard for titering prepared virus stocks must be established. At present, packaged amplicon vectors are routinely titered using reporter gene expression units to quantitate numbers of infectious amplicon virions. The strength of the promoter, sensitivity of detection of the gene product, and choice of titering cell type can greatly influence the apparent numbers of infectious virus particles. This is especially evident when comparisons are made between two amplicon vectors that possess different promoters. To this end, we have developed a new titering method based on a real-time quantitative PCR technique that allows for enumeration of transducing particles. This new approach ensures that amplicon comparison experiments are initiated with equivalent transduction units, thus allowing for a fair assessment of expression and therapeutic efficacy differences.
Department of Neurology and Center for Aging and Developmental Biology, University of Rochester School of Medicine, New York 14642, USA.
W J
WJ
Bowers
D F
DF
Howard
H J
HJ
Federoff
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D015870:Gene Expression; D018014:Gene Transfer Techniques / Q000592:standards*; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic