Oncolytic virus

An oncolytic virus is a naturally occurring or genetically modified virus that selectively replicates in and lyses cancer cells while sparing healthy tissues, thereby inducing tumor destruction and stimulating antitumor immune responses.^[1] Oncolytic viruses exploit inherent vulnerabilities in cancer cells, such as defective antiviral signaling pathways (e.g., impaired interferon responses) and overexpressed receptors, allowing preferential infection and replication within tumors.^[1] This process triggers direct oncolysis—cell lysis—releasing tumor antigens and danger signals that activate innate and adaptive immunity, including natural killer cells, T cells, and dendritic cells, to target both local and distant metastases.^[1] Additionally, engineered OVs can express immunostimulatory transgenes, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), to enhance immune infiltration into immunosuppressive tumor microenvironments.^[2] The concept of oncolytic virotherapy dates back to the early 20th century, with anecdotal reports of tumor regressions following viral infections in cancer patients, but systematic research began in the 1940s–1950s using wild-type viruses like hepatitis or adenoviruses in clinical trials.^[2] Advances in genetic engineering during the 1990s enabled the creation of tumor-selective OVs, such as the first modified herpes simplex virus (HSV-1) in 1991 for brain tumors, shifting the field toward safer, more targeted agents.^[2] By the 2000s, regulatory approvals marked a turning point: Rigvir (an echovirus type 7 enterovirus; approved in Latvia in 2004 for melanoma; suspended in 2019 due to quality control issues),^[3] followed by H101 (an adenovirus; approved in China in 2005 for head and neck cancers).^[1] To date, four oncolytic viruses have received regulatory approval worldwide, though availability varies by region: Rigvir (2004, Latvia; suspended in 2019 due to quality control issues),^[3] H101 (2005, China), talimogene laherparepvec (T-VEC, an HSV-1 variant; FDA-approved in 2015 for advanced melanoma and EMA-approved in 2016), and Delytact (G47Δ, an HSV-1; approved in Japan in 2021 for malignant glioma). No additional approvals have been granted worldwide as of November 2025.^[4] T-VEC remains the only FDA-approved oncolytic virus, administered intratumorally to express GM-CSF for immune enhancement.^[2] By 2022, over 300 clinical trials investigating oncolytic viruses had been registered globally, predominantly in phase I/II, exploring OVs in combination with immunotherapies (e.g., PD-1 inhibitors), chemotherapy, and radiation for various solid tumors, including melanoma, glioblastoma, and bladder cancer, with promising response rates in immunogenic malignancies.^[1]

Definition and Fundamentals

Core Principles of Oncolytic Activity

Oncolytic viruses are genetically modified or naturally selective viruses that preferentially replicate in tumor cells, leading to their destruction through lysis while sparing normal cells.^[1] This selective tropism arises from inherent differences between malignant and healthy tissues, allowing the viruses to serve as targeted therapeutic agents in cancer treatment.^[1] The fundamental process of oncolysis begins with viral entry into the cancer cell, followed by genome uncoating and replication using the host's cellular machinery.^[1] As viral progeny accumulate, they disrupt cellular integrity, resulting in membrane rupture and release of new infectious particles into the tumor microenvironment.^[1] This propagation amplifies the lytic effect locally, propagating the antitumor activity without systemic dissemination in healthy tissues.^[1] Cancer cells' selective vulnerability to oncolytic viruses stems from common molecular defects, such as impaired interferon (IFN) signaling pathways that normally mount an antiviral response in healthy cells.^[1] These defects, including mutations in genes like IFNAR1 or STAT1, reduce the tumor cell's ability to activate protective mechanisms like RNA interference or apoptosis upon viral infection, enabling unchecked replication.^[5] Consequently, viruses exploit these weaknesses to achieve tumor-specific amplification.^[1] The term "oncolytic" originates from the Greek "onkos," meaning mass or tumor, combined with "lytic," denoting dissolution or breakdown.^[6] Oncolytic viruses are classified based on their genetic material into DNA viruses, primarily double-stranded DNA types, and RNA viruses, mainly single-stranded RNA variants, which influences their replication strategies and engineering potential.^[1]

Selective Targeting of Cancer Cells

Oncolytic viruses exhibit selective targeting of cancer cells through exploitation of tumor-specific molecular vulnerabilities that are rare or absent in normal tissues. Many cancers harbor mutations in the RAS signaling pathway, which inactivate protein kinase R (PKR), a key antiviral defense mechanism; this allows viruses like reovirus to replicate preferentially in RAS-transformed cells while being restricted in healthy cells with intact PKR activity.^[1] Similarly, dysfunction in the p53 tumor suppressor protein, common in over 50% of human cancers, enables adenoviruses such as ONYX-015—lacking the E1B-55K gene—to replicate in p53-deficient cells but not in normal p53-proficient ones, as p53 normally promotes viral clearance.^[7] Overexpression of epidermal growth factor receptor (EGFR) in tumors like glioblastoma facilitates entry and replication of viruses such as vaccinia virus, which hijacks EGFR-RAS signaling to upregulate thymidine kinase essential for viral DNA synthesis.^[1] Receptor-mediated entry further enhances selectivity, as cancer cells often upregulate surface receptors that serve as viral attachment points. For instance, coxsackievirus A21 binds to intercellular adhesion molecule-1 (ICAM-1) and decay-accelerating factor (DAF), both overexpressed on various carcinoma cells, allowing efficient infection while sparing normal cells with lower receptor density.^[7] Herpes simplex virus type 1 (HSV-1) variants target herpesvirus entry mediator (HVEM), which is elevated in melanomas and hepatocellular carcinomas, promoting tumor-specific tropism.^[1] Adenoviruses exploit coxsackievirus and adenovirus receptor (CAR) or CD46, which are dysregulated in transformed cells, to achieve preferential entry.^[1] Cancer cells' evasion of apoptosis provides another layer of selectivity, as oncolytic viruses rely on delayed cell death to complete their replication cycle. Tumors with defective apoptotic pathways, such as those involving p53 mutations or upregulated anti-apoptotic proteins like Bcl-xL, permit viruses like Newcastle disease virus (NDV) to propagate without premature host cell suicide, contrasting with normal cells that rapidly undergo apoptosis post-infection.^[1] HSV-1 mutants, such as R3616, further capitalize on high RAS activity in cancers, which inhibits PKR and delays apoptosis via γ34.5 gene deletion effects.^[7] Tumor microenvironment factors, including hypoxia and acidosis, also favor viral propagation in malignant tissues. Hypoxic regions, prevalent in solid tumors due to poor vascularization, activate hypoxia-inducible factors (HIFs) that enhance viral gene expression; for example, adenovirus CNHK500 incorporates a hypoxia-responsive promoter (5HRE) to drive selective replication in low-oxygen environments while remaining quiescent in normoxic normal tissues.^[7] The acidic extracellular pH in tumors (typically 6.5–6.8), resulting from lactic acid accumulation via the Warburg effect, can facilitate viral entry and infectivity for certain oncolytic viruses compared to the neutral pH (7.4) of healthy tissues.^[1] The therapeutic index of oncolytic viruses—defined as the ratio of antitumor efficacy to toxicity in normal tissues—underpins their clinical viability, often exceeding that of traditional chemotherapeutics due to inherent tumor selectivity. This index is amplified by the viruses' reliance on cancer-specific defects, ensuring robust replication and lysis in tumors with minimal off-target effects, as demonstrated in preclinical models where viral titers in tumors were orders of magnitude higher than in adjacent normal cells.^[8]

