Discordance between expression and genome transfer titering of HSV amplicon vectors: Recommendation for standardized enumeration

被引:41
作者
Bowers, WJ
Howard, DF
Federoff, HJ
机构
[1] Univ Rochester, Sch Med & Dent, Ctr Aging & Dev Biol, Div Mol Med & Gene Therapy, Rochester, NY 14642 USA
[2] Univ Rochester, Sch Med & Dent, Dept Neurol, Rochester, NY 14642 USA
关键词
herpes simplex virus; amplicon; quantitative PCR; gene therapy; titering;
D O I
10.1006/mthe.2000.0039
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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.
引用
收藏
页码:294 / 299
页数:6
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