Oncolytic Adenovirus: Viruses Engineered as Potent Vehicle in Anticancer Therapy and Vaccination

Keywords

Oncolytic viruses; Adenovirus; Early antigens, Tumor suppression; Anticancer biotherapy; Anticancer vaccination

Abstract

The prospect of harnessing the cytocidal properties of viruses for targeted attacks on cancer cells, has prompted an ongoing development in the design of manipulated adenoviral vectors. The adenovirus is efficient in gene transfer, and serves as vector in clinical gene therapy. But its genome contains genes that code for products that bind to and inactivate tumor suppressor gene products, and it has been demonstrated that the deletion of the early genes produces oncolytic adenoviruses that replicate in cancer cells and kill them. New forms of oncolytic viruses with deletions of some viral genes like E1B and being armed with anticancer cDNAs contributed to enhanced cytotoxicity of adenoviral vectors. The engineering of the adenoviral vector could be useful for the development of biotherapeutic agents, as it could expand the list of anti-oncogenes used in anticancer biotherapy.

Citation

Zhang X, Zhou J, Winberg G, Huang Z and He Z. Oncolytic Adenovirus: Viruses Engineered as Potent Vehicle in Anticancer Therapy and Vaccination. SM Virol. 2019; 4(1): 1017.

Background

To date, the term “oncolytic virus” has been defined by a panel of experts as a non-pathogenic virus that specifically infects cancer cells and causes their destruction [1].

As oncolytic viruses are endowed with intrinsic anticancer activity, they are generally viewed as passive immunotherapeutics, which target the immune evasion of the malignancies and enhance antitumor immune surveillance. T he cytopathic effects exhibited by viruses after infecting cancer cells have led to their use in virotherapy. More than a century ago, it was observed that tumors spontaneously regressed in patients who had a viral infection, and this led to clinical trials that used bodily fluids containing human or animal viruses to treat cancer patients [2].

Since the discovery that some viruses are toxic and thus therapeutic to cancers, oncolytic viruses have evolved to recombinant viral strains with more specific killing potential to malignant cells, and they subsequently entered clinical trials. The recent developments have been highlighted on generation of conditional replication of the viruses in tumor, termed conditionally replicative adenoviruses (CRAd) [3,4],

expression of transgenes with therapeutic effects, and targeting and delivery of oncolytic viruses. As a human virus that exhibits tropism to virtually cells of all histologic origins, adenoviruses are used for gene transfer in the laboatories and disease therapy. The viruses have also been applied in anticancer biotherapy after being engineered to selectively replicate in the cancer cells after mutations and deletions of certain parts of the viral genome and/or by inserting target gene fragments into it to create armed adenoviruses. Oncolytic adenoviruses (OA) are genetically manipulated human adenoviruses (HAdVs) that have acquired a phenotype that enables them to infect and/or selectively replicate in tumor cells but are more restricted in normal cells [5].

Data show that manipulated adenoviruses kill cancer-derived cell lines but do not affect normal control cells, which are usually the immortalized cells of the same histologic origin [2]. To correct the genetic deficiency in malignancies, cDNA fragments that code for immunoregulators or pro apoptotic cytokines, such as melanoma differentiation-associated gene-7( MDA-7)/IL-24 [3,4], tumor necrosis factor related apoptosis inducing ligand (TRAIL) [5], and tumor suppressor gene (TSG) cDNAs [6,7,8]

or siRNA to silence anti-apoptotic genes, like survivin [9-11] have been incorporated into recombinant adenoviruses. T he adenoviral vectors expressing a tumor suppressor or cytotoxic/suicide proteins induce cell cycle arrest or a death cascade. We have constructed a recombinant adenovirus by incorpotation of a TSG frequently lost in human cancers to the viral genome by a homogenous recombination of the gragment on shuttle plasmid [12].

Expression of the transferred genes by intratumoralinjection remarkably reduced the tumor size of nude mice xenograted human nasopharyngeal carcinoma (NPC), and the in vitro study has shown that the transferred gene exertted tumor suppressive effects by enganging intracelllular signaling pathways to promote apoptosis and prevent cell cycle progression [13,14].

These strategies aim at introducing a new modality in anticancer biotherapy and have entered into preclinical studies and different phases of clinical trials. The present paper reviews the current advances in the field of oncolytic adenoviruses to assess their prospective utility as expression vectors for tumor suppressor genes (TSG) to be used in future anticancer biotherapy.