Historical Development

Early Observations and Preclinical Studies

The earliest observations of oncolytic activity date back to the early 20th century, when clinicians noted spontaneous tumor regressions in cancer patients coincident with viral infections. In 1912, Italian physician Nicola De Pace reported marked regression of cervical carcinomas in three patients following administration of live rabies virus vaccine, attributing the effect to the virus's destructive action on malignant tissue while sparing normal cells.^[9] Similar anecdotal cases emerged through the 1940s and 1950s, including remissions of leukemia and Hodgkin's lymphoma during outbreaks of measles, mumps, and influenza, prompting speculation that viruses could selectively target hyperproliferative cancer cells.^[10] These serendipitous findings, documented in case reports from institutions like Memorial Hospital in New York, laid the groundwork for intentional viral experimentation but were limited by inconsistent outcomes and lack of mechanistic understanding.^[11] Preclinical studies in the mid-20th century shifted focus to controlled animal models to validate these observations. In 1940, surgeon George T. Pack observed partial remission in a human patient with malignant melanoma following administration of rabies vaccine after a dog bite, which supported the clinical anecdotes.^[12] More systematically, Alice E. Moore at the Sloan-Kettering Institute pioneered oncolytic testing in rodents starting in 1949, demonstrating that the Russian Far East encephalitis virus completely regressed transplantable mouse sarcoma 180 tumors in vivo, with no apparent damage to healthy tissues in surviving animals.^[13] Moore's work, extended through the 1950s to viruses like vesicular stomatitis and Columbia SK, highlighted dose-dependent efficacy and tumor-specific lysis in immunocompromised mouse models, establishing viruses as viable anticancer agents.^[14] These experiments, often using subcutaneous or intraperitoneal tumor implants, reported regression rates up to 100% in select cases, though systemic toxicity remained a challenge.^[15] By the 1950s and into the 1970s, preclinical research expanded to diverse animal species and tumor types, refining the concept of spontaneous oncolysis. Studies with naturally occurring viruses, such as adenoviruses in Syrian hamsters and reoviruses in mice, confirmed selective replication in neoplastic tissues.^[10] Key figures like Chester Southam collaborated with Moore to test over 20 viral strains in rodent models, observing that oncolytic effects correlated with tumor vascularity and host immune status.^[11] Initial hypotheses posited that cancer cells' rapid division and defective antiviral responses—particularly impaired interferon production—enabled preferential viral entry and propagation, as evidenced by in vitro assays showing 10- to 100-fold higher viral yields in malignant versus normal cells.^[13] These ideas, formalized in reviews by the 1970s, emphasized metabolic vulnerabilities in transformed cells as the basis for selectivity, paving the way for targeted virotherapy without genetic modification.^[10]

Key Milestones in Clinical Translation

The clinical translation of oncolytic viruses began with exploratory human trials using wild-type viruses in the mid-20th century, gaining momentum in the 1970s through the 1990s as researchers tested their safety and preliminary efficacy in cancer patients. During the 1970s, several wild-type viruses, including adenoviruses, were administered directly to patients with various solid tumors, marking some of the earliest deliberate attempts at oncolytic virotherapy following earlier spontaneous observations. These trials, often small-scale and focused on viruses like type 7 adenovirus for conditions such as cervical cancer, demonstrated tolerable safety profiles but inconsistent antitumor responses, prompting a shift toward more controlled studies. By the 1990s, preclinical engineering efforts for herpes simplex virus (HSV) variants, such as deletions in neurovirulence genes, transitioned to Phase I trials; for instance, the first-in-human study of engineered HSV1716 began in the late 1990s for patients with recurrent malignant glioma, establishing feasibility and paving the way for subsequent generations of modified viruses.^[16][10][17] A significant milestone in the 2000s was the regulatory approval of the first oncolytic viruses, beginning with non-engineered agents and progressing to recombinant ones. In 2004, Latvia approved Rigvir, a wild-type echovirus type 7 enterovirus, for the treatment of melanoma; it was later withdrawn in 2019 due to lack of confirmatory data.^[1] In November 2005, China's State Food and Drug Administration approved H101 (Oncorine), a genetically modified E1B-deleted adenovirus, for the treatment of head and neck squamous cell carcinoma in combination with chemotherapy. This approval, based on Phase III trials showing improved response rates compared to chemotherapy alone, represented the world's first licensed recombinant oncolytic viral therapy and spurred global interest in adenovirus-based approaches. H101's success underscored the advantages of tumor-selective replication while minimizing damage to normal tissues, influencing subsequent international development efforts.^[18][19] The 2010s saw further advancements with approvals in major markets, solidifying oncolytic viruses as a viable cancer therapy modality. In October 2015, the U.S. Food and Drug Administration (FDA) approved talimogene laherparepvec (T-VEC, Imlygic), an engineered HSV-1 expressing granulocyte-macrophage colony-stimulating factor, for the local treatment of unresectable cutaneous, subcutaneous, and nodal lesions in patients with advanced melanoma recurrent after initial surgery. This approval stemmed from the Phase III OPTiM trial, which reported a 16.3% durable response rate versus 2.1% with granulocyte-macrophage colony-stimulating factor alone, marking the first oncolytic virus therapy approved by the FDA. Building on this, in June 2021, Japan's Ministry of Health, Labour and Welfare granted conditional approval to G47Δ (teserpaturev, Delytact), a triple-mutated HSV-1, for unresectable malignant glioma in patients with residual or recurrent tumors post-surgery and radiotherapy. Supported by a Phase II trial demonstrating an 84.2% one-year survival rate, this approval expanded oncolytic options for brain cancers.^[20][21][22] In the 2020s, clinical translation has accelerated with expanded trials emphasizing combination therapies and regional approvals, reflecting maturing evidence of efficacy. The approval of G47Δ in Japan further catalyzed investigations into HSV variants, with ongoing Phase III studies exploring their integration with immunotherapies; for example, the IGNYTE-3 trial, initiated in 2024 and continuing into 2025, evaluates RP1 (vusolimogene oderparepvec), an oncolytic HSV-1 armed with immune-modulating transgenes, in combination with nivolumab for advanced melanoma, aiming to enhance response durability in immunotherapy-resistant patients. These efforts, alongside Phase III investigations of other HSV-based agents in solid tumors, highlight a focus on synergistic combinations with checkpoint inhibitors and chemotherapy to broaden therapeutic impact across cancer types.^[23][24]

