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Herpes simplex virus type 1 (HSV-1) is a leading cause of infectious blindness. Current treatments for HSV-1 do not eliminate the virus from the site of infection or latent reservoirs in the trigeminal ganglia. Here, we target HSV-1 genomes directly using mRNA-carrying lentiviral particles that simultaneously deliver SpCas9 mRNA and viral-gene-targeting guide RNAs (designated HSV-1-erasing lentiviral particles, termed HELP). We show that HELP efficiently blocks HSV-1 replication and the occurrence of herpetic stromal keratitis (HSK) in three different infection models. HELP was capable of eliminating the viral reservoir via retrograde transport from corneas to trigeminal ganglia. Additionally, HELP inhibited viral replication in human-derived corneas without causing off-target effects, as determined by whole-genome sequencing. These results support the potential clinical utility of HELP for treating refractory HSK. CRISPR–Cas9 eliminates herpes simplex virus both in the corneal infection site and in the viral reservoir of the trigeminal ganglia.

We have previously shown that avirulent but replication-competent herpes simplex virus (HSV) 1716 causes cell death in human melanoma cell lines in vitro and selectively replicates in melanoma tissue in nude mice. We now present a pilot study of intratumoral injection of HSV1716 into subcutaneous nodules of metastatic melanoma in five patients with stage 4 melanoma. Two patients each received one injection, two received two injections, and one received four injections of 103 plaque-forming units HSV1716. In one patient, flattening of previously palpable tumour nodules was seen 21 days after two direct injections of HSV1716, and in injected nodules from all three patients who received two or more injections there was microscopic evidence of tumour necrosis. Immunohistochemical staining of injected nodules revealed evidence of virus replication confined to tumour cells. These findings suggest that HSV1716 is non-toxic and could be of therapeutic benefit in patients with metastatic melanoma.

Recombinant viruses are the workhorse of modern neuroscience. Whether one would like to understand a neuron’s morphology, natural activity patterns, molecular composition, connectivity or behavioural and physiologic function, most studies begin with the injection of an engineered virus, often an adeno-associated virus or herpes simplex virus, among many other types. Recombinant viruses currently enable some combination of cell type-specific, circuit-selective, activity-dependent and spatiotemporally resolved transgene expression. Viruses are now used routinely to study the molecular and cellular functions of a gene within an identified cell type in the brain, and enable the application of optogenetics, chemogenetics, calcium imaging and related approaches. These advantageous properties of engineered viruses thus enable characterization of neuronal function at unprecedented resolution. However, each virus has specific advantages and disadvantages, which makes viral tool selection paramount for properly designing and executing experiments within the central nervous system. In the current Review, we discuss the key principles and uses of engineered viruses and highlight innovations that are needed moving forward.Viral vectors are important tools for neuroscientists. In this Review, Nectow and Nestler discuss state-of-the-art recombinant viral tools, the key principles governing their selection, development and use, and how they could answer some of the most important questions in neuroscience today.

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Recent advances include preclinical proof of feasibility for a single-shot virotherapy cure, identification of drugs that accelerate intratumoral virus propagation, strategies to maximize the immunotherapeutic action of oncolytic viruses and clinical confirmation of a critical viremic threshold for vascular delivery and intratumoral virus replication. The primary clinical milestone has been completion of accrual in a phase 3 trial of intratumoral herpes simplex virus therapy using talimogene laherparepvec for metastatic melanoma. Key challenges for the field are to select 'winners' from a burgeoning number of oncolytic platforms and engineered derivatives, to transiently suppress but then unleash the power of the immune system to maximize both virus spread and anticancer immunity, to develop more meaningful preclinical virotherapy models and to manufacture viruses with orders-of-magnitude higher yields than is currently possible.

Oncolytic virotherapy holds promise for cancer treatment, but the factors determining its oncolytic activity remain unclear. Neutrophil extracellular traps (NETs) are associated with cancer progression, yet their formation mechanism and role in oncolytic virotherapy remain elusive. In this study, we demonstrate that, in glioma, upregulation of IGF2BP3 enhances the expression of E3 ubiquitin protein ligase MIB1, promoting FTO degradation via the ubiquitin-proteasome pathway. This results in increased m6A-mediated CSF3 release and NET formation. Oncolytic herpes simplex virus (oHSV) stimulates IGF2BP3-induced NET formation in malignant glioma. In glioma models in female mice, a BET inhibitor enhances the oncolytic activity of oHSV by impeding IGF2BP3-induced NETosis, reinforcing virus replication through BRD4 recruitment with the CDK9/RPB-1 complex to HSV gene promoters. Our findings unveil the regulation of m6A-mediated NET formation, highlight oncolytic virus-induced NETosis as a critical checkpoint hindering oncolytic potential, and propose targeting NETosis as a strategy to overcome resistance in oncolytic virotherapy. The m6A reader IGF2BP3 is upregulated in various cancer, including glioblastoma. Here the authors report that IGF2BP3 facilitates NETosis and glioma survival as well as resistance to oncolytic herpes simplex virotherapy.

cGAS is an important sensor of cytosolic DNA, but the mechanisms that regulate it remain largely unknown. Fan and colleagues demonstrate that cGAS and its DNA-binding activities are negatively regulated by glutamylation. Cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA during viral infection and catalyzes synthesis of the dinucleotide cGAMP, which activates the adaptor STING to initiate antiviral responses. Here we found that deficiency in the carboxypeptidase CCP5 or CCP6 led to susceptibility to DNA viruses. CCP5 and CCP6 were required for activation of the transcription factor IRF3 and interferons. Polyglutamylation of cGAS by the enzyme TTLL6 impeded its DNA-binding ability, whereas TTLL4-mediated monoglutamylation of cGAS blocked its synthase activity. Conversely, CCP6 removed the polyglutamylation of cGAS, whereas CCP5 hydrolyzed the monoglutamylation of cGAS, which together led to the activation of cGAS. Therefore, glutamylation and deglutamylation of cGAS tightly modulate immune responses to infection with DNA viruses.

