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Biotechniques. 1996 Mar;20(3):460-9.

Generation of high-titer defective HSV-1 vectors using an IE 2 deletion mutant and quantitative study of expression in cultured cortical cells.

Author information

1
Children's Hospital, Boston, MA, USA. flim@mvax.cbm.uam.es

Abstract

Vectors based on herpes simplex virus type 1 (HSV-1) show promise for gene transfer into mammalian cells because of their wide host range, efficient infection and ability to deliver genes to nondividing cells. Defective HSV-1 vectors, or amplicons, are plasmid vectors which are unable to propagate on their own but contain specific HSV-1 sequences that, in the presence of helper virus, support DNA replication and subsequent packaging into virus particles. We compared three replication-incompetent HSV-1 mutants (KOS strain 5dl1.2, strain 17 D30EBA, KOS strain d120) as the helper virus for packaging the prototype defective HSV-1 vector, pHSVlac, which uses the HSV-1 immediate-early (1E) 4/5 promoter to regulate expression of the Escherichia coli lacZ gene. Use of 5dl1.2, which contains a deletion in the IE 2 gene, consistently produced virus stocks that contained a high level of vector, undetectable levels of wild-type HSV-1 and a ratio of vector to helper greater than 1. Virus stocks prepared using 5dl1.2 were superior to those prepared using helper viruses that harbor a deletion in the IE 3 gene, either D30EBA or dl20, and supported more efficient gene transfer than possible with previously published procedures. Lactate dehydrogenase efflux assays in rat cortical cultures showed that 5dl1.2 was no more cytotoxic than either D30EBA or dl20, despite the expression of more viral genes. Rat cortical cultures infected with pHSVlac packaged with either 5dl1.2 or D30EBA were used to quantify the stability of vector expression. Our results show a decrease in the number of cells with detectable levels of beta-galactosidase to 30% of peak levels after one week, irrespective of the helper virus used. However, simultaneous superinfection with 5dl1.2, but not with either D30EBA or dl20, produced a transient increase in the number of cells expressing beta-galactosidase. Superinfection with 5dl1.2 at 9 days after gene transfer increased the number of cells expressing detectable beta-galactosidase back to peak levels, most probably because of reactivation of the IE 4/5 promoter in pHSVlac. These results thus provide the first quantitative demonstration of long-term persistence of defective HSV-1 vectors in neurons.

PMID:
8679207
DOI:
10.2144/19962003460
[Indexed for MEDLINE]
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