Mechanisms of Action

Direct Viral Replication and Lysis

Oncolytic viruses exert their primary antitumor effect through direct infection and replication within cancer cells, culminating in cell lysis and release of progeny virions to propagate the cycle. This process exploits the unique molecular defects in tumor cells, such as dysregulated signaling pathways and impaired antiviral responses, allowing selective viral propagation that spares normal cells.^[1] The cytopathic effect arises from the virus hijacking host cellular machinery for its own reproduction, leading to eventual membrane rupture and cell death.^[25] The viral life cycle in tumor cells begins with attachment, where the virus binds to specific surface receptors overexpressed on cancer cells, such as the coxsackie-adenovirus receptor (CAR) for adenoviruses or the low-density lipoprotein receptor for vesicular stomatitis virus (VSV).^[25] Entry follows via receptor-mediated endocytosis or direct membrane fusion, delivering the viral particle into the cell. Genome uncoating then occurs, releasing the nucleic acid into the appropriate cellular compartment—nucleus for DNA viruses or cytoplasm for RNA viruses—where it becomes accessible for transcription and translation.^[1] Replication ensues, with the viral genome directing the synthesis of viral proteins and nucleic acids using hijacked host ribosomes and polymerases. Assembly of new virions takes place concurrently, packaging the replicated genomes into capsids and, for enveloped viruses, acquiring lipid membranes from host organelles. Finally, lysis is triggered, often through viral-induced disruption of cytoskeletal elements and membrane integrity, releasing 100-1000 progeny virions per infected cell, depending on the virus and tumor type.^[25] Viral enzymes play a crucial role in subverting host cell machinery to prioritize virion production over normal cellular functions. For instance, in adenoviruses, early proteins like E1A act as transcriptional activators to drive viral gene expression and disrupt host cell cycle controls, while E1B inhibits apoptosis to prolong the replication window; additionally, viral proteases cleave host proteins to redirect resources toward assembly.^[1] In RNA viruses such as VSV, the large (L) protein functions as an RNA-dependent RNA polymerase to rapidly transcribe and replicate the genome, and matrix proteins contribute to suppressing host transcription, favoring viral dominance; some RNA oncolytics encode proteases that degrade host factors inhibiting replication.^[25] These enzymatic activities ensure efficient takeover, with lysis efficiency often reaching near-complete cell destruction in permissive tumor environments within 24-72 hours post-infection. Replication kinetics differ markedly between DNA and RNA oncolytic viruses, influencing their therapeutic profiles. DNA viruses, exemplified by adenovirus, replicate in the nucleus and exhibit slower kinetics, typically completing a cycle in 24-48 hours due to dependence on host DNA polymerase and phased gene expression; this allows for larger genomes and genetic modifications but may limit spread in rapidly proliferating tumors.^[1][25] In contrast, RNA viruses like VSV replicate entirely in the cytoplasm using self-encoded polymerases, enabling faster cycles of 6-12 hours and higher burst rates, which promote rapid amplification but increase risks of off-target effects if selectivity is compromised.

Indirect Immunostimulatory Effects

Oncolytic viruses exert indirect immunostimulatory effects by transforming the tumor microenvironment from an immunosuppressive "cold" state to an inflamed "hot" state, primarily through the induction of immunogenic cell death (ICD) in infected cancer cells. This process is triggered following direct viral replication and lysis, which releases damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1) and calreticulin from dying tumor cells. These DAMPs act as danger signals that promote the maturation and activation of antigen-presenting cells, including dendritic cells, thereby enhancing immune recognition of tumor antigens.^[26][27] Studies have shown that HMGB1 binds to Toll-like receptor 4 (TLR4) on dendritic cells, facilitating their recruitment and upregulation of costimulatory molecules, while calreticulin exposure on the cell surface serves as an "eat-me" signal to promote phagocytosis of tumor debris.^[28] In addition to DAMPs, oncolytic viruses release pathogen-associated molecular patterns (PAMPs), such as double-stranded RNA (dsRNA), which activate innate immune pathways. These PAMPs are recognized by pattern recognition receptors including TLRs (e.g., TLR3) and retinoic acid-inducible gene I (RIG-I), triggering signaling cascades that culminate in the production of type I interferons (IFNs), such as IFN-α and IFN-β. Type I IFNs amplify innate responses by stimulating natural killer cell activity and cytokine secretion (e.g., TNF-α and IL-12), while also priming the tumor site for subsequent adaptive immunity.^[26][27] This innate activation is crucial for breaking tumor-induced tolerance and has been demonstrated in preclinical models where RIG-I agonists derived from viral PAMPs enhanced antiviral and antitumor interferon responses.^[28] The immunostimulatory cascade extends to adaptive immunity, where dendritic cells cross-present tumor antigens derived from lysed cells to CD8+ T cells, leading to their priming, proliferation, and infiltration into the tumor. This results in a robust cytotoxic T-cell response that targets both infected and uninfected cancer cells. Furthermore, systemic immune activation can produce abscopal effects, wherein regression occurs in distant, non-injected tumors due to circulating antitumor T cells and cytokines. Clinical evidence from talimogene laherparepvec (T-VEC), an engineered herpes simplex virus, supports this, with observed responses in non-injected lesions correlating with increased T-cell infiltration.^[26][27][28] The immune response to oncolytic viruses has a dual nature: while it initially serves as a barrier through antiviral mechanisms like neutralizing antibodies and type I IFN-mediated clearance, which can limit viral spread, this same response can be harnessed to favor antitumor immunity. Engineered modifications, such as deletion of viral genes that antagonize IFN signaling or insertion of immunomodulatory transgenes (e.g., GM-CSF), help modulate this balance, reducing premature viral elimination while amplifying antitumor effects. Preclinical and early clinical data indicate that such strategies enhance T-cell persistence and overcome immunosuppressive checkpoints in the tumor microenvironment.^[26][28]

Naturally Occurring Oncolytic Viruses

Vaccinia Virus

Vaccinia virus (VACV) is a large, enveloped, double-stranded DNA virus belonging to the genus Orthopoxvirus in the family Poxviridae, characterized by a genome of approximately 190 kilobase pairs encoding around 200 genes.^[29] It exhibits a broad host range, capable of infecting a variety of mammalian species, and replicates entirely in the host cell cytoplasm, producing multiple virion forms including intracellular mature virus, cell-associated enveloped virus, and extracellular enveloped virus that facilitate systemic spread.^[29] Historically, wild-type VACV strains, such as the Lister and Western Reserve strains, were instrumental as the primary vaccine against smallpox, enabling the World Health Organization's global eradication campaign that declared the disease eliminated in 1980.^[29] As a naturally occurring oncolytic agent, wild-type VACV demonstrates preferential replication and lysis in cancer cells, particularly within hypoxic and angiogenic tumor environments, where defective interferon responses and altered signaling pathways impair antiviral defenses.^[30] This selectivity is enhanced by the virus's dependency on epidermal growth factor receptor (EGFR) activation, which is often upregulated in malignant cells and promotes VACV entry and replication.^[31] Additionally, VACV's production of extracellular enveloped virions enables efficient vascular dissemination, allowing the virus to exploit the leaky, disorganized vasculature of tumors for targeted delivery and accumulation following intravenous administration.^[30] Preclinical studies in the 1990s established the antitumor potential of wild-type VACV, demonstrating significant tumor regression in mouse models of metastatic disease, including clearance of liver metastases in colorectal cancer xenografts after systemic injection.^[30] These experiments highlighted VACV's ability to selectively colonize and destroy tumor nodules while sparing normal tissues, with high viral titers recovered from metastatic sites compared to healthy organs.^[30] For instance, intravenous delivery in immunocompetent mice led to durable remissions in established hepatic tumors, underscoring the virus's innate tropism without genetic modification.^[30]

