One branch of transduction targeting involves interactions with tumor surface markers, such as the avβ6 integrin, which is expressed in a number of aggressively transformed epithelial cancers but remains undetectable in healthy tissues (62, 63). This approach has been used to modify adenoviruses (Ad or HAdV). Vectors based on serotype 5 (HAdV-C5, Ad5) are the most commonly used. According to Bates et al. the tumor tropism of Ad5 on avβ6 was achieved by genetic insertion of a 20 amino acid peptide, A20 (NAVPNLRRGDLQVLAQKVART), which is native to foot-and-mouth disease virus, into the fiber knob DG protein of Ad5 adenovirus (62). Modified A20 viruses selectively infect cells expressing integrin avβ6 - epithelial carcinoma cells, including ovarian, breast, pancreatic and colorectal cancers (64–66). The avβ6 integrin stimulates tumor metastasis and invasion through TGF-β activation and is associated with poor prognosis (67). Therefore, targeting avβ6 integrin may be beneficial for reducing metastasis. Wild-type adenovirus 5 is characterized by poor tumor selectivity, premature sequestration, and immune inactivation, which reduces the clinical efficacy of HAdV-C5. Specifically, HAdV-C5 binds to the adenovirus CAR receptor, ubiquitously expressed in healthy cells (68), entering via avβ3/5-mediated integrin internalization (69). Additionally, HAdV-C5 interacts with clotting factor X (FX) in the blood via the hexon protein, mediating virus transduction into hepatocytes and leads to hepatotoxicity (70). These unwanted interactions were eliminated by introducing a mutation in each major capsid protein. For instance, a mutation in a fiber called KO1 is known to eliminate CAR binding (71), while RGD > RGE mutation in the penton base prevents binding to avβ3/5 integrins and the hexon responsible for FX binding was modified to prevent interaction with FX (70). In addition to targeting the tumor-associated integrin, these genetic modifications allowed efficient and selective transduction of avβ6+ and epithelial ovarian cancer cell line (EOC). It was confirmed in a human SKOV3 EOC peritoneal xenograft model in mice. Animals receiving the novel oncolytic Ad5NULL-A20 showed significantly better survival compared to animals treated with oncolytic Ad5 without the A20 insert (63). In most populations, the high seroprevalence of Ad5 significantly reduces the efficacy of oncolytics based on it (72). Therefore, Bates et al. (62) applied the same modifications for Ad5NULL-A20 to the rarer serotype HAdV-D10 isolated from the ocular mucosa of patients with conjunctivitis (73), which has a seroprevalence rate of about 10% in a European cohort. Because HAdV-D10 naturally has a very low tropism to CAR, does not utilize DSG2 as a cellular receptor, and has a low, insufficient affinity for CD46 for cell penetration, residual off-target interactions with CAR on platelets and erythrocytes are readily eliminated by KO1 modification (74), and replacement of an RGD > RGE further reduces uptake in the spleen (75). Intratumoral administration of such mutant adenoviruses (HAdV-D10.A20) to mice bearing BT20 xenografts (expressing avβ6 integrin) showed a significant reduction in tumor volume 9 days after administration, compared to placebo and wild-type HAdV-D10 serotype (62). Another example of an oncolytic virus targeting tumor markers involves the targeting of herpes simplex virus (HSV) to the epidermal growth factor receptor (EGFR). Apolloni et al. (76) engineered R-613, the first oncolytic HSV to specifically target EGFRvIII (a variant of the epidermal growth factor receptor commonly mutated in glioblastoma). Additionally, another genetically modified OV, OV-Cmab-CCL5, expresses a single-chain variant antibody fragment specific to EGFR. OV-Cmab-CCL5 is redirected to EGFR - positive glioblastoma (GBM) cells and facilitates continuous production of the cytokine CCL5 in the tumor microenvironment. In addition to efficient targeting, infection of GBM cells with OV-Cmab-CCL5 significantly enhances the migration and activation of natural killer cells, macrophages, and T cells. Furthermore, it suppresses tumor-specific EGFR signaling, reduces tumor size, and prolongs the survival of GBM carrier mice (77). A series of studies focused on retargeting the tropism of herpes simplex virus (HSV) types 1 and 2 to the human epidermal growth factor receptor 2 (HER-2) oncoprotein (78). For instance, Nanni et al. (79) targeted HSV1 to HER-2p185, which is overexpressed in ovarian and breast cancers. In the virus genome, they replaced part of the sequences encoding the receptor-binding glycoprotein gD (a major factor in HSV entry into healthy cells) with antibody fragments targeting the HER-2 oncoprotein overexpressed in human breast and ovarian cancer, thus completely eliminating the natural tropism of HSV. The resulting R-LM249 virus infects and kills exclusively tumor cells that express high levels of human HER-2. Intraperitoneal administration of R-LM249 to immunodeficient mice (Rag2-/- and Il2rg-/-) with implanted human SK-OV-3 ovarian carcinoma cells (a model that mimics the fatal condition in patients with advanced stages of the disease) significantly inhibited carcinomatosis. Sixty percent of the treated mice showed no peritoneal diffusion and the total weight of neoplastic nodules was reduced by 95%. Intraperitoneal administration of R-LM249 also suppressed the growth of HER-2+ breast cancer MDA-MB-453 cells that had metastasized to the brain and ovaries. The authors considered these findings promising for tumor therapy, although they require confirmation in clinical trials (79). The urokinase-type plasminogen activator receptor has also recently attracted interest as a target for oncolytic viruses in pancreatic cancer (80).

