Genetic engineering of OV genome to improve its anti-cancer selectivity has commenced the new era of oncolytic viral therapy. This can be traced to a publication in 1991, wherein thymidine kinase-deleted HSV-1 (dlsptk) attenuated neurovirulence in murine glioblastoma model (Martuza et al., 1991). Two recent Phase 1/2 clinical trials supports efficacy of single agent oncolytic viral therapy, in turn, backed by more than one anecdotal reports (Kelly and Russell, 2007; Park et al., 2008; Senzer et al., 2009; Eager and Nemunaitis, 2011). One of the most convincing evidence of the ability of oncolytic viruses is to efficiently and augment anticancer immunity. Intratumoral injection was provided by a trial which employed a second generation oHSV-1 Talimogene laherparepvec (T-VEC or Imlygic™) formerly called as oncoVEX GM-CSF. T-VEC was administered intratumorally which resulted in total regression of uninjected and injected lesions of 8/50 patients treated with metastatic malignant melanoma (Senzer et al., 2009).

The efficiency of OV infection is one of the major hurdle limiting the use of OV therapy for solid tumor malignancy (Shintani et al., 2011). OV therapy employs either direct inoculation or systemic administration of OV. In terms of efficacy, direct inoculation of oHSV-1 is the most reliable technique to dispense oHSV-1 to solid tumors. This is because the combination of leaky vasculature and absence of functional lymphatics in the tumor microenvironment, restricts interstitial transportation and prevents delivery of anti-tumor agents, particularly bulky virus vectors (McKee et al., 2006; Wang and Yuan, 2006). Ultrasound technique, is a therapeutically and diagnostically well established technique for over a decade now (Lindner, 2004; Kaufmann et al., 2007). Ultrasound technique apart from its conventional applications is also used to stimulate cell membrane permeabilization and sonoporation to enhance the anti-cancer efficacy of chemotherapeutic agents and to transfer plasmid DNA in-vivo and in-vitro setting (Dooley et al., 1983; Fechheimer et al., 1987). A study by Shintani et al. concluded that admission of oHSV-1 into the host Oral Squamous Cell Carcinoma (SCC) Cells was accelerated with the help of ultrasound technique. Furthermore, the presence of microbubbles improved the effect of ultrasound technique (Shintani et al., 2011). These shreds of evidence suggest that ultrasound technique can enhance the efficiency of oHSV-1 infection in OV therapy for oral SCC. Ultrasound technique also has a significant advantage over the rest in being comparatively non-invasive (Lindner, 2004; Kaufmann et al., 2007).

Newer strategies are being employed to enhance the systemic delivery of oHSV-1. One recent study on human glioma employing Gli36ΔEGFR, U87ΔEGFR and U251T3 cells demonstrated that chelating copper (Cu) could improve the efficacy by increasing serum stability of oHSV and preventing angiogenesis. The study employed three oHSVs, rHSVQ1, rQnestin34.5, and hrR3, to ensure that effect is not virus strain specific or mutation dependent (Yoo et al., 2012). The increase in Cu levels within the blood serum is noticed in several types of human tumors (Turecký et al., 1984). Cu is a vital co-factor for various angiogenic growth factors, such as angiogenin and Vascular Endothelial Growth Factor (VEGF). In addition to this, Cu is also required by tumor cells to secrete numerous angiogenic factors (Soncin et al., 1997; Hu, 1998). Besides favoring angiogenesis, Cu presence in the blood serum is found to prevent wild-type HSV infection (Shishkov et al., 1997; Panteva et al., 1998; Clewell et al., 2012). Taking into account of the fact that Cu in blood serum prevents wild-type HSV infection, this study was done to investigate whether Cu chelation improves the efficiency of oHSV by increasing its blood serum stability and anti-angiogenic effect. ATN-224, a second-generation copper chelating analog of ammonium tetrathiomolybdate reduced inhibition of oHSV mediated by blood serum. As oHSVs are delivered by intratumoral injection in the clinical setting, these results may provide some impetus for systemic delivery of oHSV as concluded by the study (Yoo et al., 2012).