Vesicular Stomatitis Virus

Vesicular stomatitis virus (VSV) is a member of the Rhabdoviridae family, characterized as a non-segmented, negative-sense single-stranded RNA virus with a compact 11 kb genome encoding five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large polymerase (L). It primarily infects livestock such as cattle, horses, and pigs, causing mild flu-like vesicular lesions, but exhibits natural attenuation in humans due to the absence of a significant reservoir and low seroprevalence, resulting in minimal pathogenicity beyond occasional lab-acquired infections. This inherent safety profile, combined with its rapid cytoplasmic replication cycle—typically completing within 6-8 hours—and broad tropism via G protein binding to low-density lipoprotein receptors or phospholipids, positions wild-type VSV as a promising naturally occurring oncolytic agent.^[32] The oncolytic selectivity of VSV stems from its exquisite sensitivity to type I interferon (IFN) signaling, a key antiviral defense pathway often defective in cancer cells. In normal cells, VSV infection triggers robust IFN production and signaling through pathways involving STAT1 and IRF proteins, leading to rapid viral clearance and minimal replication. In contrast, many tumor cells harbor mutations or epigenetic silencing in IFN-related genes (e.g., reduced expression of IFN receptors or downstream effectors like PKR), impairing this response and permitting unchecked VSV replication, cytopathic effects, and eventual cell lysis. This mechanism exploits a common vulnerability in cancers, as first demonstrated in diverse human tumor cell lines where VSV selectively induced apoptosis while sparing IFN-competent normal cells.^[33][32] Preclinical studies in the 2000s highlighted VSV's efficacy against brain tumors and metastatic disease. In orthotopic models of glioblastoma in rodents, intravenous administration of wild-type VSV resulted in selective infection and destruction of multifocal tumor masses, including invasive satellite lesions, with limited spread to surrounding normal brain tissue due to intact IFN responses in healthy neurons. Similarly, in immune-competent rat models of orthotopic hepatocellular carcinoma, intratumoral injection of VSV led to robust viral replication within tumors, significant inhibition of tumor growth, and prolonged survival without evident hepatotoxicity in normal liver cells. These findings underscored VSV's potential for treating aggressive, disseminated cancers, with viral titers reaching up to 10^8 plaque-forming units per gram of tumor tissue in responsive models.^[34][35] Early clinical insights from Phase I trials of VSV-based therapies have affirmed its safety profile and capacity for tumor localization upon intratumoral administration. In studies involving patients with hepatocellular carcinoma and other solid tumors, intratumoral injections were well-tolerated, with primarily mild flu-like symptoms and no dose-limiting neurotoxicity, alongside evidence of viral persistence and replication confined to injected lesions as monitored by imaging and biopsy. These observations validate the preclinical selectivity and support further exploration of VSV in oncolytic applications.^[36]

Reovirus

Reovirus, a member of the Reoviridae family, is a non-enveloped virus with a double-stranded RNA genome consisting of 10 segments, organized into two concentric icosahedral capsids approximately 85 nm in diameter.^[37] It is ubiquitous in the environment and commonly detected in human respiratory and gastrointestinal tracts, yet it remains non-pathogenic in immunocompetent individuals, typically causing mild or asymptomatic infections without severe disease.^{[38] [39]} This natural safety profile has positioned reovirus as a promising candidate for oncolytic virotherapy, where its selective replication in transformed cells exploits cancer-specific vulnerabilities.^[40] The oncolytic mechanism of reovirus hinges on the activation of the RAS signaling pathway, a frequent alteration in human cancers. In normal cells, reovirus replication is inhibited by the double-stranded RNA-activated protein kinase (PKR), which phosphorylates eIF2α to block viral protein synthesis.^[41] However, RAS transformation dysregulates this antiviral response, allowing efficient viral replication; specifically, activated RAS enhances endosomal acidification, promoting proteolytic uncoating of the viral outer capsid by lysosomal cathepsins and facilitating subsequent viral disassembly, genome release, and progeny production.^{[42] [43]} This pathway-specific selectivity enables reovirus to target tumors with RAS mutations or upstream activations, such as those in EGFR signaling, without harming healthy tissues.^[44] Preclinical evidence from the 1990s and 2000s established reovirus's efficacy against RAS-driven tumors in mouse models. In a seminal 1998 study, reovirus type 3 Dearing strain selectively replicated in and cleared v-ras-transformed mouse 3T3 cell tumors implanted subcutaneously in severe combined immunodeficiency mice, demonstrating tumor regression without systemic toxicity. For glioma, a 2001 xenograft model using human U-118MG cells in athymic mice showed that intratumoral reovirus injection led to complete tumor clearance in 50% of treated animals, correlating with RAS pathway activation and viral replication within tumors.^[45] Similarly, in prostate cancer models during the early 2000s, reovirus treatment of androgen-independent PC3 xenografts in nude mice resulted in significant tumor growth inhibition and extended survival, attributed to RAS-mediated viral oncolysis.^[46] Translational progress includes early-phase clinical trials assessing reovirus safety and activity in specific cancers. A phase I trial in children with relapsed or refractory extracranial solid tumors administered intravenous reolysin (a clinical-grade reovirus formulation) at escalating doses, confirming tolerability with no maximum tolerated dose reached and evidence of viral clearance from blood within days, supporting further pediatric evaluation.^[47] In head and neck cancers, a phase I/II trial combining reolysin with carboplatin and paclitaxel in patients with recurrent or metastatic squamous cell carcinoma reported an objective response rate of 28% and stable disease in 50%, with viral replication detected in tumor biopsies, indicating preliminary antitumor efficacy.^[48]

Other Wild-Type Examples

Senecavirus A, a porcine picornavirus also known as Seneca Valley virus (SVV-001), shows oncolytic potential through its receptor ANTXR1 (TEM8), which is upregulated in various cancers including sarcomas.^[49] Preclinical models have highlighted its activity against sarcomas and skin cancers, where it induces tumor cell lysis and systemic antitumor immunity without significant toxicity to normal tissues.^[50] It was the first picornavirus evaluated in human clinical trials for oncolytic use, primarily targeting neuroendocrine tumors but with broader preclinical promise. As of 2025, a phase I/II trial is evaluating SVV-001 in combination with nivolumab and ipilimumab for high-grade neuroendocrine tumors.^[51][52] RIGVIR, an adapted wild-type echovirus type 7 (ECHO-7), was approved in Latvia in 2004 for the treatment of melanoma and local skin/subcutaneous metastases, marking one of the earliest regional authorizations for a naturally oncolytic virus; however, its marketing authorization was suspended in Latvia in 2019 due to discrepancies in confirmatory data.^[53][3] Its selectivity for melanoma cells arises from preferential replication in tumors expressing intercellular adhesion molecule-1 (ICAM-1), leading to direct cytolysis and improved survival in retrospective studies of surgically treated patients.^[54][55] Semliki Forest virus (SFV), an alphavirus, demonstrates rapid oncolysis in preclinical models of neuroblastoma due to its high replication rate and induction of apoptosis in tumor cells bearing GD2 antigens.^[56] While effective in lysing neuroblastoma cell lines and enhancing immune responses in vitro, human clinical data remain limited, with most studies confined to animal models and early vector evaluations.^[57][58] Among naturally occurring oncolytic viruses, RNA viruses predominate due to their rapid replication, error-prone genomes that facilitate tumor adaptation, and inherent immunostimulatory properties that amplify antitumor effects.^[1] Regional approvals, such as RIGVIR's in Latvia, underscore the potential for localized deployment of unmodified strains, though global adoption has been slower pending further validation.^[53]

