**4. Overview of the Current Progress in OV Therapy for PDAC**

In 2015, the FDA approved Talimogene laherparepvec (T-VEC; Imlygic™), a genetically modified herpes simplex virus, to treat melanoma [119]. T-VEC is the first and still the only FDA-approved OV. However, numerous OVs are currently in preclinical studies

and clinical trials for various malignancies, including PDAC. Tables 1 and 2 summarize the results of preclinical studies (Table 1) and clinical trials (Table 2) of various oncolytic viruses against pancreatic cancer.

Preclinical studies demonstrated that a wide range of different viruses could be efficient OVs against PDAC (Table 1).

Additionally, gene therapy targets, such as oncogene knockdown, insertion of functional tumor-suppressor genes, and expression of functional RNAs also demonstrate improved cancer-killing efficacy when combined with OV. One method uses adenoviruses and adeno-associated viruses to deliver apoptotic genes to tumor cells. Such gene therapy using Adenovirus subtype 5 mediates rat insulin promoter directed thymidine kinase (A-5-RIP-TK)/ganciclovir (GCV) gene therapy resulting in significantly enhanced cytotoxicity to both Panc1 and MiaPaCa2 pancreatic cancer cells in vitro [176]. Another review explored the potential use of OV expressing functional p53 [117]. Another method would use OV to deliver siRNA transgenes for oncogenetic knockdown, such as ONYX-411 siRNAras expressing a mutant K-ras siRNA which significantly reduced K-ras mRNA expression at 48 h posttreatment and improved oncolytic activity [134]. The inclusion of an endostatin-angiostatin fusion gene in VVhEA also showed significant antitumor potency in vivo [152].

There have been many experiments screening for more effective virotherapies within available libraries, and modulated viruses such as the adenovirus Ad∆CAR-SYE has been shown to significantly suppress tumor growth, and complete regression of tumors was observed in vivo [129]. In addition, more efficient and tumor-specific targeting peptides and OV could be identified by using additional libraries, and modifications to existing OV based on these findings are also promising. Such modifications have been shown to be effective, with the adenoviruses VCN-01 variants ICOVIR-15K and ICOVIR-17 [130]. As discussed previously, the ability of the OV to modulate the ECM was observed, as tumors treated with VCN-01 showed a dramatic decrease in the intratumoral HA content [130]. Other adenoviral variants such as Ad5PTDf35(pp65) have also demonstrated T-cell stimulation and dendritic cell (DC) modulation to increase efficient transduction within a human context [133].

Combinatorial treatments of chemotherapies, or chemovirotherapy, such as OV paired gemcitabine, have demonstrated improved oncolytic capabilities in vitro and in vivo than either treatment on their own. In vitro and in vivo studies showed that myxoma virus (MYXV) and gemcitabine therapies can be combined sequentially to improve the overall survival in intraperitoneal dissemination (IPD) models of pancreatic cancer [177]. The addition of chemotherapies to OV therapy using a combination of an oncolytic herpes simplex virus-1 mutant NV1066 with 5-FU increased viral replication up to 19-fold compared with cells treated with virus alone, and similar results were achieved by the addition of gemcitabine [125]. Similarly, oVV-Smac combined with gemcitabine greater cytotoxicity and potentiated apoptosis [148]. H-1PV combined with cisplatin, vincristine or sunitinib induced effective immunostimulation via a pronounced DC maturation, better cytokine release and cytotoxic T-cell activation [154]. The addition of gene targets alongside chemovirotherapy has also shown greater cytotoxic efficiency, as with VV-ING4 in combination with gemcitabine [150]. Even in cell lines that demonstrate resistance to viral infection, resistance can be broken with simultaneous treatments. Viruses like VSV rely on nonspecific interactions with the cell surface during the earliest stages of infections, and polycations have been shown to improve viral production and increase oncolysis by increasing the amount of virus interacting with cells during attachment [60]. Additionally, the use of JAK inhibitors like ruxotinilib and IKK inhibitors like TPCA-1 have also been shown to increase viral reproduction and oncolysis [56,60]. Other potential combinatorial treatment regimens could include radiovirotherapy or chemoradiotherapy.





hPDAC = human PDAC, mPDAC = mouse, SC = subcutaneous injection; OT = orthotopic injection, IP = intraperitoneal injection; IV = intravenous injection.



PFU = plaque-forming unit.

Some of these treatment methodologies are already being tested in clinical trials. Table 2 describes the OV currently being tested in clinical trials, and while some are still underway, OV including ONYX-15, AD5-yCD, and T-VEC are well-tolerated, and in some cases, biologically active, either alone or in combination chemovirotherapies [167–171,174].
