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 develo pment of gene-based therapy for neurodegenerative diseases. The plasmid-bas ed amplicon vector system allows for facile introduction of transcription u nits, possesses the potential for carrying gene inserts up to approximately 130 kb in length, and can be packaged into infectious virus devoid of cont aminating cytotoxic helper virus. For accurate assessments to be made regar ding vector comparison and improvements in vector design, a standard for ti tering prepared virus stocks must be established. At present, packaged ampl icon vectors are routinely titered using reporter gene expression units to quantitate numbers of infectious amplicon virions. The strength of the prom oter, sensitivity of detection of the gene product, and choice of titering cell type can greatly influence the apparent numbers of infectious virus pa rticles. This is especially evident when comparisons are made between two a mplicon vectors that possess different promoters. To this end, we have deve loped a new titering method based on a real-time quantitative PCR technique that allows for enumeration of transducing particles. This new approach en sures that amplicon comparison experiments are initiated with equivalent tr ansduction units, thus allowing for a fair assessment of expression and the rapeutic efficacy differences.