An important drawback of systemic OV delivery is off target viral replication in patients who are immune compromised. To tackle off target viral replication, one effective approach adopted is retargeting the virus infectivity to attain infection of target cell selectively, see Table 1. Retargeting is done by either, i) Modifying present viral coat proteins or glycoproteins to include single chain antibodies (scFv) or peptide ligands that attach to the required receptor. ii) Utilizing soluble adapters which identify both the oncolytic virus and an exclusive receptor on the target cell. iii) Inserting glycoproteins having distinct host range from other viruses. For retargeting of HSV, several stratagems are employed considering the challenges involved in retargeting HSV. Figure 1 shows process of systemic delivery of oncolytic HSV-1 to the tumor cells by retargeting, which includes eliminating the ingress using the native gD receptors of HSV at the same time attaining infections similar to the ones attained with wt-HSV on vulnerable cells. (Goins et al., 2016). A recent study demonstrated that nectin-1:scFv anti-CEA adapter (soluble bridging molecule) enhances transduction efficacy and produces three times reduction in tumor volume when compared to no adaptor control animals bearing human gastric carcinoma MKN45 tumors in the flank. Infection of HSV-resistant Chinese hamster ovary (CHO) cells expressing ectopic carcinoembryonic antigen (CEA) and vector alone resistant nectin-1/CEA-harboring human gastric carcinoma cells was successfully demonstrated by this adapter (Baek et al., 2011). Another interesting study which employed vascular stomatitis virus-G glycoprotein (VSV-G) to replace either HSV-1 gB (Tang et al., 2001) or gD (Anderson et al., 2000) displayed promising results. When VSV-G was replaced with gD, the entry proficiency of the resultant pseudotyped virus relegated, besides it entered the cell exclusively through endocytosis and most of the virions were trapped and destroyed through the acidic pH of the endosomal pathway. In contrast, when VSV-G was replaced with HSV-1 gB, entry of HSV-1 was comparable with that of wt-HSV in rat striatum. Here VSV-G acts like a fusogen for HSV-1 entrance in the cell due to its structural correspondence to HSV-1 gB. This report gives strong evidence that the HSV-1 entry machinery can be engineered by substituting different glycoproteins with functional substitutes. These in turn produce infectious virions that bring about the cell entry guided by alternate proteins in the cells expressing complementary receptors (Goins et al., 2016).

Retargeted oHSVs have been proven efficacious when it comes to systemic delivery of oHSV. To further increase the effectiveness of retargeted oHSVs, the viral protein cargo is packaged with the help of carrier cells to deliver it effectively to the tumor site. The early research employed irradiated tumor cell, which was later discontinued taking in safety considerations (Munguia et al., 2008; Park et al., 2008; Russell and Peng, 2008; Willmon et al., 2009). The packaging of oHSVs with the carrier can be done either by loading onto the cells or by incorporating into the cell's compartments. The virus particles can be delivered by the carrier cells in three ways, (a) Tumor microenvironment can be used to attract carrier cells. (b) Cytokine-induced killer carrier cells or tumor specific carrier T cells can be used to target tumor cells particularly. (c) In the third method the tumor cells are not targeted by the carrier cell, but the tumor location is targeted e.g., lymph nodes. Mesenchymal stem cells appear to be promising carriers belonging to the former group wherein they are accumulated in the tumor stroma due to the tumor microenvironment having the hypoxic environment and inflammatory cytokine expression associated with the tumor. Limitations being the prevention of virus infection to the carrier cells due to the oHSV-1 specificity and mesenchymal stem cell transferred adoptively may itself feed the tumor (Willmon et al., 2009). A study in 2015 showed delivery of R-LM249 via MSCs is achievable and efficient. One problem faced was MSCs could be barely infected with HER-2 retargeted R-LM249, but the problem was solved by forcibly infecting MSCs with retargeted R-LM249. Also, therapeutic results were reaped with single i.v dose of R-LM249 infected MSCs in lung and brain metastases. The study concluded achievable and efficient delivery of carrier-mediated (MSCs) delivery of retargeted oHSV (Leoni et al., 2015).

