Herpes simplex virus (HSV) has several potential advantages as a vector for delivering genes to the nervous system. The virus naturally infects and remains latent in neurons and has evolved the ability of highly efficient retrograde transport from the site of infection at the periphery to the site of latency in the spinal ganglia. HSV is a large virus, potentially allowing the insertion of multiple or very large transgenes. Furthermore, HSV does not integrate into the host chromosome, removing any potential for insertional activation or inactivation of cellular genes. However, the development of HSV vectors for the central nervous system that exploit these properties has been problematical. This has mainly been due to either vector toxicity or an inability to maintain transgene expression. Here we report the development of highly disabled versions of HSV-1 deleted for ICP27, ICP4, and ICP34.5/open reading frame P and with an inactivating mutation in VP16. These viruses express only minimal levels of any of the immediate-early genes in noncomplementing cells. Transgene expression is maintained for extended periods with promoter systems containing elements from the HSV latency-associated transcript promoter (J. A. Palmer et al., J. Virol. 74:5604-5618, 2000). Unlike less-disabled viruses, these vectors allow highly effective gene delivery both to neurons in culture and to the central nervous system in vivo. Gene delivery in vivo is further enhanced by the retrograde transport capabilities of HSV. Here the vector is efficiently transported from the site of inoculation to connected sites within the nervous system. This is demonstrated by gene delivery to both the striatum and substantia nigra following striatal inoculation; to the spinal cord, spinal ganglia, and brainstem following injection into the spinal cord; and to retinal ganglion neurons following injection into the superior colliculus and thalamus.

We have previously developed an oncolytic herpes simplex virus-1 based on a clinical virus isolate, which was deleted for ICP34.5 to provide tumor selected replication and ICP47 to increase antigen presentation as well as tumor selective virus replication. A phase I/II clinical trial using a version of this virus expressing granulocyte macrophage colony-stimulating factor has shown promising results. The work reported here aimed to develop a version of this virus in which local tumor control was further increased through the combined expression of a highly potent prodrug activating gene [yeast cytosine deaminase/uracil phospho-ribosyltransferase fusion (Fcy::-Fur)] and the fusogenic glycoprotein from gibbon ape leukemia virus (GALV), which it was hoped would aid the spread of the activated prodrug through the tumor. Viruses expressing the two genes individually or in combination were constructed and tested, showing (a) GALV and/or Fcy::Fur expression did not affect virus growth; (b) GALV expression causes cell fusion and increases the tumor cell killing at least 30-fold in vitro and tumor shrinkage 5-to 10-fold in vivo; (c) additional expression of Fcy::Fur combined with 5-fluorocytosine administration improves tumor shrinkage further. These results indicate, therefore, that the combined expression of the GALV protein and Fcy::Fur provides a highly potent oncolytic virus with improved capabilities for local tumor control. It is intended to enter the GALV/Fcy::Fur expressing virus into clinical development for the treatment of tumor types, such as pancreatic or lung cancer, where local control would be anticipated to be clinically advantageous. (Cancer Res 2006; 66(9): 4835-42)

This approach is therefore beneficial in increasing localised TNFalpha expression as compared to the use of non-replicative approaches, and combines the effects of TNFalpha with oncolytic virus replication which is expected to further enhance the efficacy of radiotherapy in a combined treatment approach.

Background A replication-competent, attenuated, oncolytic herpes simplex virus-1, OncoVEXGALV/CD, has previously been engineered to express a fusogenic protein from the gibbon ape leukemia virus and cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT) which converts fluorocytosine (5-FC) to 5-fluorouracil (5-FU). OncoVEXGFP is an analogous vector that expresses enhanced green fluorescent protein. Methods We assessed the ability of OncoVEXGALV/CD and OncoVEXGFP to infect, replicate within, and lyse four head and neck squamous carcinoma (HNSCC) cell lines in vitro. The effects of adding 5-FC with OncoVEXGALV/CD were evaluated. Results HNSCC was permissive to GFP expression in100% of cells by OncoVEXGFP at a multiplicity of infection (MOI) of 1 after 48 hours, and supported logarithmic viral replication. Virus caused >60% cell death six days after exposure to virus at MOI 0.1 in three of the four cell lines. 5-FC failed to enhance cytotoxicity induced by OncoVEXGALV/CD at MOI 0.1. However, for the least sensitive SCC25 cell line, virus at MOI 0.01 was cytotoxic to only 4% of cells after six days, but was cytotoxic to 35% of cells with 5-FC. Conclusions OncoVEXGALV/CD efficiently infects, replicates within and lyses HNSCC at relatively low viral doses. Prodrug conversion by CD did not enhance therapy at viral doses which cause efficient cytotoxicity, but may have beneficial effects in less sensitive cell lines at low viral doses.

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13139 HSV in which ICP34.5 is deleted directs tumour selective cell lysis and has proven safe in Phase I clinical trials. To produce oncolytic HSV with enhanced anti-tumour properties, we have deleted ICP34.5 from a clinical isolate of HSV-1, which enhances the tumour cell killing capabilities of the virus, deleted ICP47 (which blocks antigen presentation), and inserted the gene encoding GM-CSF. This aimed to maximize anti-tumour immune responses following intra-tumoural injection and provide an in situ, patient-specific, anti-tumour vaccine, combined with oncolysis. In vivo, both injected and non-injected tumours could be cured and animals were then protected against tumour cell challenge. A Phase I clinical trial with the virus (OncoVEXGM-CSF) has been conducted including patients with cutaneous or sub-cutaneous deposits of a number of tumour types (Lead Investigator: Professor Charles Coombes, Hammersmith Hospital, London). This demonstrated the virus to have a good safety profile, the main side effects being ‘flu-like symptoms, similar to those which have previously been observed with other oncolytic products. Virus replication and GM-CSF expression was demonstrated together with inflammation, flattening and necrosis of injected lesions which was in some cases considerable and which was also in some cases observed in lesions which had not themselves been injected. In all cases where necrosis was observed in biopsies, this correlated with areas of staining for HSV, suggesting the virus had caused the effect. Following this promising data, Phase II studies are underway in multiple tumour types. In addition to OncoVEXGM-CSF, further versions of OncoVEX expressing other active genes have been constructed and tested in pre-clinical models. These include a virus expressing TNF∝, intended to be synergistic with radiotherapy, and versions of the virus expressing a pro-drug activating gene combined with the delivery of a fusogenic glycoprotein designed to maximize the properties of the virus for local tumour control. Each of these have shown promising results in pre-clinical tumour models, including in combination with chemotherapy and radiotherapy where benefits which are at least additive have been demonstrated. [Table: see text]