Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Optimal delivery of RNA interference by viral vectors for cancer therapy.

In recent years, there has been a surge in the innovative modification and application of the viral vector-based gene therapy field. Significant and consistent improvements in the engineering, delivery, and safety of viral vectors have set the stage for their application as RNA interference (RNAi) delivery tools. Viral vector-based delivery of RNAi has made remarkable breakthroughs in the treatment of several debilitating diseases and disorders (e.g., neurological diseases); however, their novelty has yet to be fully applied and utilized for the treatment of cancer. This review highlights the most promising and emerging viral vector delivery tools for RNAi therapeutics while discussing the variables limiting their success and suitability for cancer therapy. Specifically, we outline different integrating and non-integrating viral platforms used for gene delivery, currently employed RNAi targets for anti-cancer effect, and various strategies used to optimize the safety and efficacy of these RNAi therapeutics. Most importantly, we provide great insight into what challenges exist in their application as cancer therapeutics and how these challenges can be effectively navigated to advance the field.

Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

Single-dose replicating poxvirus vector-based RBD vaccine drives robust humoral and T cell immune response against SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines.

Extracellular Vesicles and Viruses: Two Intertwined Entities.

Viruses share many attributes in common with extracellular vesicles (EVs). The cellular machinery that is used for EV production, packaging of substrates and secretion is also commonly manipulated by viruses for replication, assembly and egress. Viruses can increase EV production or manipulate EVs to spread their own genetic material or proteins, while EVs can play a key role in regulating viral infections by transporting immunomodulatory molecules and viral antigens to initiate antiviral immune responses. Ultimately, the interactions between EVs and viruses are highly interconnected, which has led to interesting discoveries in their associated roles in the progression of different diseases, as well as the new promise of combinational therapeutics. In this review, we summarize the relationships between viruses and EVs and discuss major developments from the past five years in the engineering of virus-EV therapies.

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Characterization of Critical Determinants of ACE2-SARS CoV-2 RBD Interaction.

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. In this study, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2, by utilizing our recently developed NanoBiT technology-based biosensor as well as pseudotyped-virus infectivity assays. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, efficacy of competitive inhibitors, as well as neutralizing antibody activity. We also look at the implications the mutations may have on virus transmissibility, host susceptibility, and the virus transmission path to humans. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.

Concise Review: Targeting Cancer Stem Cells and Their Supporting Niche Using Oncolytic Viruses.

Cancer stem cells (CSCs) have the capacity to self-renew and differentiate to give rise to heterogenous cancer cell lineages in solid tumors. These CSC populations are associated with metastasis, tumor relapse, and resistance to conventional anticancer therapies. Here, we focus on the use of oncolytic viruses (OVs) to target CSCs as well as the OV-driven interferon production in the tumor microenvironment (TME) that can repress CSC properties. We explore the ability of OVs to deliver combinations of immune-modulating therapeutic transgenes, such as immune checkpoint inhibitor antibodies. In particular, we highlight the advantages of virally encoded bi-specific T cell engagers (BiTEs) to not only target cell-surface markers on CSCs, but also tumor-associated antigens on contributing components of the surrounding TME and other cancer cells. We also highlight the crucial role of combination anticancer treatments, evidenced by synergy of OV-delivered BiTEs and chimeric-antigen receptor T cell therapy. Stem Cells 2019;37:716-723.

Differential recruitment of E3 ubiquitin ligase complexes regulates RET isoform internalization.

The RET receptor tyrosine kinase is implicated in normal development and cancer. RET is expressed as two isoforms, RET9 and RET51, with unique C-terminal tail sequences that recruit distinct protein complexes to mediate signals. Upon activation, RET isoforms are internalized with distinct kinetics, suggesting differences in regulation. Here, we demonstrate that RET9 and RET51 differ in their abilities to recruit E3 ubiquitin ligases to their unique C-termini. RET51, but not RET9, interacts with, and is ubiquitylated by CBL, which is recruited through interactions with the GRB2 adaptor protein. RET51 internalization was not affected by CBL knockout but was delayed in GRB2-depleted cells. In contrast, RET9 ubiquitylation requires phosphorylation-dependent changes in accessibility of key RET9 C-terminal binding motifs that facilitate interactions with multiple adaptor proteins, including GRB10 and SHANK2, to recruit the NEDD4 ubiquitin ligase. We showed that NEDD4-mediated ubiquitylation is required for RET9 localization to clathrin-coated pits and subsequent internalization. Our data establish differences in the mechanisms of RET9 and RET51 ubiquitylation and internalization that may influence the strength and duration of RET isoform signals and cellular outputs.This article has an associated First Person interview with the first authors of the paper.

Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 µ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.

Loss of Tumour Suppressor TMEM127 Drives RET-mediated Transformation Through Disrupted Membrane Dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTK) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumour pheochromocytoma (PCC) can be caused by activating mutations of the RET receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumour suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability, and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Syngeneic mouse model of human HER2+ metastatic breast cancer for the evaluation of trastuzumab emtansine combined with oncolytic rhabdovirus.

Background: Established mouse models of HER2+ cancer are based on the over-expression of rodent Neu/Erbb2 homologues, which are incompatible with human HER2 (huHER2) targeted therapeutics. Additionally, the use of immune-deficient xenograft or transgenic models precludes assessment of native anti-tumour immune responses. These hurdles have been a challenge for our understanding of the immune mechanisms behind huHER2-targeting immunotherapies. Methods: To assess the immune impacts of our huHER2-targeted combination strategy, we generated a syngeneic mouse model of huHER2+ breast cancer, using a truncated form of huHER2, HER2T. Following validation of this model, we next treated tumour-bearing with our immunotherapy strategy: oncolytic vesicular stomatitis virus (VSVΔ51) with clinically approved antibody-drug conjugate targeting huHER2, trastuzumab emtansine (T-DM1). We assessed efficacy through tumour control, survival, and immune analyses. Results: The generated truncated HER2T construct was non-immunogenic in wildtype BALB/c mice upon expression in murine mammary carcinoma 4T1.2 cells. Treatment of 4T1.2-HER2T tumours with VSVΔ51+T-DM1 yielded robust curative efficacy compared to controls, and broad immunologic memory. Interrogation of anti-tumour immunity revealed tumour infiltration by CD4+ T cells, and activation of B, NK, and dendritic cell responses, as well as tumour-reactive serum IgG. Conclusions: The 4T1.2-HER2T model was used to evaluate the anti-tumour immune responses following our complex pharmacoviral treatment strategy. These data demonstrate utility of the syngeneic HER2T model for assessment of huHER2-targeted therapies in an immune-competent in vivo setting. We further demonstrated that HER2T can be implemented in multiple other syngeneic tumour models, including but not limited to colorectal and ovarian models. These data also suggest that the HER2T platform may be used to assess a range of surface-HER2T targeting approaches, such as CAR-T, T-cell engagers, antibodies, or even retargeted oncolytic viruses.

A T cell-targeted multi-antigen vaccine generates robust cellular and humoral immunity against SARS-CoV-2 infection.

SARS-CoV-2, the etiological agent behind the coronavirus disease 2019 (COVID-19) pandemic, has continued to mutate and create new variants with increased resistance against the WHO-approved spike-based vaccines. With a significant portion of the worldwide population still unvaccinated and with waning immunity against newly emerging variants, there is a pressing need to develop novel vaccines that provide broader and longer-lasting protection. To generate broader protective immunity against COVID-19, we developed our second-generation vaccinia virus-based COVID-19 vaccine, TOH-VAC-2, encoded with modified versions of the spike (S) and nucleocapsid (N) proteins as well as a unique poly-epitope antigen that contains immunodominant T cell epitopes from seven different SARS-CoV-2 proteins. We show that the poly-epitope antigen restimulates T cells from the PBMCs of individuals formerly infected with SARS-CoV-2. In mice, TOH-VAC-2 vaccination produces high titers of S- and N-specific antibodies and generates robust T cell immunity against S, N, and poly-epitope antigens. The immunity generated from TOH-VAC-2 is also capable of protecting mice from heterologous challenge with recombinant VSV viruses that express the same SARS-CoV-2 antigens. Altogether, these findings demonstrate the effectiveness of our versatile vaccine platform as an alternative or complementary approach to current vaccines.

Engineering Rapalog-Inducible Genetic Switches Based on Split-T7 Polymerase to Regulate Oncolytic Virus-Driven Production of Tumour-Localized IL-12 for Anti-Cancer Immunotherapy.