The adenoviral genomic composition and genetic basis of using adenovirus as anticancer therapy agent

Viruses encode for specific genes to kill the infected cells by multiple mechanisms including apoptosis triggering; it is induced directly by viral genomic products or through activation of death receptors and p53. HAdVs can infect a broad range of human cells with high efficiency and achieve high levels of transgene expression. Moreover, the viral genome adenovirus (Ad) is genetically stable and the inserted foreign genes are generally maintained without change through successive rounds of viral replication. These features make Ad vectors attractive in gene therapy. The replication cycle of HAdVs is divided into two stages: the early stage and the late stage. During early stage, the viral proteins are expressed from six distinct early regions, to function as modifiers of the microenvironment so as to favor the replication of the adenoviruses. In the later stages of viral infection, the viral progeny is formed after assembly with structural proteins, viral load is increased to a threshold, and viruses then exploit apoptotic machinery to facilitate the efficient viral progeny release and spread. T he early regions of Ad genome include E1A, E1B, E2A, E2B, E3 and E4.

In adenovirus, apoptosis inducing proteins include, E1A 12S and 13S proteins, E3, E4 of adenovirus [15]. The adenovirally encoded apoptotic regulators also function to escape inflammatory reactions and host’s immune responses.

E1A, a pro-apoptotic adenoviral protein

E1A is a nuclear protein and a known regulator of gene expression. Although E1A does not bind to DNA directly, it interacts with a large number of cellular proteins functioning as transcription regulators [16]. Activation of c-Jun may be the result of transcriptional regulation, e.g. the coactivator p300 can participate in transcription of the jun gene [17,18]. CRAds based on the deletion of E1A has been generated, with introduction of a mutation in the pRb-binding domain of E1A. It has been shown to replicate in tumor cells with disrupted Rb signaling pathway [19,20].

These CRAds with manipulation in a single adenoviral antigen exhibited potential for cancer therapy, but they still replicate and cause some cytopathic effects in normal cells in vitro [21,22]. E1B, a viral genomic product that binds and interferes the activities of tumor suppressors T he 55K proteins encoded by early regions 1B (E1B-55K) from HAdV types 2, 5 and 12 contribute to complete cell transformation by antagonizing host apoptosis and growth arrest. In the case of Ad2/5 E1B-55K products, these growth-promoting activities correlate with their ability to direct transcriptional repression of p53 responsive promoters by binding to p53 [23]. Together with human papillomavirus (HPV) encoding p53 inhibitor protein E6, E1B also abrogates transcriptional induction of downstream pro-apoptotic factors by p53 through molecular interactions [24,25]. CRAd, which are capable of cancer-selective replication and oncolysis, have received widespread attention as potentially ideal tools for anticancer biotherapy. ONYX-015(dl1520), is a CRAd with E1B deletion based cancer selective replicative potential and a mutant adenovirus created by deletion in the gene coding for E1B that enables selective replication in malignant cells with dysfunctional p53 signaling pathway [26-29]. Clinical trails of ONYX-015 in combination with chemotherapy have yielded remarkably good efficacy and safety in patients with head and neck cancers [30].

As for hepatobiliary cancers, the clinical trial of intralesional ONYX- 015 showed sufficient safety but limited therapeutic effects [31]. These studies suggest that for biliary cancers, further efforts to develop CRAds are warranted, so as to exert more selective replication and effective oncolysis than ONYX-015. Ad Max, the Ad5 based adenoviral vector we used as expressor of tumor suppressor BLU [12], is known to be disrupted in E1 and E3 [32,33]. E1B expression was still detected on immunoblotting. Several strains of siRNAs have been designed by searching for the AA dinucleotides in the full length sequence of the E1B 55K coding gene (nucleotides 21093509 in Human adenovirus C serotype 5, complete genome; AY339865.1, GenBank), Each AA and the 3’ adjacent 19 nucleotides were chosen as potential siRNA target sites. T hey were listed in Table 1

Table 1: Sequence of anti E1B siRNAs used in the present study.

The oligonucleotides were synthesized and tested for the potential of E1B silencing. Data showed that some of the fragments remarkably enhanced cytotoxicity when introduced to host cells (Figure 1).