A significant number of studies have focused on the construction of viral chimeras (hybrids) - the combination of viral particles and proteins of different viruses to improve the properties of oncolytic viruses (OVs) (81). Chimerism can include any transgenic viruses with combined properties, including “fiber mosaicism” (82), pseudotyping - exchange of envelope glycoproteins or whole capsids (83) ( Figure 3 ) and/or xenotype switching (interspecies combinations of viral capsid components) (84). Vesicular stomatitis virus (VSV) is a good example of an animal virus that has been successfully engineered for safe therapeutic applications in oncology. VSV is capable of infecting a broad spectrum of cell types and demonstrates rapid replication kinetics, which enhances its capacity to disseminate within tumors and induce robust oncolysis. Importantly, VSV is non-pathogenic to humans and exhibits preferential replication in malignant cells due to the impairment of type I interferon (IFN) signaling pathways in cancer cells (85, 86).

One of the most popular trends is the creation of adenovirus hybrid fibers. Adenoviruses attach to the target cell via the knob domain of the trimeric capsid protein fiber. This domain is structurally conserved across different Ads serotypes but utilizes several different receptors. The interaction of Ads with the receptor depends on both fiber length and flexibility (87). For example, fiber length influenced the bio distribution of Ads in mouse models of ovarian cancer, increasing the tumor-to-liver transduction ratio about 10-fold when HAdV-5-based virus was used with a shorter fiber from HAdV-3 to HAdV-5/3 (88). Replacing the HAdV-5 fiber with the HAdV-3 fiber changes not only the tropism to cellular receptors, but also significantly improves binding and cell penetration (89). For example, transduction levels of HAdV-5/3 primary melanoma cells increased by three orders of magnitude compared to HAdV-5 controls (90). Similarly HAdV-5/16 and HAdV-5/50 chimeras show specific tropism to pancreatic cancer cells (91). When combined with transcription control elements, these fiber chimeric viruses exhibit high specificity to target cells, such as the Ad targeting melanoma shown by Rivera et al. (92). Besides HAdV-5 chimeras, HAdV-35 chimeras with other serotypes are popular (93). It is also possible to replace the fiber entirely, provided that it can be incorporated into the capsid. For example, the TRAIL-armed (tumor necrosis factor-related apoptosis-inducing ligand) oncolytic virus HAdV-5/35 induced superior tumor shrinkage after both systemic and intraperitoneal administration in in vivo models of cervical cancer (94). An Ad5LacZ vector, incorporating a mutation in the hexon variable region (HVR7) and pseudo typed with the Ad35 Ad5CMV-HCR5*7*E451Q/F35++ fiber (termed Ad5T*F35++), devoid of FX binding and redirected from CAR to CD46, demonstrated significantly higher transduction of EOC cells compared to the parental Ad5 vector (95). Fiber chimerism can be used to modify transduction specificity in a more indirect way. For example, HAdV-41 has features of both long and short fiber phenotypes. Cell binding and transduction depend exclusively on the long fiber. Ads with short fibers cannot bind to receptors and are eliminated in vitro and in vivo. If HAdV-5 is modified with short fiber HAdV-41 (HAdV-5/41s), it will lead to disadaptation of such viruses (96). As a result, “blind” adenoviruses can function as a framework for retargeting novel receptors (97). The capsid components of viruses can be replaced not only by equivalent domains of species-specific serotypes, but also by components of viruses of other species. The strategy of obtaining interspecific hybrids has been termed “xenotype switching”. Thus, the transduction efficiency and tropism of adenoviruses in vivo were improved by using knob domains from Ad dogs - CAdV-2 (84), or by fiber exchange with Ad sheep - OAdV-7 (84). A HAdV-5-based vector with a chimeric fiber incorporating a HAdV-2 site and a bovine BAdV-4 (Ad5FB4) fiber is of interest, as this fiber binds to membrane proteins of the B7 family overexpressed by murine leukemia cells derived from dormant tumors (98). The possibility of modulating tropism by creating chimeras has also been considered in other viral systems. Pseudotyping of adeno-associated viruses (AAV) to expand their tropism typically involves incorporating a vector genome derived from AAV2 into the capsid of another serotype, such as AAV5 (99). This combination allows for enhanced transduction during gene transfer into the lungs (100). Additionally, Tyrosine residues (101) and other kinase targets such as serine, threonine, and lysine on the AAV capsid can enhance in vitro transduction efficiency and also lead to improved transgene expression (102). Incorporating designed ankyrin repeat proteins (DARPins) (103) or a nanobody (104) into the AAV capsid has enhanced specific targeting of B cells (103). The pseudotyping strategy has also been applied to retroviral and lentiviral vectors, which show low transduction efficiency. Increased specificity can be achieved by exchanging glycoproteins with other enveloped viruses. The first of such a chimera was Moloney Murine Leukemia Virus, pseudotyped with the glycoprotein of vesicular stomatitis virus (VSV-G) (83), characterized by high transduction efficiency and broad tropism. Other glycoproteins for pseudotyping have origins in Sindbis virus (105), paramyxovirus (106), rabies virus (107), and measles virus (MV) (108). Pseudotyping with MV-glycoproteins opens the possibility of antibody-mediated retargeting and enables specific transduction of both antigen-expressing target cells in mixed cell cultures and resting primary lymphocytes (109). In addition, MV-glycoproteins have been utilized to repurpose VSV for virotherapy, in which broad VSV-G-mediated tropism is disadvantageous (110).

Another approach to achieving virus specificity is biochemical retargeting, which involves conjugation with cell-targeting ligands. This includes, for example, the use of bispecific antibodies, biotin-avidin molecular bridges, and pegylation. This approach stands out because it does not lead to complications associated with genetic modifications and allows targeting the virus to several cell receptors simultaneously. The main disadvantage of the method is that the modification is limited to one generation of the virus and is not transmitted to the offspring after replication (111). One example of such modification, Ad5 targets the EGFR, which is overexpressed in many types of head and neck squamous cell carcinoma (HNSCC) (112). Upon binding to one of its seven native ligands, EGFR is activated, dimerises and promotes cell proliferation, contributing to tumor growth (113). To achieve specific targeting of EGFR by human adenovirus type 5 (HAdV-5)-based vectors, the affinity ligand EGFR affilin was covalently attached at a specific position to either the fiber knob or the hexose capsid protein. In vitro and in vivo studies investigated the transduction of EGFR-specific cancer cells as well as susceptibility to natural sequestration mechanisms, pharmacokinetics and biodistribution profiles of affilin-decorated vectors (114). In vitro studies provided results supporting the concept that covalent attachment of a receptor-specific affilin to the adenoviral capsid provides an efficient and versatile tool for adenoviral vectors to target cancer-specific receptors. However, in the case of EGFR as a vector target, transduction into non-target tissues and the low availability of the receptor in tumor tissues prevented effective tumor transduction by affilin-decorated vectors, making EGFR a difficult receptor to target with adenoviral vectors (114). A similar approach to biochemical targeting is the modification of tumor ligands to facilitate interaction with OVs. One of such modifications is sialylation, the enrichment of tumor surface glycans with sialic acid residues. In a study on breast cancer (115) using nanobiophysical approaches, it was shown that overexpression of α-sialylated glycans, by altering their mechanical properties, enabled reovirus attachment and infection in a serotype-dependent manner. Co-injection of α-sialylated glycans and reovirus increased reovirus infectivity in malignant cells. This study confirmed both the use of reoviruses as oncolytic agents in nanomedicine and the role of α-sialylated glycans as adjuvants in enhancing oncolysis, opening new perspectives in oncolytic cancer therapy (115). Recent reviews discuss the topic of cell modification in great detail, particularly in relation to enhancing the efficacy of oncolytic viruses (116).