The approval of different cytokines as anti-neoplastic agents has been challenged by dose-limiting toxicities. Although reducing dose levels affords improved tolerability, efficacy is precluded at these suboptimal doses. Strategies combining cytokines with oncolytic viruses have proven to elicit potent survival benefits in vivo, despite promoting rapid clearance of the oncolytic virus itself. Herein, we developed an inducible expression system based on a Split-T7 RNA polymerase for oncolytic poxviruses to regulate the spatial and temporal expression of a beneficial transgene. This expression system utilizes approved anti-neoplastic rapamycin analogues for transgene induction. This treatment regimen thus offers a triple anti-tumour effect through the oncolytic virus, the induced transgene, and the pharmacologic inducer itself. More specifically, we designed our therapeutic transgene by fusing a tumour-targeting chlorotoxin (CLTX) peptide to interleukin-12 (IL-12), and demonstrated that the constructs were functional and cancer-selective. We next encoded this construct into the oncolytic vaccinia virus strain Copenhagen (VV-iIL-12mCLTX), and were able to demonstrate significantly improved survival in multiple syngeneic murine tumour models through both localized and systemic virus administration, in combination with rapalogs. In summary, our findings demonstrate that rapalog-inducible genetic switches based on Split-T7 polymerase allow for regulation of the oncolytic virus-driven production of tumour-localized IL-12 for improved anti-cancer immunotherapy.

Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies.

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design.

Fatty acid transport protein inhibition sensitizes breast and ovarian cancers to oncolytic virus therapy via lipid modulation of the tumor microenvironment.

Introduction: Adipocytes in the tumour microenvironment are highly dynamic cells that have an established role in tumour progression, but their impact on anti-cancer therapy resistance is becoming increasingly difficult to overlook. Methods: We investigated the role of adipose tissue and adipocytes in response to oncolytic virus (OV) therapy in adipose-rich tumours such as breast and ovarian neoplasms. Results: We show that secreted products in adipocyte-conditioned medium significantly impairs productive virus infection and OV-driven cell death. This effect was not due to the direct neutralization of virions or inhibition of OV entry into host cells. Instead, further investigation of adipocyte secreted factors demonstrated that adipocyte-mediated OV resistance is primarily a lipid-driven phenomenon. When lipid moieties are depleted from the adipocyte-conditioned medium, cancer cells are re-sensitized to OV-mediated destruction. We further demonstrated that blocking fatty acid uptake by cancer cells, in a combinatorial strategy with virotherapy, has clinical translational potential to overcome adipocyte-mediated OV resistance. Discussion: Our findings indicate that while adipocyte secreted factors can impede OV infection, the impairment of OV treatment efficacy can be overcome by modulating lipid flux in the tumour milieu.

Oncolytic virus driven T-cell-based combination immunotherapy platform for colorectal cancer.

Colorectal cancer is the third most diagnosed cancer and the second leading cause of cancer mortality worldwide, highlighting an urgent need for new therapeutic options and combination strategies for patients. The orchestration of potent T cell responses against human cancers is necessary for effective antitumour immunity. However, regression of a limited number of cancers has been induced by immune checkpoint inhibitors, T cell engagers (TCEs) and/or oncolytic viruses. Although one TCE has been FDA-approved for the treatment of hematological malignancies, many challenges exist for the treatment of solid cancers. Here, we show that TCEs targeting CEACAM5 and CD3 stimulate robust activation of CD4 and CD8-positive T cells in in vitro co-culture models with colorectal cancer cells, but in vivo efficacy is hindered by a lack of TCE retention in the tumour microenvironment and short TCE half-life, as demonstrated by HiBiT bioluminescent TCE-tagging technology. To overcome these limitations, we engineered Bispecific Engager Viruses, or BEVirs, a novel tumour-targeted vaccinia virus platform for intra-tumour delivery of these immunomodulatory molecules. We characterized virus-mediated TCE-secretion, TCE specificity and functionality from infected colorectal cancer cells and patient tumour samples, as well as TCE cytotoxicity in spheroid models, in the presence and absence of T cells. Importantly, we show regression of colorectal tumours in both syngeneic and xenograft mouse models. Our data suggest that a different profile of cytokines may contribute to the pro-inflammatory and immune effects driven by T cells in the tumour microenvironment to provide long-lasting immunity and abscopal effects. We establish combination regimens with immune checkpoint inhibitors for aggressive colorectal peritoneal metastases. We also observe a significant reduction in lung metastases of colorectal tumours through intravenous delivery of our oncolytic virus driven T-cell based combination immunotherapy to target colorectal tumours and FAP-positive stromal cells or CTLA4-positive Treg cells in the tumour microenvironment. In summary, we devised a novel combination strategy for the treatment of colorectal cancers using oncolytic vaccinia virus to enhance immune-payload delivery and boost T cell responses within tumours.

CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

Virally programmed extracellular vesicles sensitize cancer cells to oncolytic virus and small molecule therapy.

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or "cancer-killing" viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.