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• W2053207943 endingPage "1097" @default.
• W2053207943 startingPage "1087" @default.
• W2053207943 abstract "Herpes simplex virus type 1 (HSV-1) represents an attractive vehicle for a variety of gene therapy applications. To render this virus safe for clinical use, its cytotoxic genes must be removed without losing its ability to express transgenes efficiently. Our vectors are deleted for the essential immediate early genes ICP4 and ICP27. These genes are controlled by unique promoters having enhancer elements responsive to a viral structural protein VP16. The expression of these genes occurs prior to the activation of all other lytic functions and is thus required to initiate and complete the virus replication cycle. For large scale manufacture of clinical grade vectors, efficient cell lines must be generated that express the essential viral gene products in trans during vector propagation. Here we describe methods for engineering HSV-1 production cell lines that improve vector growth by altering the kinetics of complementing gene expression. We examined the ability of Vero cells independently transduced with ICP4 and ICP27 under transcriptional control of their respective promoters to support the growth of a replication defective vector (JDTOZHE), deleted for ICP4, ICP27 and approximately 20 kb of internal elements that are not required for virus growth in Vero cells. Vector yield on this cell line was 3 logs lower than wild-type virus grown on Vero cells. To understand the mechanism underlying poor vector yield, we examined the expression of ICP4 and ICP27 during virus complementation. While ICP27 was expressed immediately on vector infection, the expression of ICP4 was considerably delayed by 8-10 h, suggesting that the ICP4 promoter was not adequately activated by VP16 delivered by the infectious vector particle. Use of the ICP0 promoter to express ICP4 from the cellular genome resulted in higher induction levels and faster kinetics of ICP4 expression and a 10-fold improvement in vector yield. This study suggests that vector complementation is highly dependent on the kinetics of complementing gene expression and can lead to large differences in vector yield." @default.
• W2053207943 created "2016-06-24" @default.
• W2053207943 creator A5000108324 @default.
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• W2053207943 creator A5074227657 @default.
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• W2053207943 date "2009-03-01" @default.
• W2053207943 modified "2023-09-27" @default.
• W2053207943 title "Engineering cell lines for production of replication defective HSV-1 gene therapy vectors" @default.
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• W2053207943 doi "https://doi.org/10.1002/bit.22123" @default.
• W2053207943 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18828174" @default.
• W2053207943 hasPublicationYear "2009" @default.
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