Transcriptional targeting is most often achieved by placing critical parts of the viral genome under the control of tumor-specific promoters, which are active in tumors but inactive in most normal tissues. Cancer-specific promoters are primarily applicable to engineer DNA-genome OVs (e.g. herpesviruses, adenoviruses). For example, the human epidermal growth factor receptor 2 (HER-2) promoter is often used to regulate the replication of herpes simplex virus (HSV) in glioma cells (79). For HSV-based oncolytics, the detargeting/retargeting strategies used so far are based on genetic modifications of glycoprotein D (gD), where specific amino acid residues (30, 40) are deleted and amino acid 38 is replaced with a single-chain fragment of the scFv antibody to the cancer marker HER-2 (117). HER-1 is also highly suitable for regulation by the surviving promoter (118). Transcriptional control of the viral alpha 4 gene encoding infected cell protein-4 (ICP4) by the Survivin/BIRC5 cellular promoter conferred a tumor cell-restricted replicative potential to the virulent genome of HSV-1 (118). Sasso et al. generated a dual-regulated HSV-1 in which tumor cell-restricted replicative potential was combined with selective transduction via ERBB2 HER-2 receptor retargeting (119). In addition to targeting replication in cancer cells, detargeting the tropism of HSV from natural herpesvirus entry mediator (HVEM) and nectin receptors is used to prevent off-target and extra-tumor infections (120). Combination strategies, including transcriptional retargeting/detargeting and post-transcriptional retargeting, have proven to be highly effective in achieving tumor specificity while preserving virulence of the virus (121). Expression of HSV proteins (such as γ34.5,ICP4, ICP27 and UL8) outside the tumor can be prevented by inserting targeted miRNA sequences or tissue-specific promoters into the virus genome (122). For example, due to the hybrid nestin enhancer-HSP68 minimal promoter, expression of the virulence gene γ34.5 was restricted to nestin-positive glioblastoma cells (123).

The cell mass of solid tumors develops predominantly in a hypoxic environment, therefore, a number of studies (as shown in Table 3 ) have focused on regulating the expression of the toxic adenovirus E1A protein through regulatory elements responsive to hypoxia. The E1A gene was placed under the control of various tissue-specific tumor promoters for controlled cytotoxic effects ( Table 3 ). One of such promoters is the human telomerase promoter (human telomerase reverse transcriptase hTERT, see Table 3 ), as telomerase activity is often higher in cancer cells than in normal cells (133). Another example is the replacement of the E1A promoter with the promoter/enhancer of the melanocyte- and melanoma-specific tyrosinase gene (137). At the same time, mutations were introduced into the E1A gene itself that prevented interaction with the retinoblastoma protein pRb and the coactivating protein p300, thus excluding virus replication in normal cells (AdTyrΔΔ2Δ24). As a result, AdTyrΔΔ2Δ24 showed Ad-specific replication, and killed in vitro exclusively melanoma cells. AdTyrΔΔ2Δ24 was further enhanced by incorporating a tyrosinase promoter to trigger the E4 gene (Ad2Xtyr), and the efficacy of this approach was demonstrated in a number of organotypic in vitro cultures (137). More examples of promoters used for tumor-selective replication of Ads and their expression of E1A are listed in Table 3 .

