Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.

Progress and problems with the use of viral vectors for gene therapy

Herpes simplex virus (HSV) infections of humans have been documented since the advent of writing. The spectrum of disease was expanded to include primary and recurrent infections of mucous membranes (gingivostomatitis, herpes labialis, and genital HSV infections), keratoconjunctivitis, neonatal HSV infection, visceral HSV infections of the immunocompromised host, HSV encephalitis, Kaposi's varicella-like eruption, and an association with erythema multiforme. Cumulative experience suggests that factors associated with pregnancy may place both the mother and fetus at increased risk for severe infection, possibly because of altered cell-mediated immunity. The major risk to the fetus is with primary or initial genital HSV infection of the mother. PCR evaluation of cerebrospinal fluid can be utilized to monitor therapeutic outcome in patients with herpes simplex encephalitis. The use of HSV for gene therapy heralds a new era of herpes biology, the conversion of a hazardous foe into a user-friendly surgical tool. Asymptomatic shedding of virus can continue despite clinically effective suppression with acyclovir, so the possibility of person-to-person transmission persists. Newborns with HSV infections can be classified as having disease that is localized to the skin, eyes, and mouth; affects the central nervous system (CNS); or is disseminated. Drug resistance was considered rare and resistant isolates were thought to be less pathogenic until a series of acyclovir-resistant HSV isolates from patients with AIDS were characterized. The risk of nephrotoxicity can be minimized by administering acyclovir by slow infusion and ensuring adequate hydration.

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Herpes Simplex Viruses

G207 is a conditionally replicating derivative of herpes simplex virus (HSV) type-1 strain F engineered with deletions of both γ134.5 loci and a lacZ insertion disabling the UL39 gene. We have demonstrated the efficacy of G207 in treating malignant glial tumors in athymic mice, as well as the safety of intracerebral G207 inoculation in mice and in Aotus nancymai. We sought to determine the safety of G207 inoculation into cerebral malignant glial tumors in humans. Criteria for inclusion into this dose-escalation study were the diagnosis of histologically proven malignant glioma, Karnofsky score ⩾70, recurrence despite surgery and radiation therapy, and an enhancing lesion greater than 1 cm in diameter. Serial magnetic resonance images were obtained for volumetric analysis. The trial commenced at a dose of 106 plaque forming units (p.f.u.) inoculated at a single enhancing site and was completed when the 21st patient was inoculated with 3 × 109 p.f.u. at five sites. While adverse events were noted in some patients, no toxicity or serious adverse events could unequivocally be ascribed to G207. No patient developed HSV encephalitis. We found radiographic and neuropathologic evidence suggestive of anti-tumor activity and long-term presence of viral DNA in some cases.

Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial.

Oncolytic viruses can kill tumour cells through a dual mechanism of action; the direct lysis of cells, and the induction of an immune response. The first oncolytic virus has been approved in China, and another has been recommended for approval in the United States. This Review discusses the biology of oncolytic viruses as well as key oncolytic viruses in clinical development, and investigates the challenges associated with developing oncolytic viruses as a new therapeutic modality for cancer.

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Oncolytic viruses: a new class of immunotherapy drugs

We have created a double mutant of the herpes simplex virus (HSV) type 1 (termed G207) with favourable properties for treating human malignant brain tumours: replication–competence in glioblastoma cells (and other dividing cells), attenuated neurovirulence, temperature sensitivity, ganciclovir hypersensitivity, and the presence of an easily detectable histochemical marker. G207 has deletions at both γ34.5 (RL1) loci and a lacZ gene insertion inactivating the ICP6 gene (UL39). G207 kills human glioma cells in monolayer cultures. In nude mice harbouring subcutaneous or intracerebral U–87MG gliomas, intraneoplastic inoculation with G207 causes decreased tumour growth and/or prolonged survival. G207 is avirulent upon intracerebral inoculation of mice and HSV–sensitive non–human primates. These results suggest that G207 should be considered for clinical evaluation in the treatment of glioblastomas.

Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas

Malignant gliomas are the most common malignant brain tumors and are almost always fatal. A thymidine kinase-negative mutant of herpes simplex virus-1 (dlsptk) that is attenuated for neurovirulence was tested as a possible treatment for gliomas. In cell culture, dlsptk killed two long-term human glioma lines and three short-term human glioma cell populations. In nude mice with implanted subcutaneous and subrenal U87 human gliomas, intraneoplastic inoculation of dlsptk caused growth inhibition. In nude mice with intracranial U87 gliomas, intraneoplastic inoculation of dlsptk prolonged survival. Genetically engineered viruses such as dlsptk merit further evaluation as novel antineoplastic agents.

https://science.sciencemag.org/content/sci/252/5007/854.full.pdf

Experimental therapy of human glioma by means of a genetically engineered virus mutant

We have shown that a drug-resistant mutant from a clinical isolate of herpes simplex virus contains a single point mutation in the DNA polymerase gene that confers resistance to both acyclovir and foscarnet. The mutated amino acid is located within a distinct conserved region shared among alpha-like DNA polymerases which we designate region VII. We infer that these conserved sequences are directly or indirectly involved in the recognition and binding of nucleotide and PPi substrates.

A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance.

Eucaryotic, viral, and bacteriophage DNA polymerases of the alpha-like family share blocks of sequence similarity, the most conserved of which has been designated region I. Region I includes a YGDTDS motif that is almost invariant within the alpha-like family and that is similar to a motif conserved among RNA-directed polymerases and also includes adjacent amino acids that are more moderately conserved. To study the function of these conserved amino acids in vivo, site-specific mutagenesis was used to generate herpes simplex virus region I mutants. A recombinant virus constructed to contain a mutation within the nearly invariant YGDTDS motif was severely impaired for growth on Vero cells which do not contain a viral polymerase gene. However, three recombinants constructed to contain mutations altering more moderately conserved residues grew on Vero cells and exhibited altered sensitivities to nucleoside and PPi analogs and to aphidicolin. Marker rescue and DNA sequencing of one such recombinant demonstrated that the region I alteration confers the altered drug sensitivity phenotype. These results indicate that this region has an essential role in polymerase function in vivo and is involved directly or indirectly in drug and substrate recognition.

Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition.

From marker rescue, sequencing, transcript, and latency analyses of the thymidine kinase-negative herpes simplex virus mutant dlsactk and studies using the thymidine kinase inhibitor Ro 31-5140, we infer that the virus-encoded thymidine kinase is required in murine trigeminal ganglia for acute replication and lytic gene expression, for increasing the numbers of cells expressing latency-associated transcripts, and for reactivation from latent infection.

Herpes simplex virus thymidine kinase and specific stages of latency in murine trigeminal ganglia.

Two specific inhibitors of herpes simplex virus thymidine kinase, N2-phenyl-2'-deoxyguanosine and N2-(m-trifluoromethylphenyl)guanine, were tested for their ability to inhibit the reactivation of virus from explant cultures of latently infected murine trigeminal ganglia. Both compounds significantly diminished the frequency of reactivation compared with that of untreated controls.

K. L. Ruffner

Papers

Experimental therapy of human glioma by means of a genetically engineered virus mutant

A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance.

Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition.

Herpes simplex virus thymidine kinase and specific stages of latency in murine trigeminal ganglia.

Specific inhibitors of herpes simplex virus thymidine kinase diminish reactivation of latent virus from explanted murine ganglia.