Synergistic effects of deleting multiple nonessential elements in nonreplicative HSV-1 BAC genomic vectors play a critical role in their viability.

Nonreplicative Herpes simplex virus type-1 (HSV-1) genomic vectors have already entered into clinical trials for neurological gene therapy thanks to their scalable growth in permissive cells. However, the small transgene capacity of this type of HSV-1 vectors currently used in the clinic represents an important limiting factor as a gene delivery system. To develop high-capacity nonreplicative genomic HSV-1 vectors, in this study we have characterized a series of multiply deleted mutants which we have constructed in bacterial artificial chromosomes (BACs), removing up to 24 kb of unstable or dispensable genomic sequences to allow insertion of transgenes up to this size. We show that synergistic effects of deletions of: the HSV-1 replication origins oriS and oriL, the HSV-1 internal repeat region, the remaining ICP4 gene copy and the genes encoding for ICP27, UL56, UL55, can severely reduce the growth of these HSV-1 vectors. Given that several of these elements have been characterized as 'non-essential' for viral growth in cell culture by single-deletion experiments of wild-type HSV-1, our study highlights the need to re-evaluate their functional contribution in the context of multiply deleted nonreplicative HSV-1 genomic vectors. Our BAC mutants described here can serve as useful starting platforms to accelerate HSV-1 vector development.