A Novel Multiply-Mutated HSV-1 Strain for the Treatment of Human Brain Tumors

Abstract

A promising approach for the therapeutic treatment of brain tumors utilizes replication-competent, neuroattenuated herpes simplex virus-1 (HSV-1) mutants. This approach requires mutation of HSV-1 to eliminate killing of normal, nondividing cells of the brain (e.g., neurons). We have generated a HSV-1 double-mutant, designated 3616UB, by interrupting the uracil DNA glycosylase (UNG) gene in a previously studied ICP34.5 mutant, R3616. The HSV-1-encoded UNG gene is required for efficient HSV-1 replication in nondividing cells, but is dispensable for replication in rapidly dividing cells. The specific function of the HSV-1 ICP34.5 gene is not completely clear, but it is thought to be necessary for viral replication in cells of the nervous system, because, when mutated, the resultant viral strains are fully neuroattenuated. Strain 3616UB did not replicate in primary neuronal cultures in vitro or in mouse brain, but efficiently killed six of six human tumor cell lines within 6 days in vitro and successfully infected and replicated within brain tumor xenografts. The potential safety of 3616UB for human use is enhanced by an unexpected hypersensitivity to the antiherpetic drug ganciclovir. These data suggest that 3616UB may be effective for the treatment of human brain tumors. Intratumoral injection of 3616UB into human medulloblastoma or angiosarcoma xenografts established in severe combined immunodeficient (SCID) mice produced significant growth arrest and some tumor regressions. Strain 3616UB was as effective as R3616 in this therapy study and did not cause any obvious distress in the treated animals. Together, the data show that 3616UB is a very safe alternative to other HSV-1 mutants because the presence of two mutations reduces the possibility of recombinational events in situ that could lead to the generation of virulent viral progeny during 3616UB therapy. Neuroattenuated HSV-1 mutants have great potential as antineoplastic therapy in animal models of brain tumors, but some safety concerns remain regarding their use in humans. We present a novel HSV-1 strain, designated 3616UB, that was engineered to be a safer alternative to HSV-1 mutants that carry a single neuroattenuating mutation. Strain 3616UB, derived from HSV-1 strain F, lacks both copies of the ICP34.5 gene and the uracil DNA glycosylase gene. Strain 3616UB has the ability to replicate in and destroy human tumor cell lines and infects and spreads through human medulloblastoma xenografts established in immunodeficient rodents. This virus, however, does not replicate in cultured neurons and, following direct intracranial injection of rodents, does not cause any apparent neurotoxicity. When tested for therapeutic benefit in human tumor xenografts, strain 3616UB showed significant potential as a therapy vehicle. Additionally, 3616UB is hypersensitive to ganciclovir, a feature that may enhance the therapeutic benefit of HSV-1 3616UB therapy.