Engineered Oncolytic Viruses

Attenuation and Safety Modifications

Attenuation of oncolytic viruses involves genetic modifications to diminish their virulence in healthy tissues, thereby enhancing safety for therapeutic applications while retaining the capacity for selective replication and lysis within tumor cells.^[1] Common strategies include the deletion or mutation of viral genes critical for efficient replication in normal host cells, which exploits inherent differences in cellular machinery between malignant and non-malignant tissues.^[59] These modifications reduce the risk of uncontrolled viral spread, a primary concern with wild-type viruses that can cause severe infections in immunocompetent individuals.^[60] A prominent example is the deletion of the ICP34.5 gene (also known as γ34.5) in herpes simplex virus type 1 (HSV-1), which encodes a neurovirulence factor that promotes viral protein synthesis shutdown evasion in normal cells. This deletion attenuates the virus by impairing its replication in neurons and other healthy cells, thereby blocking neurovirulence, but allows propagation in transformed cells with disrupted interferon signaling pathways.^[61] Similarly, in adenoviruses, deletion of the E1B-55kDa gene product, as in the dl1520 (also called ONYX-015) construct, impairs late viral mRNA nuclear export in normal cells, allowing selective replication in tumor cells with altered RNA processing pathways. Although originally attributed to p53 deficiencies, the mechanism is p53-independent.^[62] This modification results in reduced viral yield in healthy cells compared to tumor cells, as demonstrated in preclinical models. Safety profiling of attenuated oncolytic viruses typically employs multi-step growth curve assays to quantify replication kinetics, revealing significantly slower viral propagation in normal cells versus robust expansion in tumor cells over multiple cycles.^[59] For instance, ICP34.5-deleted HSV-1 shows orders-of-magnitude lower titers in primary human fibroblasts than in glioma cell lines after successive infections. These assays, often complemented by animal models assessing biodistribution and toxicity, confirm the attenuation's effectiveness in limiting off-target effects.^[63] Regulatory oversight, particularly from the U.S. Food and Drug Administration (FDA), mandates comprehensive preclinical data on attenuation to ensure minimal pathogenicity, including shedding studies that monitor viral excretion and environmental transmission risks to verify containment in patients.^[64] FDA guidelines emphasize demonstrating reduced replication in non-target tissues through in vitro and in vivo assays, alongside genetic stability assessments, to mitigate potential adverse events before advancing to clinical trials.^[63] Such requirements have facilitated the safe development of attenuated viruses like dl1520, which underwent rigorous evaluation for off-target replication prior to human testing.^[65]

Tumor-Specific Targeting Strategies

Tumor-specific targeting strategies in oncolytic viruses aim to enhance selectivity by confining viral replication, entry, or propagation predominantly to malignant cells, thereby minimizing damage to healthy tissues. These approaches leverage genetic and molecular engineering to exploit tumor-associated molecular alterations, such as aberrant gene expression or receptor overexpression, building on the inherent selectivity of some viruses while improving precision. Key methods include transcriptional control, modification of viral entry mechanisms, arming with homing transgenes, and directed evolution techniques.^[1] Transcriptional targeting restricts viral gene expression to tumor cells by placing essential viral genes under the control of tumor-specific promoters. These promoters, such as the human telomerase reverse transcriptase (hTERT) promoter or the survivin promoter, are active in cancer cells due to their reliance on dysregulated pathways like telomere maintenance and anti-apoptotic signaling, but remain quiescent in normal cells. For instance, in adenoviral vectors, the E1A gene—crucial for viral replication—has been driven by the E2F-1 promoter to target retinoblastoma (Rb) pathway-defective tumors, demonstrating selective replication in glioma cells. Similarly, prostate-specific antigen (PSA) promoters have been used in adenoviruses to limit replication in prostate cancer models. This strategy has been pivotal in engineering viruses like CG0070, where the E1A gene is controlled by the E2F-1 promoter, enhancing safety in clinical settings.^[7][1][66] Retargeting viral entry, also known as transductional targeting, involves modifying the viral capsid or envelope proteins to redirect attachment and infection toward tumor-specific receptors while ablating binding to normal cell receptors. This is achieved through genetic alterations, such as incorporating ligands or single-chain antibodies into viral surface proteins. A prominent example is the retargeting of herpes simplex virus (HSV) glycoprotein D to the urokinase plasminogen activator receptor (uPAR), which is overexpressed on various tumor cells, allowing selective entry into malignant tissues like gliomas. In adenoviruses, fiber knob domain modifications have been used to target integrins or other tumor-associated receptors, reducing off-target effects in non-cancerous cells. Poliovirus variants have also been engineered to exploit CD155, a receptor upregulated in many cancers, for enhanced tropism. These modifications improve the therapeutic index by ensuring viruses primarily infect and lyse tumor cells.^[7][1][66] Arming oncolytic viruses with transgenes that promote tumor homing further refines specificity by enabling the virus or its progeny to navigate toward or within the tumor microenvironment. This involves inserting genes that encode factors inhibiting angiogenesis or recruiting immune cells to tumor sites, such as interleukin-12 (IL-12), which suppresses vascular endothelial growth factor (VEGF) and enhances localized immune responses. For example, the adenovirus VG161, armed with IL-12, has shown targeted accumulation in hepatocellular carcinoma models by disrupting tumor vasculature and promoting T-cell infiltration. Similarly, HSV-based G47Δ-mIL12 incorporates murine IL-12 to drive selective immune-mediated homing and clearance of tumor cells in glioblastoma. These transgene insertions are designed to amplify the virus's natural tropism without broadly disseminating to healthy tissues.^[1][66] Directed evolution selects for oncolytic virus variants with improved tumor tropism through iterative passaging in tumor cell cultures under selective pressure, mimicking natural adaptation to favor replication in malignant environments. This process generates mutants with enhanced entry efficiency or replication kinetics specific to tumor defects, such as deletions in non-essential genes that confer tumor selectivity. For instance, HSV-1 strain HF10, derived through serial passaging, lacks UL43 and UL49.5 genes, resulting in robust replication and spread in tumor xenografts while sparing normal cells. In adenoviruses like ONYX-015, directed approaches have refined E1B deletions for p53-deficient tumor targeting. This method has yielded high-impact variants used in preclinical models, emphasizing empirical optimization over rational design alone.^[7][1][66]