We have previously demonstrated safety of G207, a doubly mutated (deletion of both γ134.5 loci, insertional inactivation of UL39) herpes simplex virus (HSV) for patients stereotactically inoculated in enhancing portions of recurrent malignant gliomas. We have now determined safety of two inoculations of G207, before and after tumor resection. Inclusion criteria were histologically proven recurrent malignant glioma, Karnofsky score ≥70, and ability to resect the tumor without ventricular system breach. Patients received two doses of G207 totaling 1.15 × 109 plaque-forming units with 13% of this total injected via a catheter placed stereotactically in the tumor. Two or five days later, tumor was resected en bloc with catheter in place. The balance of G207 dose was injected into brain surrounding the resection cavity. Six patients with recurrent glioblastoma multiforme were enrolled. Two days after the second G207 inoculation, one patient experienced transient fever, delirium, and hemiparesis, which entirely resolved on high-dose dexamethasone. No patient developed HSV encephalitis or required treatment with acyclovir. Radiographic and neuropathologic evidence suggestive of antitumor activity is reported. Evidence of viral replication was demonstrated. G207 appears safe for multiple dose delivery, including direct inoculation into the brain surrounding tumor resection cavity.

We recently demonstrated that GGAA promoters (GGAAprom) can be used to drive the expression of therapeutic genes (i.e., Cas9) in vitro and in vivo in a highly specific way in Ewing sarcoma cells. In this study, we take advantage of this strategy to express the suicide gene thymidine kinase from herpes simplex virus (HSV-TK), a suicide gene frequently used as an antitumoral therapy in clinical settings. The expression of HSV-TK in Ewing sarcoma cells under the control of the GGAA promoter resulted in cell death in the presence of ganciclovir both in vitro and in vivo. Interestingly, in vitro studies revealed a strong bystander effect. In preclinical models, the tumor volume of Ewing sarcoma xenografts expressing HSV-TK was dramatically reduced upon valganciclovir treatment. When adenoviral vectors were used as suitable therapeutics for clinical translation, HSV-TK expression in Ewing sarcoma cells reduced cell viability in vitro and reduced tumor growth in vivo. Interestingly, the death of Ewing sarcoma cells was accompanied by massive infiltration of the residual tumor by immune cells. Our results demonstrate that suicide genes can be delivered in a specific way to Ewing sarcoma cells by combining Ewing-specific GGAA promoters and adenoviral vectors, paving the way for the development of cancer gene therapies based on the selective expression of genes with therapeutic potential.

Oncolytic viruses are smart therapeutics against cancer due to their potential to replicate and produce the needed therapeutic dose in the tumor, and to their ability to self-exhaust upon tumor clearance. Oncolytic virotherapy strategies based on the herpes simplex virus are reaching their thirties, and a wide variety of approaches has been envisioned and tested in many different models, and on a range of tumor targets. This huge effort has culminated in the primacy of an oncolytic HSV (oHSV) being the first oncolytic virus to be approved by the FDA and EMA for clinical use, for the treatment of advanced melanoma. The path has just been opened; many more cancer types with poor prognosis await effective and innovative therapies, and oHSVs could provide a promising solution, especially as combination therapies and immunovirotherapies. In this review, we analyze the most recent advances in this field, and try to envision the future ahead of oHSVs.

Within the oncolytic virus field, the extent of virus replication that is essential for immune stimulation to control tumor growth remains unresolved. Using infected cell protein 0 (ICP0)-defective oncolytic Herpes simplex virus type 1 (HSV-1) and HSV-2 viruses (dICP0 and dNLS) that show differences in their in vitro replication and cytotoxicity, we investigated the inherent features of oncolytic HSV viruses that are required for potent antitumor activity. In vitro, the HSV-2 vectors showed rapid cytotoxicity despite lower viral burst sizes compared to HSV-1 vectors. In vivo, although both of the dICP0 vectors initially replicated to a similar level, HSV-1 dICP0 was rapidly cleared from the tumors. In spite of this rapid clearance, HSV-1 dICP0 treatment conferred significant survival benefit. HSV-1 dICP0–treated tumors showed significantly higher levels of danger-associated molecular patterns that correlated with higher numbers of antigen-presenting cells within the tumor and increased antigen-specific CD8+ T-cell levels in the peripheral blood. This study suggests that, at least in the context of oncolytic HSV, the initial stages of immunogenic virus replication leading to activation of antitumor immunity are more important than persistence of a replicating virus within the tumor. This knowledge provides important insight for the design of therapeutically successful oncolytic viruses.