Pre-treating blood serum with cyclophosphamide/venom of cobra to exhaust blood serum factors like complement proteins and IgM to which HSV binds readily decreases sequestration and increases systemic availability (Chiocca et al., 1999; Ikeda et al., 2000; Wakimoto et al., 2002). A recombinant HSV-1, hrR3, with U_L39 deleted, selectively replicates in cancerous cells with significant anticancer activity and results in better survival in animal models of brain (Mineta et al., 1994; Jacobs et al., 2001), pancreas (Kasuya et al., 1999), colon (Yoon et al., 2000) and liver cancers (Pawlik et al., 2000). It was conjectured that a strong immune suppressant like cyclophosphamide might ad interim repress systemic immunity for viruses, permitting better replication of the therapeutic virus and augment the anticancer effect (Ikeda et al., 1999). Co-administration of cyclophosphamide, an antineoplastic and immune suppressive agent, proved to be efficacious as it improved the anticancer activity of hrR3 (Ikeda et al., 1999; Wakimoto et al., 2004).

In the case of solid tumor malignancies, it is not considered advantageous to dispense OV by systemic route considering that the tumor is usually isolated from general systemic circulation and delivering the OV systemically subjects patient's full body to the virus inflicting the entire body to the danger of undesirable clinical circumstances. As cancer patients already have suppressed systemic immunity, administration of virus might trigger clinical complications which might not appear in healthy animals (Coffey and Thompson, 2002). Intratumoral injection of oHSV-1 is one of the most reliable techniques of administering oHSV-1 (Shintani et al., 2011). It was believed that injecting OV intratumorally results in the generation of more virus particles by the malignant cells and the newly generated virus particles shortly replicate in every individual tumor cell by expanding through the tumor. Soon it was found that reovirus killed malignant cells and brought about local necrosis on single intratumoral injection while the tumor cells distant from the injection site continued to proliferate indicating the spread of the virus was not efficient enough. These finding sparked the need to developed new strategies of intratumoral injection to increase virus spread. One such strategy was drawn up to deliver HSV-1 vectors. The strategy consisted of OV delivery to multiple tumor sites or the OV could be injected intratumorally at a single site but in a volume large enough which is capable of getting to more malignant cells. When the OV is being injected at multiple sites inside the tumor mass, it is advised that at least 3–5 sites are injected. In the case of single injection at a single site, the composition of OV should be between 10 and 100% of the tumor mass volume (Coffey and Thompson, 2002). The tumor microenvironment is dense in connective tissue (Sauthoff et al., 2003; Yun, 2008). Losartan, U.S FDA approved Ag-II antagonist improves the reach of oHSV into the tumor mass via disturbing transforming growth factor beta 1 (TGF-β1) signaling and subsequently declining production of stromal collagen in the tumor microenvironment of human breast, pancreatic and skin desmoplastic tumor models in mice (Diop-Frimpong et al., 2011).

A neoteric approach to enhance oHSV-1 development in malignant cells is by using chemical sensitizers. Small molecules as chemical sensitizers neutralize the residual antiviral activities within the tumor cells that are resistant to the OV. Histone deacetylase (HDAC) inhibitors were shown to repress residual Interferon (IFN) responsiveness by the tumor cell in turn enhancing the efficacy of oHSV-1 with no compromise in the oHSV-1 specificity (Chang et al., 2004; Nguyên et al., 2008; Otsuki et al., 2008; MacTavish et al., 2010). A couple of small molecules, dilazep and dipyridamole, were identified in a high throughput screening with attenuated HSV lacking ribonucleotide reductase. Both the small molecules improved virus growth and decreased tumor growth in athymic mice bearing subcutaneous Du145 tumors by inhibiting cellular equilibrative nucleoside transporter-1 (ENT-1) eventually resulting in induction of cellular ribonucleotide reductase (Passer et al., 2010).