Figure 1: siRNAs against adenoviral E1B enhanced adenoviral cytotoxicity to NPC derived HNE1 cells. The cells previously infected with recombinant BLU Ad5 were transfected with anti E1B siRNAs designed as described in the text. Viability was assayed by co-incubating with CCK-8 kit and registered with an ELISA reader. The numbers below the x-axis depicted the species of oligonucleotides tested: 1. Scramble RNA; 2. AS22A5P; 3. AS22A5Q; 4. AS22A5R; 5. AS22A5S; 6. AS22A5T. Data was derived from at three independent tests.

The silencing of oncogene in cancer cells mediated by siRNA could be utilized as a therapeutical approach. When a fragment of short hairpin RNA (shRNA) against mutant K-ras was introduced to an oncolytic adenovirus ONYX-411, a 10-fold increase of the growth inhibition potency has been observed in cancer cells [34]. It has been noted that improvement of gene knockdown, curtail of emergence of viral escape mutants could be achieved by delivery of multiple shRNA delivery [35]. 

The E2 region of the viral genome encodes multiple proteins, including E2A functioning as a DNA binding protein (DBP). It possesses specific affinity for single stranded viral DNA, and the binding plays a role in the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role to activate the major late promoter (MLP) [36]. New generations of Ad5 based vectors have been introduced by additionally deleting viral genes, so as to attenuate their expression to avoid leakage problems. Vectors with E1, E2A/B, E3 and E4 deletions in different combinations showed low in vitro cytotoxicity and higher stability in vivo, but it remains to demonstrate whether the new second and third generation vectors significantly prolong transgene expression in vivo [ 37,38]. Immunoregulatory functions of E3 proteins HAdVs are grouped into species A (HAdV-A) through species G (HAdV-G), and the level of sequence diversity is as high as 40% [39]. The size and composition of the E3 region differs considerably among Ad species, and the E3 transcription unit of HAdVs encodes proteins with immunoregulatory activities; Differential immunomodulatory functions encoded in early transcription unit 3 (E3) may play an important role in disease [40-42]. Common immune evasion functions in E3 proteins from species C have been described. Ads express species C E3 protein and enable the viruses to evade recognition and elimination by the host immune system by various mechanisms. An E3 protein, E3/19K retains MHC class I molecules and MHC class I-related chain A and B in the endoplasmic reticulum of infected cells, thereby suppressing recognition by cytotoxic T lymphocytes [43-46] and activation of natural killer (NK) cells [47,48]. It has been reported that an E3 protein E3/49K from species D targeted uninfected cells; it specifically binding lymphocytes and that cell surface protein tyrosineCD45 was identified as its receptor [49]. Such functions would be important for the utility of species D Ads as vectors for vaccination and gene therapy in humans, given that these have a number of favorable features [50]. Other common E3 proteins (E3/10.4K–14.5K) down-regulate various apoptosis receptors from the cell surface or affect TNF-α–induced signaling [45,51].

Disruption in this genomic portion may enhance host antiviral immunity against tumor cells harboring the mutant adenovirus during the therapy. Adenovirus E4 open reading frame 4 (E4orf4) T he protein encoded by adenovirus E4 open reading frame 4 (E4orf4), protein (14-kDa) is a multifunctional viral regulator which functions to regulate gene expression at multiple steps, alternative splicing events, phosphorylation of viral and cellular proteins and protein translation [52-55]. Its expression of E4orf4 at high levels also induces caspase- and p53-independent, non-classical apoptosis in many human tumor cells [56,57]. Oncogenic transformation of primary cells sensitizes the host to cell death induced by E4orf4 induced.

Oncolytic adenoviruses: adenoviruses manipulated to be conditionally replicated in tumor cells

The replication of adenovirus depends on the entry of the host into the S phase of cell cycle. A tumor suppressor, retinoblastoma tumor suppressor (pRb) prevents the entry to S phase. Within the E1A molecule, there is a product of the retinoblastoma susceptibility gene binding site [58,59]. E1A gene is responsible for inactivation of several proteins, including retinoblastoma, allowing entry into S-phase. When E1A binds the protein pRb, transcription factor E2F1 dissociates and the host cells are prompted to enter the S phase. The adenovirus with deletion of the pRb-binding E1A has reduced replication potential in normal cells with intact pRb function, but the replicative ability in cancer cells is unaffected [60,61]. The Ad delta-24 with the deletion of the binding motif with 24 amino acid residues shows an enhanced efficacy in treating glioma-carrying mutant pRb [62]. 