Host cell proteases are determinants for the replication and pathogenicity of enveloped viruses (111). For example, host proteases activate the envelope protein ENV inHIV-1, the spike glycoprotein of SARS-CoV-2. The proteolytic cleavage of the envelope liberates the functional domains, allowing membrane fusion and entry into the cell (138). The proteases that are overexpressed by cancer cells can be targeted by oncolytic viruses, such as matrix metalloproteinases (MMPs). By attaching blocking ligands to MMP-cleavable linkers at the amino (N)-ends of retroviral glycoproteins, viral transduction was inhibited, providing evidence in favor of MMP activation. The common furin proteinases and/or tryptase from Clara airway secretory cells typically cleave the MV and Sendai virus fusion proteins specifically (139, 140). After reprogramming MV from these proteinases to MMP with minimal structural changes to the fusion protein, recombinant MV expressing the modified fusion protein (MV-MMP) did not multiply or exhibit cytotoxic effects in cells that did not express MMP. When injected into MMP-positive subcutaneous tumors in mice, MV-MMP maintained its complete oncolytic activity. After cerebral injection, MV-MMP did not infect or kill susceptible mice like wild-type MV did, indicating the increased safety of recombinant virus for the nervous system (141). Targeting oncolytic viruses to MMP-positive cancer cells at the level of viral particle activation may be safe and beneficial, as demonstrated by these experiments. Urokinase-type plasminogen activator is another protease that can be targeted in invasive metastatic cancer cells (142).

MicroRNAs (miRNAs) are a class of short (∼22 nucleotides) non-coding RNA molecules that play a crucial role in epigenetic regulation (143). In normal cells, miRNAs regulate processes, such as cell differentiation, proliferation, apoptosis, and other processes. Changes in the expression profile of microRNAs have been found in the development and progression of most malignant neoplasms. They can act as both oncogenes and tumor growth suppressors (144). To date, numerous microRNAs have been identified with both increased and decreased expression in tumors of different locations. Individual microRNAs and their combinations have been proposed as diagnostic markers and prognostic factors, and their potential use as tumor growth inhibitors is being actively explored (145). The strategy for limiting viral pathogenicity to healthy tissues involves using miRNA from normal cells to restrict viral replication. This is done by interacting with miR-specific tissue sites (miRNA-TS) inserted into the viral genome. For example, members of the picornavirus family are promising candidates for cancer treatment due to their potent oncolytic activity. However, their replication is not limited to tumor cells and can occur in a variety of normal tissues. To enhance the safety of these OVs, miRNA-TS, which are expressed in normal tissues but are absent (or minimally present) in cancer cells, were inserted into the 3’ non-coding regions of the viral genome. In normal tissue, miRNAs inhibit viral activity via miRNA-TS in the viral genome by translationally repressing or catalytically degrading viral RNAs (146). This inhibition does not occur in cancer cells, where these miRNAs are absent or depressed at low levels.

Another example of miRNA targeting is the oncolytic vesicular stomatitis virus, which is highly tumor-specific due to its susceptibility to the interferon response in normal tissues. However, VSV is highly neurotoxic not only in rodents but also in non-human primates (147). Therefore, it is necessary to restrict VSV replication in the brain without altering the oncolytic effect of the virus. Kelly et al. (148) inserted a miRNA-125 targeting site into the L gene, which completely rescued cell viability in astrocytes. In contrast, inserting it into the M gene only resulted in a 30% rescue. Impressively, 90% of the mice infected with this recombinant virus survived without exhibiting any neurotoxic effects, and the virus’s oncolytic activity against other cancer cells remained unaffected (149). Additionally, another studydescribes how miRNA-TS can be easily inserted into the RNA of the Coxsackievirus B3 (CVB3) genome using fusion cloning technology (150). See more examples of miRNA and OVs in Table 4 .

A relatively recent non-transduction targeting method has been found in the application of recombination of oncolytic viruses. The method is based on the regulation of gene expression by exposure of transduced cells to light waves of a certain spectrum. The genetic construct responsible for the mechanism for implementing this method is called the optogenetic module (OM). Optogenetic modules incorporated into recombinant oncolytic viruses can help improve tumor selectivity, reduce toxicity, and regulate the expression of immunomodulators, tumor suppressor genes, tumor-associated antigens, and microRNAs. Controlling expression with specific wavelengths of light may, in the future open up new, safe options for treating recurrent cancers with oncolytic viruses. Viral vectors have previously integrated various types of OM. In the study by Hagihara and colleagues, they developed a tumor-specific replication-competent adenovirus in which the expression of adenovirus early region genes 1A (E1A) and 1B (E1B), essential for viral replication, is driven by the human telomerase reverse transcriptase (hTERT) promoter element (183). The E1A and E1B genes were inserted downstream of the Gal4 upstream activator sequence (UASG). When exposed to blue light, the GAVPO complex, comprising the smallest light-oxygen-volt (LOV) protein, VVD, Gal4 (Gal4 residues 1–65), and the p65 activation domain — homodimerizes, allowing Gal4 to bind UASG and subsequently triggers the expression of E1A and E1B (183). The resulting photoactivatable oncolytic adenovirus (paOAd) was evaluated in cancer cell lines (A549, H1299, HepG2) and in a subcutaneous xenograft model (HepG2 or H1299 cells and Rag2/Il2rg double knockout mice). PaOAd demonstrated an oncolytic effect equal to that of the parent adenovirus. A pronounced cytopathic effect induced by paOAd was detected in TERT-positive human small intestinal organoids (SIO). Despite the positive results, the hTERT promoter is not specific for cancer cells, raising concerns about the safety of viral replication in non-cancerous cells (183).