Enhancements for Therapeutic Efficacy

To enhance the therapeutic efficacy of oncolytic viruses beyond direct cell lysis, engineers insert genetic payloads that induce additional antitumor mechanisms, such as localized toxicity, vascular disruption, or imaging-guided monitoring, while leveraging prior tumor-specific targeting to ensure selective delivery.^[67] These modifications amplify immune responses and tumor destruction without compromising viral replication. One key approach involves incorporating suicide genes, which convert non-toxic prodrugs into cytotoxic agents within infected tumor cells. The herpes simplex virus thymidine kinase (HSV-TK) gene is a prominent example, where the viral enzyme phosphorylates ganciclovir (GCV) into a monophosphate form that is further activated by cellular kinases into a triphosphate analog, incorporating into DNA and causing chain termination and apoptosis. This system exhibits a potent bystander effect, as the toxic metabolites diffuse to neighboring uninfected tumor cells, enabling broader killing even if viral infection is incomplete. Preclinical studies with HSV-TK-armed adenoviruses in prostate cancer models demonstrated significant tumor regression when combined with GCV, with up to 80% reduction in tumor volume compared to virus alone.^[67] A phase II trial of Ad5-yCD/mutTKSR39rep-ADP combined with intensity-modulated radiation therapy (IMRT) for intermediate-risk prostate cancer showed safety and a 42% relative reduction in positive prostate biopsies at 2 years compared to IMRT alone.^[68] Anti-angiogenic payloads further boost efficacy by disrupting tumor vasculature, starving hypoxic regions and potentiating viral spread. Oncolytic viruses engineered to express endostatin, a fragment of collagen XVIII that inhibits vascular endothelial growth factor (VEGF) signaling and endothelial cell migration, have shown marked antitumor activity in preclinical models.^[69] For instance, an oncolytic herpes simplex virus (HSV-Endo) delivering endostatin suppressed angiogenesis and reduced tumor burden by 70% in human colon cancer xenografts.^[69] Similarly, thrombospondin-1, which binds CD36 on endothelial cells to block proliferation and induce apoptosis, has been incorporated into oncolytic adenoviruses, leading to normalized tumor vessels and enhanced viral replication in head and neck squamous cell carcinoma models.^[70] A vaccinia virus variant expressing an endostatin-angiostatin fusion protein achieved complete tumor eradication in 50% of treated pancreatic cancer xenografts, outperforming unarmed controls.^[71] Reporter genes enable non-invasive monitoring of viral biodistribution and replication kinetics, optimizing dosing and assessing treatment response in real time. Green fluorescent protein (GFP) facilitates fluorescence imaging to visualize viral spread in superficial or explanted tumors, as demonstrated in oncolytic adenovirus models where GFP expression correlated with infection sites in murine xenografts.^[72] Luciferase reporters, such as firefly luciferase, support bioluminescence imaging (BLI) for deeper tissue penetration, quantifying viral persistence with high sensitivity; in reovirus studies, luciferase-armed variants revealed peak replication at 48 hours post-infection in glioma models.^[73] These tools have accelerated preclinical optimization, with BLI detecting as few as 10 infected cells and guiding clinical translation by confirming tumor-selective localization. For radioenhancement, the sodium iodide symporter (NIS) gene is inserted to mediate uptake of radioiodides like iodine-131 (¹³¹I), concentrating radiation within tumors for synergistic virotherapy. NIS, a transmembrane glycoprotein, actively transports iodide into infected cells via the sodium gradient, enabling SPECT/PET imaging and targeted radiotherapy.^[74] In oncolytic measles virus (MV-NIS), expression in glioblastoma xenografts resulted in 50-fold higher ¹³¹I accumulation compared to non-transduced cells, enhancing cell killing through beta-particle emission and the crossfire effect on adjacent stroma.^[75] This radiosensitization extended median survival in murine models from 25 to 45 days, with dosimetry confirming therapeutic doses without systemic toxicity.^[75] Similar efficacy was observed in herpes simplex virus-NIS constructs for hepatocellular carcinoma, where ¹³¹I uptake amplified oncolysis by 2-3 fold. Recent advances include the use of CRISPR/Cas9 to engineer more precise modifications in oncolytic viruses, such as enhanced immune-modulating payloads or tumor-specific insertions, and innovative platforms like engineered bacteria to temporally control OV release and activity, demonstrating improved efficacy in preclinical solid tumor models as of 2025.^[76][77]

Clinical Applications

Approved Therapies

Talimogene laherparepvec (T-VEC), marketed as Imlygic, is a genetically modified herpes simplex virus type 1 (HSV-1) engineered to selectively replicate in melanoma cells and express granulocyte-macrophage colony-stimulating factor (GM-CSF) to enhance antitumor immunity. The U.S. Food and Drug Administration (FDA) approved T-VEC in October 2015 as the first oncolytic viral therapy for the local treatment of unresectable cutaneous, subcutaneous, and nodal lesions in patients with melanoma recurrent after initial surgery. It is administered via intratumoral injection, starting with an initial dose of up to 4 mL of 10^6 PFU/mL followed by 4 mL of 10^8 PFU/mL three weeks later, and then every two weeks until no injectable lesions remain or intolerance occurs. In the pivotal phase III OPTiM trial, T-VEC demonstrated a durable response rate of 16.3% compared to 2.1% with subcutaneous GM-CSF alone, with responses lasting at least six months; median overall survival was 23.3 months versus 18.9 months, establishing its clinical benefit in advanced melanoma.^[21][78] Oncorine (H101) is a recombinant adenovirus type 5 with a deletion in the E1B-55K gene, enabling selective replication in p53-deficient tumor cells commonly found in head and neck cancers. The China State Food and Drug Administration (SFDA, now National Medical Products Administration) approved H101 in November 2005 for the treatment of late-stage refractory head and neck squamous cell carcinoma. It is typically administered intratumorally in combination with chemotherapy, such as cisplatin and 5-fluorouracil, at doses of 1 × 10^12 viral particles per injection for up to six cycles. A phase III randomized trial showed that H101 combined with chemotherapy achieved an objective response rate of 78.8% versus 39.6% with chemotherapy alone, with significant improvements in short-term survival and quality of life metrics, supporting its approval for enhancing tumor regression and patient outcomes in this indication.^[79][80] Teserpaturev (G47Δ), marketed as Delytact, is a third-generation oncolytic HSV-1 with triple mutations (deletions in ICP6, ICP34.5, and insertion of LacZ in ICP34.5) to improve tumor selectivity, enhance antitumor immunity, and ensure safety by preventing replication in normal cells. Japan's Ministry of Health, Labour and Welfare granted conditional approval for G47Δ in June 2021 for the treatment of recurrent or residual malignant glioma in patients over 5 years old, following surgical resection or radiation, as the first oncolytic virus approved in that country. The therapy is delivered via stereotactic intratumoral injection at a dose of 3 × 10^8 PFU/mL, up to 10 times over several months, often post-standard care like temozolomide. In a phase II single-arm trial of 19 patients with recurrent glioblastoma, G47Δ achieved a one-year survival rate of 84.2%, exceeding the historical 15% rate, with a favorable safety profile showing no severe adverse events related to viral replication and only mild flu-like symptoms in most cases.^[22][81] RIGVIR is a naturally occurring, non-genetically modified echovirus type 7 (ECHO-7) strain adapted for oncolytic activity, selectively targeting and lysing melanoma cells while sparing healthy tissue. It received national approval in Latvia, an EU member state, in 2004 for the treatment of melanoma skin cancer stages IB-IIC, as well as local treatment of skin and subcutaneous metastases from advanced melanoma. Administered intramuscularly or subcutaneously at doses escalating from 10^6 to 10^10 TCID50 weekly, then biweekly for maintenance, RIGVIR was used in over 500 melanoma patients in Latvia, with post-marketing data indicating prolonged overall survival in early-stage patients—such as a 4- to 6.6-fold reduction in disease progression risk for stage IIB compared to untreated cohorts—without reported serious adverse effects. However, in 2019, Latvia's State Agency of Medicines withdrew RIGVIR's registration due to insufficient evidence from randomized controlled trials demonstrating clinical efficacy and safety.^[53][82][83]