The oncolytic adenoviruses are converted from non-oncogenic adenoviruses, of serotypes 2 and 5 through certain manipulations. The mutant viruses have gone through three generations of development as defined by the mutations introduced [63]. And the modification of adenoviruses include: Attenuation In this category, adenoviral vectors of three generations have developed. During the process of viral replication, tumor cells are killed by ablation of the viral vectors. The first generation oncolytic Ads are administered in combination with chemotherapy and/or irradiation to achieve efficacy, wtih accetable level of safety. Oncolytic Ads of second generation are armed with therapeutic transgenes, to enhance the efficacy by increasing virion release, spread and effect of bystander. Oncolytic Ads of third generation are modified in viral capsid for transductional detrageting of normal cells but targeting cancer cells. Targeting A conditionally replicative adenovirus (CRAd) was created by deleting a 24 base pair deletion in the retinoblastoma(Rb)-binding domain of the E1A protein (Ad5- Δ24E3). Whenever the pRb binding site on Adv early antigen interfring with cell cycle is manipulated, induction of S-phase in host cells is disabled [64], and retinoblastoma is silenced, this restricts Ad5-Δ24E3 to replication only in proliferating cells, such as tumour cells. Manipulations introduced to Adv genome to produce oncolytic virions Delta-24-RGD, a tumor-selective, replication-competent adenovirus with augmented cellular infectivity [65,66].

The deletion of RGD motif was introduced in a mutant viral E1A. The mutant AdV with Delta-24-RGD selectively replicates in tumor cells lyse the malignant cells in which pRb is inactivated. Delta-24-RGD’s augmented infectivity is due to an insertion of an RGD-motif in the f iber knob, allowing for integrin-mediated infection, independent of cocksackie-adenovirus receptors (CAR) [67]. Intratumoral injection of Delta-24-RGD to xenografted human gliomas resulted in a remarkably longer survival than controls [68,69].

Onyx-015 as previously mentioned, was originally named Ad2/5 dl1520 [29,30,70]. It is an experimental oncolytic virus created by genetically engineering [29,30]. The E1B-55kDa gene has been deleted allowing the virus to selectively replicate in and lyse p53 deficient cancer cells [70]. ONYX-015 is an E1B-55kDa gene-deleted adenovirus which selectively replicates in and p53-deficient cancer cells and lyses them. Clinical trial suggested that tissue destruction was also highly selective, most in tumor lesions; significant tumor regression occurred in over 20% of evaluable patients; no toxicity to injected normal peritumoral tissues was demonstrated. That ONYX 015-induced necrosis in p53 mutant tumors more likely than were p53 wild-type tumors. The data implied a feasibility of modifying genome region coding for anti-apoptotic E1B as a promising approach to generate OA.

Adenovirus armed with anticancer cDNA: an ideal anticancer agent needs to be specific in action and should not harm the normal cells

To date, cancer is recognized as a disorder of multiple genetic defects, the development of which is aided by compromised immune surveillance and dysregulation in programmed cell death (PCD) [71,72].

Different abnormalities in PCD may promote carcinogenesis by hampering host immune surveillance, and also renders cancer cells resistant to antitumor therapies including chemo- and radiotherapy, as the cell killing in such context is mediated by activation of apoptosis. The inherited defects contributing to the immune evasion and resistance to therapy can be corrected by transfer of cDNAs with tumor suppressive activity. Cancer-targeting gene virotherapy (CTGVT) is an approach that uses an oncolytic adenoviral vector containing antitumor gene fragments as a combination of gene therapy and oncolytic adenovirus. When armed with some anticancer cDNA or siRNAs, coding for pro-drug converting enzymes, immunoregulatory cytokines, or pro-apoptotic proteins, an adenovirus could be adapted as a gene therapy vector. Apoptosis is a precise cell death process inside the human body. Extrinsic or death receptor-induced apoptosis has been intensively studied. It is triggered by ligation of death receptors to death ligands [73].

One of the death ligands, TRAIL has gained much attentionas a targeted therapeutic candidate and has therefore entered the Phase I clinical trial TRAIL has been known for its preferential killing of malignantly transformed cells, and low cytoxicity has been demonstrated [74-76].

The intratumor administration of an adenoviral recombinant of TRAIL has proved to possess therapeutic effect in the primary and metastatic models of xenografted human tumors in mice. The E1B deleted oncolytic adenovirus ZD55 armed with the death ligand TRAIL and a second mitochondrial activating component (Smac) contributed to the complete eradication of human hepatoma xenograft in nude mice [77].