Another group of researchers developed an alternative Opt/Cas-Ad system designed to regulate and enhance a tumor suppressor gene (184). Opt/Cas-Ad is based on a recombinant modified adenoviral vector (AdK7) carrying dCas9 accompanied by a Cry2-CIBN module (Cry2: Cryptochrome-2; CIBN: N-terminal fragment of calcium and integrin-binding protein 1, dCas9: catalytically inactive Cas9). Optically controlled Opt/Cas-Ad increased the expression level of Dkk-3 mRNA under blue light illumination and induced Dkk-3-mediated apoptosis in a PC3 cell xenograft tumor model. As a result, the growth of subcutaneous PC3 tumors in mice was effectively suppressed by oncolytic virus and spatiotemporal light gene expression. The authors suggest that the Opt/Cas-Ad system could improve the efficacy and safety of current virotherapy, as well as expand the therapeutic potential of OM for cancer treatment (184).

However, the prevalence of endogenous antibodies against adenoviruses makes their systemic administration difficult for cancer treatment (185), so the use of other potential oncolytic viruses are preferable. For example, in a different approach, the photo-dimerizing proteins pMag and nMag Magnet (186) were inserted into two flexible polymerase (L) domains of measles virus (MeV) or rabies virus (RABV), thereby reducing the catalytic activity of the polymerase. Blue light-induced heterodimerization of pMag and nMag proteins promotes the formation of the ribonucleoprotein complex (RNA-dependent RNA polymerase, nucleocapsid and cofactor proteins) and triggers viral replication. Recombinant MeV and RABV containing photocontrolled L protein and EGFP showed accelerated replication when activated by blue light. The antitumor potential of MeV with photocontrolled viral L-polymerase (rMeV-EGFP-LDMH) was demonstrated in a xenograft model (MDA-MB-468 cells and BALB/c nu/nu mice). Intratumoral injection of rMeV-EGFP-LDMH, followed by blue light illumination, showed significant suppression of tumor growth with 100% survival in the treated mice (186). Although the virus models discussed demonstrate promising results, a key limitation of these optogenetic modules is their reduced efficacy in clinical applications due to the poor tissue penetration of blue light. In contrast, optogenetic modules that utilize red-shifted light (e.g., REDMAP, iLight, PhyA-FHY1, PhyB-PIF, BphS) offer a more promising and viable alternative, as they can penetrate tissues more effectively (187). Genetic modifications and chemical approaches have been used to improve viral cellular entry through the cancer cell-specific receptors. These modifications are meant to add ligands to the virus for improving the binding to the receptors of the cancer cells, and to block the binding to normal cells. There are some challenges for both approaches, for example, the ligand incompatibility with the symmetrical icosahedral form of some viruses. Both strategies can be used in combination (111).

Prescribing oncolytic viruses (OVs) to patients might still be challenging due to delivery issues. Most OVs require direct injection into tumors (intratumoral delivery) to maximize effectiveness and minimize off-target effects. This method is feasible for accessible tumors like melanoma but is difficult for deep-seated or hard-to-reach tumors, requiring specialized skills and equipment. Intravenous delivery is being explored but faces issues, such as rapid clearance by the immune system and unpredictable dosing at the tumor site.

Pre-existing immunity is another hurdle that may limit OVs’ applications. Some patients may have pre-existing immunity to the virus being used, which can neutralize the OV before it reaches the tumor, reducing therapeutic efficacy.