A key hallmark of cancer, immune escape by the tumors, presents a valuable target in the field novel cancer therapeutics. HSV-1 armed with GM-CSF (T-VEC) proved to be successful in treating cancers all due to the recruitment of immune cells coupled with confined oncolytic activity (Burke, 2010; Melcher et al., 2011). Phase I clinical trial of HSV armed with GM-CSF was conducted in patients with either cutaneous or s.c gastrointestinal, breast and head and neck deposits of cancer resulted in extensive immune cell infiltration as revealed by post-treatment biopsies (Hu et al., 2006). A range of cytokines have been used with oHSV to modify and enhance antitumor immunity including GM-CSF, IL12, IFN-α and Tumor necrosis factor (TNF-α) but GM-CSF has reproduced the most reliable and best results. IL-12, IFN-α and TNF-α have also shown promise in preclinical cancer studies (Liu et al., 2003). A study in murine HER-2/neu breast cancer cells, TUBO, in-vitro and in-vivo employed HSV-1 and HSV-2 ICP0 mutants to investigate the effect of oncolytic viruses on the immune system. The concluded, extended replication of the oncolytic virus within the tumor cells is insignificant, but the initial stages of immunogenic virus replication are more important which leads to activation of antitumor immunity (Workenhe et al., 2014). A study on A20 lymphoma tumors in BALB/c mice reported that oncolytic HSV-1 (GM-CSF inserted into ICP34.5/ICP47 deleted strain of HSV-1, JS1) serves as patient-specific tumor vaccine by the liberation of tumor antigens when administered intratumorally. The virus had antitumor immune effects on both injected and uninjected tumors (Liu et al., 2003). A recent interesting study reported that oHSV-2 can induce cell death of colorectal cancer cells both in-vitro and in-vivo along with recruiting adaptive immune response (Yin et al., 2017).

HSVs are “armed” with genes that can enhance antitumor cytolytic activity. Most commonly oHSV-1 is armed with immunostimulatory molecules. Apart from immunostimulatory molecules oHSV-1 is also armed with prodrug converting enzymes, angiogenesis inhibitors, suicide genes and fusogen membrane glycoproteins (Shen and Nemunaitis, 2006), see Table 1. (i) Oncolytic HSV-1 armed with Immunomodulatory molecules- oHSV-1 armed with soluble B7-1, IL-12 or IL-18 were developed and displayed similar replication profile as to the wt-HSV in-vitro. The in-vivo efficacy was evaluated in A/J mice bearing syngeneic, poorly immunogenic tumors of Neuro2a neuroblastoma. IL-12 happened to have the greatest efficacy among the three expressed by G207 (double mutated oHSV-1). The triple combination proved to be more efficient than either double or single combination of the armed oncolytic viruses (Ino et al., 2006). (ii) Oncolytic HSV-1 armed with anti-angiogenic molecule- rQt3, an oHSV-1 armed with metalloproteinases 3 tissue inhibitor. rQt3 produced immature collagen extracellular matrix increasing peak infection level of the virus with a remarkable reduction in tumor vascular density resulting in decreased endothelial progenitor cell circulation and delayed the growth of the tumor in athymic mice carrying malignant peripheral nerve sheath or human neuroblastoma tumor (Mahller et al., 2008). (iii) Oncolytic HSV-1 armed with fusogenic membrane glycoprotein: arming oHSV-1 with fusogenic membrane glycoproteins might result in syncytium formation by the neighboring uninfected cells, in turn, facilitating the killing of the tumor cell. There are some toxicity concerns on the uninfected tissues (Todo, 2008). (iv) Oncolytic HSV-1 armed with Suicide genes/Pro-drug converting enzymes: an oHSV-1 (MGH2) genetically modified to express two prodrug-activating genes, secreted human intestinal carboxylesterase and CYP2B, wherein each yields active metabolite of irinotecan and cyclophosphamide. The study concluded, MGH2 proves to be effective in preclinical glioma models in combination with chemotherapeutic agents (Tyminski et al., 2005). (v) Oncolytic HSV armed for in-vivo non-invasive imaging. Two oHSV-1 were created expressing firefly luciferase with either immediate early 4/5 promoter or gC promoter for non-invasive in-vivo imaging. Athymic mice carrying subcutaneous Gli36ΔEGFR glioma tumors were then studied with super sensitive charged coupled device camera for luciferase expression. Luciferase expressed by IE4/5 promoter was linked with oHSV-1 infection as IE4/5 promoter acts immediately post viral infection. Luciferase expressed by strict late gC promoter is associated with viral replication as strict late g/C promotor, late in the replication cycle (Yamamoto et al., 2006).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.