The extrinsic apoptotic pathway is regulated by inhibitor proteins, including the cellular FLICE/caspase-8 inhibitor protein (cFLIP), which negatively regulates the activation of caspase-8 as a decoy binding partner [78]. The coding gene of cFLIP produces up to eleven isoforms of transcripts by alternative splicing, and three c-FLIP protein isoforms derived from distinct mRNA splice variants have been identified, namely c-FLIPL, c-FLIPS, and c-FLIPR [79]. The long c-FLIP isoform, c-FLIPL , is structurally similar to procaspase-8, with two tandem DEDs at its N-terminus and a catalytically inactive caspase-like domain at its C-terminus [80]. It has been reported that silencing c-FLIPL in ovarian cancer cells with RNAi induced apoptosis leading to significantly decreased tumor development and reduced cellular proliferation in vivo [79,80]. This suggests that recombinant adenovirus incorporating anti FLIP RNAi may exert anticancer efficacy. One application of oncolytic adenovirus would be transfer of siRNA targeted against anti-apoptotic molecules to enhance host antitumor immunity. We have shown that BLU/ZMYND10, encoded by a TSG mapped on a chromosomal region, 3p21, frequently lost in variety of human tumors inhibited NF-kB signaling, and hence the anti-apoptotic factor it transcriptionally induces, e.g. cIAP-2, cFLIP, to promote TRAIL trigering apoptosis when BLU is transferred by AdV 5 vector [13]. T hus tumor killing potential of the oncolytic adenoviruses could be enhanced by genetic modifications with insert of sequences coding for (1) enzymes converting an innocuous pro-drug into a cytotoxic agent [81,82];

(2) proteins that (at least theoretically) selectively induces a programmed death response in malignant cells, since tumor growth frequently depends on the defects in the apoptotic machinery [83]; or (3) short hairpin RNAs that interfere with the activities of factors required for the survival of transformed, but not normal cells [84].

The efficacy of adenoviral therapeutic recombinants

Several mutant genetic lesions within adenoviral genome leading to oncolytic activities have been described [85,86].

The production of oncolytic adenoviruses involves the deletion of the E1 through E4 genes, and disable the functions necessary for replication in normal cells but still permits replication of the mutant viruses with cytocidal effects in cancer cells [87]. ONYX-015 and ZD55 were both generated by deleting the E1B55 kd gene. In cancer patients, durable regressions are achieved in combination of recombinant adenovirus administration with chemotherapy (e.g., cisplatin) [88,89]. In line with this, the combined administration of ONYX-015 and cisplatin has provided potent antitumor activity, but further improvement of the oncolyticadenovirus using a single virotherapeutic agent is expected. An armed therapeutic oncolytic adenovirus system, the ZD55 strain is not only devoid of the E1B 55-kd gene, similar to ONYX-015, but is also incorporated with foreign genes with antitumor activity in the viral genome, e.g. TRAIL. IL-24 or SOCS3 [6, 90, 91].

The ZD55 strain exhibits similar cytopathic effects and replication kinetics with ONYX-015 in vitro; however, the inserted gene is expressed and the expression level increases with replication of the virus. ZD55 not only replicates selectively in tumor cells and lyses them, but also releases the therapeutically active antitumor agents to the tumor microenvironment. Manipulated oncolytic adenoviruses in which viral and/or cellular genes are placed under the control of artificial tumor specific promoters have been developed and show promising results in anticancer therapy in animal models. Based on the fact that a fetal protein, alpha-fetoprotein (AFP) is switched on during the hepatocarcinogenesis, AFP promoter was used to control the expression of the E1A viral gene in hepatocellular carcinoma (HCC) cells. The manipulation achieved preferential replication of the pro apoptotic E1A gene over-expressing Ad in AFP-producing HCC cells [92].

Midkine mRNA is over-expressed in osteosarcoma cell lines, and an oncolytic adenovirus has been constructed by inserting the midkine promoter upstream of the coding portion of the adenoviral genes, to drive the expression of the adenoviral antigens [83].

