Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers.
Gene Ther
; 8(2): 111-20, 2001 Jan.
Article
in En
| MEDLINE
| ID: mdl-11313780
ABSTRACT
Recently developed helper virus-free methods of herpes simplex virus (HSV) amplicon vector packaging provide stocks that are virtually devoid of the cytotoxic component normally associated with traditional helper virus-based packaging methods. These approaches involve cotransfection of amplicon plasmid DNA with either a five-cosmid set or a bacterial artificial chromosome (BAC) that contains the HSV genome without its cognate pac signals. Helper virus-free amplicon packaging produces low-titer stocks (<10(5) expressing particles/ml) that exhibit a high frequency of pseudotransduction. In an effort to enhance amplicon titers, we introduced in trans a genomic copy of the virion host shutoff (vhs) protein-encoding gene UL41 into both cosmid- and BAC-based packaging strategies. Cotransfection of this plasmid with the amplicon and packaging reagents results in a 10-fold higher amplicon titer, and stocks that do not exhibit the pseudotransduction phenomenon. To further enhance packaging efficiency, the HSV transcriptional activator VP16 was introduced into packaging cells 1 day before the packaging components. Pre-loading of packaging cells with VP16 led to an additional enhancement of amplicon titers, an effect that did not occur in the absence of vhs. Increased helper virus-free amplicon titers resulting from these modifications will make in vivo transduction experiments more feasible.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Viral Proteins
/
Genetic Therapy
/
Herpes Simplex Virus Protein Vmw65
/
Herpesvirus 1, Human
/
Genetic Vectors
Limits:
Animals
Language:
En
Journal:
Gene Ther
Journal subject:
GENETICA MEDICA
/
TERAPEUTICA
Year:
2001
Document type:
Article
Affiliation country:
United States