Investigational Therapies

As of 2025, the clinical trial landscape for oncolytic viruses encompasses nearly 190 registered investigations globally, with a substantial focus on rare and refractory solid tumors, including hepatocellular carcinoma, glioblastoma, and pediatric malignancies. These trials predominantly evaluate engineered viruses in phases I and II, emphasizing safety, antitumor efficacy, and immune activation in patients who have progressed on standard therapies.^[84][85] VG161, a recombinant oncolytic herpes simplex virus type 1 (HSV-1) armed with interleukin-12 (IL-12), IL-15/IL-15 receptor α fusion protein, and a PD-L1 blocking peptide, is under evaluation in phase I/II trials for advanced primary liver cancer refractory to prior therapies. In a phase I study of 35 patients with heavily pretreated hepatocellular carcinoma (HCC), intratumoral VG161 administration yielded an objective response rate (ORR) of 17.14% and a disease control rate (DCR) of 60%, with a median overall survival of 9.4 months and no dose-limiting toxicities observed.^[86][87] Ongoing phase II efforts, including combinations with immune checkpoint inhibitors like camrelizumab, report median progression-free survival of 6.3 months and 6-month overall survival rates of 87.5% in second-line HCC settings, highlighting VG161's potential to remodel the tumor immune microenvironment.^[88][89] Pexa-Vec (JX-594), an engineered vaccinia virus featuring thymidine kinase gene deletion and granulocyte-macrophage colony-stimulating factor (GM-CSF) insertion, demonstrated encouraging phase II results in advanced HCC, including statistically significant overall survival improvements with intratumoral dosing followed by best supportive care. However, the subsequent phase III PHOCUS trial, which tested sequential Pexa-Vec plus sorafenib versus sorafenib alone in 600 patients, failed to meet its primary endpoint of overall survival benefit, leading to halted development despite prior successes in immune activation and tumor necrosis.^[90][91][92] MG1MA3-CA, a replication-competent oncolytic Maraba rhabdovirus expressing the MAGE-A3 tumor antigen, is being assessed in phase I/II trials for incurable, advanced MAGE-A3-positive solid tumors such as melanoma and breast cancer. Early phase I data from 41 patients revealed safe intravenous and intratumoral delivery, with antitumor immunity evidenced by MAGE-A3-specific CD8+ T-cell responses exceeding 1% of circulating T cells in 50% of evaluated cases, correlating with clinical benefit in select responders.^[93][94] Emerging candidates in 2025 include VSV-IFNβ-NIS, an armed vesicular stomatitis virus expressing interferon-β and the sodium iodide symporter, advanced to multiple phase I trials for solid tumors, with ongoing evaluations in glioblastoma to enhance tumor selectivity and imaging capabilities while minimizing neurotoxicity. Similarly, parvovirus H-1PV, known for its non-pathogenic profile and oncolytic effects via cell cycle arrest and immune stimulation, has progressed to phase II trials in recurrent glioblastoma and other adult malignancies, building on preclinical data demonstrating cytotoxicity in medulloblastoma models through downregulation of oncogenic pathways like MYC and NFIA. Recent phase III advancements include cretostimogene grenadenorepvec (CG0070), which showed a 75.2% complete response rate in non-muscle invasive bladder cancer in 2024, and CAN-2409, which met its primary endpoint of improved disease-free survival in prostate cancer in late 2024.^{[1][95][96][97][98][99]}

Combination with Other Cancer Treatments

Integration with Immunotherapies

Oncolytic viruses (OVs) induce immunogenic cell death in tumor cells, releasing tumor-associated antigens and damage-associated molecular patterns that trigger innate and adaptive immune responses, thereby transforming immunosuppressive "cold" tumors into inflamed "hot" ones responsive to immune checkpoint inhibitors (ICIs) such as PD-1/PD-L1 blockers.^[100] This inflammation enhances T-cell infiltration and activation, countering the tumor microenvironment's inhibitory signals and amplifying the efficacy of ICIs.^[101] For instance, talimogene laherparepvec (T-VEC), an engineered herpes simplex virus, has been combined with pembrolizumab in trials demonstrating this priming effect in melanoma patients.^[102] Key clinical trials underscore this synergy, notably the phase Ib portion of MASTERKEY-265, where T-VEC plus pembrolizumab yielded an objective response rate (ORR) of approximately 62% in patients with unresectable stage IIIB-IV melanoma, surpassing historical pembrolizumab monotherapy rates of around 33-40%.^[103] Long-term follow-up from this trial reported a complete response rate of 43% and a 4-year overall survival of 71.4%, with durable responses in over 90% of responders.^[104] Although the subsequent phase III MASTERKEY-265 did not meet its primary endpoint for progression-free survival improvement (ORR 48.6% vs. 41.3% for placebo plus pembrolizumab), it confirmed the combination's tolerability and suggested benefits in subgroups with injectable lesions.^[105] Mechanistically, OVs promote tumor-infiltrating lymphocyte (TIL) expansion and neoantigen release, which bolsters the antitumor activity of chimeric antigen receptor (CAR)-T cells by improving their trafficking and persistence within solid tumors.^[106] This antigen spillover also augments cancer vaccine efficacy, as demonstrated in preclinical models where OV-induced neoantigens enhanced T-cell priming and systemic immunity when paired with mRNA vaccines.^[107] Such effects address CAR-T limitations in immunosuppressive environments, leading to improved tumor clearance in combination settings.^[108] As of 2025, emerging combinations integrate OVs with bispecific antibodies targeting immune checkpoints or tumor antigens, enhancing T-cell redirection and OV-mediated lysis in refractory cancers.^[109] Additionally, pairings with engineered cytokine therapies, such as IL-2 variants, amplify OV-stimulated immune responses while minimizing systemic toxicity, as seen in ongoing trials for solid tumors where these approaches yield higher response durability.^[110] In 2025 updates, pelareorep combined with atezolizumab and chemotherapy in first-line pancreatic ductal adenocarcinoma showed promising progression-free survival improvements in phase II (BRACELET-1).^[111] These advances highlight OVs' role in broadening immunotherapy applicability beyond checkpoint inhibition.^[4]

Synergy with Chemotherapy and Radiation

Oncolytic viruses (OVs) enhance the efficacy of chemotherapeutic agents by sensitizing tumor cells to drug-induced cytotoxicity, often through disruption of cell cycle checkpoints and promotion of apoptosis. For instance, OVs can exploit chemotherapy-induced DNA damage to amplify viral replication and tumor cell death, while certain chemotherapeutics suppress antiviral immune responses, allowing greater OV propagation within the tumor microenvironment. This bidirectional synergy overcomes resistance mechanisms, such as cell cycle arrest, where OVs interfere with repair pathways, making cells more vulnerable to agents like cisplatin.^[112][113] A representative example is the combination of reovirus (pelareorep) with gemcitabine in pancreatic adenocarcinoma, where the virus sensitizes cancer cells by inducing mitotic catastrophe and enhancing drug uptake via upregulated receptors, leading to improved antitumor activity. In a phase II trial, this regimen yielded a median overall survival of 10.2 months (95% CI 6.6-14.1) compared to historical controls of 6.7 months, with manageable toxicity profiles including fatigue and nausea. Similarly, oncolytic adenoviruses paired with cisplatin demonstrate enhanced replication through upregulation of cellular factors like GADD34, resulting in greater tumor regression in preclinical models of head and neck cancer.^[114][113] OVs also synergize with radiation therapy by leveraging the DNA damage response (DDR) to boost viral replication and oncolysis. Radiation induces double-strand breaks that activate DDR pathways, which OVs exploit to increase progeny virus production and extend tumor cell killing beyond direct lysis. In herpes simplex virus (HSV)-based therapies, viral proteins like ICP0 degrade DNA repair enzymes such as DNA-PKcs, further potentiating radiotherapy effects in radioresistant tumors like glioma. A phase I trial of oncolytic HSV-1 (G207) administered 24 hours prior to 5 Gy radiation in recurrent glioblastoma showed safety with no encephalitis cases and radiographic responses in 3 of 9 patients, achieving a median survival of 7.5 months. Early clinical data from such OV-radiation combinations demonstrate safety and potential efficacy in recurrent high-grade gliomas.^[115][116] Clinical evidence supports optimized timing in OV-chemotherapy and OV-radiation combinations to maximize synergy while minimizing off-target effects. For example, pre-treatment with OVs 24-48 hours before chemotherapy or radiation allows viral replication to peak during peak drug or radiation exposure, enhancing local tumor control. In ovarian cancer, a phase 1b trial of oncolytic adenovirus TILT-123 combined with pegylated liposomal doxorubicin (a doxorubicin formulation) evaluates safety and preliminary efficacy in platinum-resistant cases, building on preclinical data showing increased apoptosis and immune activation. Such protocols have demonstrated response rates up to 54% in phase II studies of adenovirus virotherapy with chemotherapy in similar settings.^{[116][117][118]} As of 2025, emerging trends focus on nanoparticle-based delivery systems to refine OV combinations with chemotherapy and radiation, enabling timed release and reduced systemic toxicity. Nanoparticles encapsulating viral genomes or whole viruses respond to tumor-specific triggers like hypoxia or pH, achieving controlled dissemination and minimizing immune clearance, with preclinical models showing over 80% tumor regression and 90% lower hepatic uptake compared to free viruses. These formulations support precise scheduling of OV deployment prior to chemo-radiation cycles, potentially broadening applicability in solid tumors while lowering adverse events like fever and chills.^[119]