Infectivity and in vitro cytocidal effects of the engineered virus were significantly enhanced in target cells of osteosarcoma. These findings indicate that gene manipulation allows tailored virotherapy and facilitates more effective treatments for osteosarcoma. NPC is a malignant tumor that is endemic to certain regions in the world, including Alaska, North Africa, southeast and southern China [93]. In its undifferentiated form NPC is tightly associated with latent infection of a lymphotropic human herpesvirus, the Epstein-Barr virus (EBV); therefore, EBV-dependent transcriptional targeting has been exploited in NPC biotherapy. The viral replication initiation site of EBV, OriP, was used to construct a conditionally replicating adenovirus, termed adv. OriP. E1A [17]. Extensive cell death was observed in EBV-positive NPC cells, but no cytoxicity was noted in a panel of EBV-negative cells, which were derived from NPC and fibroblasts from the nasopharynx. Adenoviral replication was demonstrated only in EBV-positive cells, and a combination of local irradiation and adenoviral administration caused the xenografted tumors to completely disappear within two weeks. Similar effects were observed in metastatic gastrointestinal cancer treated with a virus in which the therapeutic genes were controlled by the carcinoembryonic antigen (CEA) promoter [94]. Recombinant adenoviruses combined with other tumor-specific promoters, like human TERT [95,96] or E2F [97] ,

showed effects in a broad range of cancer types

Conclusion

1. As an efficient tool to transfer gene, adenovirus could be manipulated as a potent vector in anticancer therapy and vaccination by modifying its genomic components.

2.The successful construction of several strains of oncolytic adenoviruses has suggested that viral vectors specifically replicating in malignant cells, and armed with anticancercDNA could achieve therapeutic effects. As a disease of genetic deficiency, cancer could be treated with a biotherapeutic approach by introducing different genes to correct a variety of functional defects.

3. Oncolytic adenoviruses with inserted anticancer cDNA would expand the list of therapeutic target genes and improve the efficacy of biotherapy.

Availability of data and material

The sequence of adenoviral E1B coding gene used in the present study is available in complete genome of Human adenovirus C serotype 5,AY339865.1 in Gen Bank,NCBI (Human adenovirus C serotype 5,complete genome-Nucleotide–NCBI. http://www.ncbi.nlm.nih.gov/nuccore/33465830/). 

Acknowledgements

The work conducted in our laboratory is supported by Provincial Medical Research Fund of Guangdong (2014A267 and 2018A358 to XZ). Our work on enhancement of adenoviral toxicity by silencing of E1B was supported by an International Cancer Research Exchange and Transfer of Technology (ICRETT) fellowship (ICR/2015/391721) to XZ by UICC.

References

1. Galluzzi L, Vacchelli E, Bravo-San Pedro JM. Classification of current anticancer immunotherapies. Oncotarget. 2014; 5: 12472-12508.

2. Alemany R, Balague C, Curiel DT. Replicative adenoviruses for cancer therapy. Nat Biotechnol. 2000; 18: 723-727.

3. Dash R, Dmitriev I, Su Z-z, Bhutia SK, Azab B, Vozhilla, et al. Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) improves therapeutic efficacy in low CAR prostate cancer cells. Cancer Gene Ther. 2010; 17: 447-456.

4. Kreis S, Philippidou D, Margue C, Behrmann I. IL-24: a classic cytokine and/ or a potential cure for cancer? J Cell Mol Med. 2008; 12: 2505-2510.

5. Dong F, Wang L, John J. Davis JJ, Hu W, Zhang L, et al. Eliminating Established Tumor in nu/nu NudeMice by a Tumor Necrosis Factor-A Related Apoptosis-Inducing Ligand Armed Oncolytic Adenovirus. Clin Cancer Res. 2006; 12: 5224-5230.

6. Ma J, He X, Wang W, Huang Y, Chen L, Cong W, et al. E2F promoter regulated oncolytic adenovirus with p16 gene induces cell apoptosis and exerts antitumor effect on gastric cancer. Dig Dis Sci. 2009; 54: 1425-1431. 7. Wang X, Su C, Cao H, Li K, Chen J, Jiang L, et al. A novel triple-regulated oncolytic adenovirus carrying p53 gene exerts potent antitumor efficacy on common human solid cancers. Mol Cancer Ther. 2008;

7: 1598-1603.

8. Xie M, Niu JH, Chang Y, Qian QJ, Wu HP, Li LF, et al. A novel riple-regulated oncolytic adenovirus carrying PDCD5 gene exerts potent antitumor efficacy on common human leukemic cell lines. Apoptosis. 2009; 14:1086-1094.

9. Zhang R, Ma L, Zheng M, Ren J, Wang T, Meng Y, et al. Survivin knockdown by short hairpin RNA abrogates the growth of human hepatocellular carcinoma xenografts in nude mice. Cancer Gene Ther. 2010; 17: 275-288.

