Improved Packaging System for Generation of High-Level Noncytotoxic HSV-1 Amplicon Vectors Using Cre-loxP Site-Specific Recombination to Delete the Packaging Signals of Defective Helper Genomes

Abstract

Amplicons are promising helper-dependent HSV-1-derived vectors that allow the transfer and expression of very large foreigner DNA into dividing and quiescent cells. We had already described an approach to prepare large amounts of high-titer amplicon vectors, using Cre-loxP site-specific recombination system to delete the packaging ("a") signals of an HSV-1 recombinant helper virus (HSV-1 LaL). Amplicon vectors prepared using such a system showed a level of contamination with helper particles lower than 1%. The residual helper particles generated by this system are, however, replication-competent, thus precluding their use in gene therapy. To avoid such potential spread of residual particles, we present here the development of a defective Cre-loxP-based helper virus (HSV-1 LaLΔJ), deleted of the genes encoding ICP4 and ICP34.5 proteins from the helper genome, in addition to the native "a" signals. HSV-1 LaLΔJ carries a single floxed "a" signal in gC locus. To produce HSV-1 LaLΔJ and to prepare the amplicon vectors, we have constructed two novel cell lines expressing the essential ICP4 protein, either alone or in combination with Cre recombinase. These cell lines were conceived to complement ICP4 while minimizing the probability of generating replication-competent particles. In this paper we present results demonstrating that the novel helper system allows ready production of large amounts of high-titer amplicon vectors. Residual helper particles generated still do not exceed 0.5% of the viral population and can grow only in cells expressing ICP4. Amplicon vectors produced with this method showed no cytotoxicty for infected cells.