Challenges and Future Directions

Current Limitations and Obstacles

One of the primary limitations of oncolytic virus therapy is the host's pre-existing antiviral immunity, which often neutralizes the virus before it can effectively reach and replicate within tumors. Neutralizing antibodies against common oncolytic viruses, such as herpes simplex virus (HSV), are present in more than 50% of the population, significantly impairing initial and repeat dosing by promoting rapid viral clearance through complement activation and adaptive immune responses.^[1] This issue is particularly pronounced for viruses like adenovirus type 5, where seroprevalence can reach 50-90% in adults, limiting systemic delivery and therapeutic efficacy in seropositive patients.^[120] High levels of these antibodies have been associated with reduced overall survival, such as a median of 12.5 months in patients with high neutralizing antibody titers compared to 21.2 months in those with low titers.^[121] Delivery challenges further constrain the clinical utility of oncolytic viruses, especially in solid tumors where poor vascularization and dense extracellular matrix impede viral penetration and spread. Intratumoral injection, the predominant administration route, often fails to achieve uniform distribution due to desmoplastic barriers, as seen in pancreatic ductal adenocarcinoma, resulting in limited exposure to metastatic or deep-seated lesions.^[1] Systemic routes like intravenous delivery exacerbate risks of off-target effects and toxicity, including liver accumulation in adenoviruses and potential environmental shedding, with HSV DNA detected in 37% of post-injection wound exudates across trials.^[121] These penetration issues contribute to inconsistent antitumor responses, compounded by immune clearance mechanisms that reduce viral bioavailability.^[76] Tumor heterogeneity poses a significant barrier to broad efficacy, as genetic and microenvironmental variations lead to differential viral replication and immune activation across cancer types. In immunosuppressive tumor microenvironments, such as those with high interferon-stimulated gene expression, response rates drop markedly to 15% compared to 45% in low-expression settings, due to enhanced antiviral defenses that hinder oncolysis.^[121] Similarly, markers like AXL-high expression correlate with only 15% objective response rates, reflecting resistance from regulatory T cells and cytokine suppression that dampen antitumor immunity.^[121] This variability underscores the therapy's challenges in uniformly addressing diverse solid tumors, including those with "cold" immune profiles resistant to viral-induced inflammation.^[76] Regulatory and manufacturing obstacles also impede widespread adoption, with good manufacturing practice (GMP) production facing scalability limitations from reliance on adherent cell cultures that yield insufficient titers for large-scale clinical use.^[122] Biodistribution concerns, including uncontrolled viral dissemination, necessitate rigorous monitoring, as regulatory bodies like the FDA require isolation protocols for shedding exceeding 3 log10 copies per milliliter.^[121] These hurdles, including complex purification and storage requirements under cold chain conditions, delay progression from preclinical to commercial stages.^[1]

Emerging Advances and Research Trends

Recent advances in oncolytic virus (OV) engineering have leveraged CRISPR/Cas9 technology to enable precise arming of viral genomes, facilitating multi-gene insertions that enhance therapeutic potency through synthetic biology approaches. For instance, in large-genome viruses such as herpes simplex virus type 1 (HSV-1), CRISPR/Cas9 allows for the targeted insertion of multiple transgenes, including immune-modulating cytokines and tumor-specific suicide genes, while minimizing off-target effects and improving replication specificity in cancer cells.^[123] This method has been refined with in vitro protocols like Advanced viral genome in vitro Cas9 editing (AdVICE), which supports traceless manipulation of DNA virus genomes up to several megabases, enabling the creation of next-generation OVs with integrated synthetic circuits for conditional activation.^[124] Such innovations address previous limitations in viral payload capacity and safety, paving the way for more robust antitumor responses. Personalized OV therapies are gaining traction through patient-derived models and AI-optimized engineering, allowing for tailored virus selection and modification based on individual tumor profiles. Patient-derived organoids and xenografts serve as platforms to screen OV candidates, identifying those with optimal replication and lysis in specific tumor microenvironments, thereby improving efficacy predictions before clinical use.^[125] Complementing this, AI-driven models integrate multi-omics data to simulate viral dynamics and optimize genome edits, such as predicting immune evasion strategies or enhancing tropism for heterogeneous tumors; for example, hybrid ecological models using time-delayed equations forecast OV-tumor interactions with high accuracy.^[126] These approaches, highlighted in recent reviews, accelerate the transition from bench to bedside by enabling rapid iteration of viral constructs matched to patient genetics.^[127] Novel OV platforms inspired by bacteriophages and incorporating RNA switches are emerging to achieve tighter control over conditional replication, expanding therapeutic versatility. Bacteriophage-inspired designs draw from phage display and lytic cycles to engineer hybrid vectors that selectively target cancer cell surface markers, delivering payloads without infecting healthy tissues; recent studies demonstrate their use in disrupting tumor-promoting biofilms and eliciting antitumor immunity in solid malignancies.^[128] Meanwhile, RNA switch mechanisms, such as those responsive to oncogenic mRNAs or tumor-specific metabolites, regulate viral gene expression for replication only in malignant cells—for instance, engineered influenza A viruses with chemogenetic switches show selective activation in glioma models, reducing systemic toxicity.^[129] These platforms represent a shift toward modular, programmable OVs that integrate synthetic regulatory elements for enhanced precision. As of 2025, the OV pipeline reflects a surge in global clinical trials, with approximately 150 active studies worldwide—roughly double the number initiated around 2020—driven by expanded manufacturing capabilities and regulatory support.^[85] This growth underscores an increased focus on challenging indications, including liquid tumors like leukemia and lymphoma, where engineered OVs such as modified adenoviruses are being tested for intravascular delivery to disseminated cells.^[130] Similarly, trials targeting brain metastases have proliferated, with OVs like DNX-2401 combined with checkpoint inhibitors showing promising infiltration and response rates in glioblastoma models, addressing barriers like the blood-brain barrier through intratumoral or convection-enhanced delivery.^[131] These trends signal a maturing field poised for broader integration into multimodal cancer regimens.