10. Han Z, Lee S, Je S, Eom CY, Choi HJ, Song JJ, Kim JH. Survivin silencing and TRAIL expression using oncolytic adenovirus increase anti-tumorigenic activity in gemcitabine-resistant pancreatic cancer cells. Apoptosis. 2016; 21: 351-364.

11. Tanaka T, Uchida H. Inhibition of Survivin by Adenovirus Vector Enhanced Paclitaxel-induced Apoptosis in Breast Cancer Cells. Anticancer Res. 2018; 38: 4281-4288

12. Zhang X, Liu H, Li B, Huang P, Shao J, He Z. Tumor suppressor BLU inhibits proliferation of nasopharyngeal carcinoma cells by regulation of cell cycle, c-Jun N-terminal kinase and the cyclin D1 promoter. BMC Cancer. 2012; 12: 267.

13. Zhou J, Huang Z, Wang Z, Liu S, Grandien A, Ernberg I, et al. Tumor suppressor BLU promotes TRAIL-induced apoptosis by downregulating NF-κB signaling in nasopharyngeal carcinoma. Oncotarget. 2014; 8: 43853 43865.

14. Zhang X, Song-Jun Shao SJ, Zhou J, Xiao-Wu LiXW, He Z, Huang Z. Tumor suppressor BLU exerts growth inhibition by blocking ERK signaling and disrupting cell cycle progression through RAS pathway interference. Int J clin exp Pathol. 2018; 11: 158-168.

15. Marcellus RC, Teodoro JG, Wu T, Brough DE, Ketner G, Shore GC, et al. Adenovirus type 5 early region 4 is responsible for E1A-induced p53 independent apoptosis. J Virol. 1996; 70: 6207-6215.

16. Frisch SM, Mymryk JS. Adenovirus-5 E1A: paradox and paradigm. Nature Rev Molec Cell Biol. 2002; 3: 441-452.

17. Cha EJ, Oh BC, Wee HJ, Chi XZ, Goh YM, Lee KS, et al. E1A physically interacts with RUNX3 and inhibits its transactivation activity. J Cell Biochem. 2008; 105: 236-244.

18. Kitabayashi I, Eckner R, Arany Z, Chiu R, Gachelin G, Livingston DM, et al. Phosphorylation of the adenovirus E1A-associated 300 kDa protein in response to retinoic acid and E1A during the differentiation of F9 cells. EMBO J. 1995; 14: 3496-3509.

19. Heise C, Hermiston T, Johnson L. An adenovirus E1A mutant that demonstrates potent and selective systemic anti-tumoral efficacy. Nat Med. 2000; 6: 1134-1139.

20. Sánchez-Prieto R, Quintanilla M, Cano A. Carcinoma cell lines become sensitive to DNA-damaging agents by the expression of the adenovirus E1A gene. Oncogene. 1996; 13: 1083-1092.

21. Chia MC, Shi W, Li JH, Sanchez O, Strathdee CA, Huang D, et al. A conditionally replicating adenovirus for nasopharyngeal carcinoma gene therapy. Mol Ther. 2004; 9: 804-817.

22. Fueyo J, Gomez-Manzano C, Alemany R. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene. 2000; 19: 2-12.

23. Berk AJ. Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus. Oncogene. 2005;

24: 7673-7685. 24. Lechner MS, Laimins LA. Inhibition of p53 DNA binding by human papillomavirus E6 proteins. J Virol. 1994; 68: 4262-4273.

25. Li Q, Zhao LY, Zheng Z, Yang H, Santiago A, Liao D. Inhibition of p53 by adenovirus type 12 E1B-55K deregulates cell cycle control and sensitizes tumor cells to genotoxic agents.J Virol. 2011; 85: 7976-7988.

26. Rothmann T, Hengstermann A, Whitaker NJ, Scheffner M, Hausen H. Replication of ONYX-015, a potential anticancer adenovirus, is independent of p53 status in tumor cells. J Virol. 1998; 72: 9470-9478.

27. Harada JN, Berk AJ. p53-Independent and-dependent requirements for E1B55K in adenovirus type 5 replication. J Virol. 1999; 73: 5333-5344.

28. Heise C, Jhoannes AS, Williams A, McCormick F, Hoff DDV, Kirn DH. ONYX 015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nat Med. 1997; 3: 639-645.

29. Khuri FR, Nemunaitis J,Ganly I. A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5- f luorouracil in patients with recurrent head and neck cancer. Nat Med. 2000; 6: 879-885.

