"Armed" oncolytic herpes simplex viruses for brain tumor therapy

Abstract

Genetically engineered, conditionally replicating herpes simplex viruses type 1 (HSV-1) are promising therapeutic agents for brain tumors and other solid cancers. They can replicate in situ, spread and exhibit oncolytic activity via a direct cytocidal effect. One of the advantages of HSV-1 is the capacity to incorporate large and/or multiple transgenes within the viral genome. Oncolytic HSV-1 can therefore be "armed" to add certain functions. Recently, the field of armed oncolytic HSV-1 has drastically advanced, due to development of recombinant HSV-1 generation systems that utilize bacterial artificial chromosome and multiple DNA recombinases. Because antitumor immunity is induced in the course of oncolytic activities of HSV-1, transgenes encoding immunomodulatory molecules have been most frequently used for arming. Other armed oncolytic HSV-1 include those that express antiangiogenic factors, fusogenic membrane glycoproteins, suicide gene products, and proapoptotic proteins. Provided that the transgene product does not interfere with viral replication, such arming of oncolytic HSV-1 results in augmentation of antitumor efficacy. Immediate-early viral promoters are often used to control the arming transgenes, but strict-late viral promoters have been shown useful to restrict the expression in the late stage of viral replication when desirable. Some armed oncolytic HSV-1 have been created for the purpose of noninvasive in vivo imaging of viral infection and replication. Development of a wide variety of armed oncolytic HSV-1 will lead to an establishment of a new genre of therapy for brain tumors as well as other cancers.

Figure 1

A schema describing the T-BAC system for constructing “armed” oncolytic HSV-1 with the G47Δ backbone. The desired transgene for “arming” is inserted into the multiple cloning site of the shuttle vector (SV-01). The first step is to insert the entire sequence of the shuttle vector into the loxP site of T-BAC by a Cre-mediated recombination, followed by an electroporation into E. coli. The second step is to co-transfect the co-integrate with a plasmid expressing FLP onto Vero cells to excise the BAC sequence flanked by the FRT sites. The objective armed oncolytic HSV-1 appear as GFP-negative and lacZ-positive virus plaques. Non-recombined viruses do not appear, due to the presence of the lambda stuffer sequence (lmd) causing an oversize of the genome.

Figure 2

Concept of antitumor efficacy augmentation using oncolytic HSV-1 armed with an immunostimulatory gene. When oncolytic HSV-1 armed with the IL-12 gene infects tumor cells, IL-12 is secreted in the course of viral replication and stimulates the immune cells. In addition to direct tumor cell killing via viral replication and spread, tumor cells are destroyed by augmented antitumor immune responses, resulting in enhanced antitumor activities.

Figure 3

Structures of representative armed oncolytic HSV-1. The HSV-1 genome consists of long and short unique regions (U_L and U_S) each bounded by terminal (T) and internal (I) repeat regions (R_L and R_S). Armed oncolytic HSV-1 created by using the T-BAC (or G47Δ-BAC) system has the backbone structure of G47Δ, a third-generation oncolytic HSV-1. It has triple deletions in the γ34.5, ICP6 and α47 genes. The transgene is inserted into the deleted ICP6 locus. As a marker, it also expresses the LacZ gene driven by the ICP6 promoter. Armed oncolytic HSV-1 created by using the HSVQuik system has the backbone structure similar to G207 or MGH1, second-generation oncolytic HSV-1. It has double deletions in the γ34.5 and ICP6 genes. The transgene is inserted into the deleted ICP6 locus. As a marker, it also expresses the GFP gene driven by the ICP6 promoter. The OncoVEX series has the backbone structure of a second-generation oncolytic HSV-1 with double deletions in the γ34.5 and α47 genes. The transgene is inserted into the deleted γ34.5 loci.