Abstract

Conditionally replicating herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Certain antitumor functions may be added to oncolytic activities of recombinant HSV-1 vectors by inserting transgenes into the viral genome. Because conventional homologous recombination techniques had required time-consuming processes to create "armed" oncolytic HSV-1 vectors, we established an innovative construction system using bacterial artificial chromosome and two recombinase systems (Cre/loxP and FLPe/FRT). Using G47Delta, a safe and efficacious oncolytic HSV-1 with triple gene mutations, as the backbone, this system allowed a rapid generation of multiple vectors with desired transgenes inserted in the deleted ICP6 locus. Four oncolytic HSV-1 vectors, expressing murine interleukin 18 (mIL-18), soluble murine B7-1 [B7-1-immunoglobulin (B7-1-Ig)], both, or none, were created simultaneously within 3 months. In vitro, all newly created recombinant vectors exhibited virus yields and cytopathic effects similar to the parental G47Delta. In two immunocompetent mouse tumor models, TRAMP-C2 prostate cancer and Neuro2a neuroblastoma, the vector expressing both mIL-18 and B7-1-Ig showed a significant enhancement of antitumor efficacy via T-cell-mediated immune responses. The results show that "arming" with multiple transgenes can improve the efficacy of oncolytic HSV-1 vectors. The use of our system may facilitate the development and testing of various armed oncolytic HSV-1 vectors.

Highlights

  • The key to developing useful oncolytic herpes simplex virus type 1 (HSV-1) vectors is to acquire high antitumor efficacy without compromising safety, obtaining as wide therapeutic window as possible

  • G207 is one of the first oncolytic HSV-1 vectors used in clinical trials [1] and has deletions in both copies of the c34.5 gene and a lacZ insertion inactivating the ICP6 gene [2]

  • Transfections were done on Vero cells by using 0.9 Ag of DNA composed of a 1:1:1 mixture of G47D DNA purified by Na/I method, pBAC-ICP6EF, and pBAC-ICP6EF linearized with Asc I digestion, with Lipofectamine (Invitrogen, Carlsbad, CA)

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Summary

Introduction

The key to developing useful oncolytic herpes simplex virus type 1 (HSV-1) vectors is to acquire high antitumor efficacy without compromising safety, obtaining as wide therapeutic window as possible. We used BAC and two recombinase systems (Cre/loxP and FLPe/FRT) to develop a method that allowed a rapid, reliable, and simultaneous construction of multiple ‘‘armed’’ oncolytic HSV-1 vectors using G47D as the backbone. Required is the shuttle vector (pVec9), a replication-conditional plasmid that contains the lacZ gene (without a promoter), loxP and FRT sites, a CMV promoter, and a multiple cloning site where the desired transgene is cloned (Fig. 1C).

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