30. Ries S, KornWM. ONYX-015: mechanism of action and clinical potential of a replication-selective adeno-virus. Br J Cancer. 2002; 86: 5-11

31. Makower D, Rozenblit A, Kaufman H. Phase II clinical trialo fintra lesional administration of the oncolytic adenovirus ONYX-015 in patients with hepatobiliary tumors with correlative p53 studies. Clin Cancer Res. 2003; 9: 693-702.

32. Ng P, Parks RJ, Cummings DT, Evelegh CM, Sankar U, Graham FL. A high efficiency Cre/loxP-based system for construction of adenoviral vectors. Hum Gene Ther. 1999; 10: 2667-2672.

33. Branton PE, Bayley ST, Graham FL. Transformation by human adenoviruses. Biochim Biophys Acta. 1985; 780: 67-94.

34. Shiver JW, Fu L, Chen DR, Casimiro ME, Davies RK, Evans, ZQ, et al. Replication-incompetent adenoviral vaccine vector elicits effective anti immunodeficiency-virus immunity. Nature. 2002; 415: 331-335.

35. Zhang YA, Nemunaitis J, Samuel SK, Chen P, Chen Y, Tong AW. Antitumor Activity of an Oncolytic Adenovirus-Delivered Oncogene Small Interfering RNA. Cancer Res 2006; 66: 9736-9743.

36. Lambeth LS, Smith CA. Short hairpin RNA-mediated gene silencing. Methods Mol Biol. 2013; 942: 205-232.

37. Lusky M, Christ M, Rittner K, Dieterle A, Dreyer D, Mourot B, et al. E1/E2 in vitro and in vivo biology of recombinant adenovirus vectors with E1 or E1/E4 deleted. J Virol. 1998; 72: 2022-2032.

38. Chinnadurai G. Control of apoptosis by human adenovirus genes. Semin Virol 1998; 8: 399-408.

39. Weaver EA, Hillestad ML, Khare R, Palmer D, Ng P, Barry MA. Characterization of species C human adenovirus serotype 6 (Ad6). Virology. 2011; 19-27.

40. Burgert H-G. Subversion of host defense mechanisms by adenoviruses. Curr Top Microbiol Immunol. 2002; 269: 273-318.

41. Gregory SM, Nazir SA, Metcalf JP. Implications of the innate immune response to adenovirus and adenoviral vectors. Future Virol. 2002; 6: 357 374.

42. Windheim M, Hilgendorf A, Burgert H-G. Immune evasion by adenovirus E3 proteins: Exploitation of intracellular trafficking pathways. Curr Top Microbiol Immunol. 2004. 273: 29-85.

43. Burgert H-G, Kvist S. An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell. 1985; 41: 987 997.

44. Sester M, Ruzsics Z, Mackley E, Burgert H-G. The transmembrane domain of the adenovirus E3/19K protein acts as an ER retention signal and contributes to intracellular sequestration of MHC class I molecules. J Virol. 2013; 87: 6104-6117.

45. Horwitz MS, Efrat S, Christen U, Von Herrath MG, Oldstone MB. Adenovirus E3 MHC inhibitory genes but not TNF/Fas apoptotic inhibitory genes expressed in beta cells prevent autoimmune diabetes. Proc Natl Acad Sci USA. 2009; 106: 19450-19454.

46. Ruzsics Z. Transposon-assisted cloning and traceless mutagenesis of adenoviruses: Development of a novel vector based on species D. J Virol. 2006; 80: 8100-8113.

47. McSharry BP. Adenovirus E3/19K promotes evasion of NK cell recognition by intracellular sequestration of the NKG2D ligands major histocompatibility complex class I chain-related proteins A and B. J Virol. 2008; 82: 4585-4594.

48. Sester M, Koebernick K, Owen D, Ao M, Bromberg Y, May E, et al. Conserved amino acids within the adenovirus 2 E3/19K protein differentially affect downregulation of MHC class I and MICA/B proteins. J Immunol. 2010; 184: 255-267.

49. Windheim M, South combe JH, Kremmer E, Chaplin L , Urlaub D, Falk CS, et al. A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions. Proc Natl Acad Sci USA. 2013; 100: 4884-4893

50. McNees AL, Gooding LR. Adenoviral inhibitors of apoptotic cell death. Virus Res. 2002; 88: 87-101.

51. Elsing A, Burgert HG. The adenovirus E3/10.4K-14.5K proteins down modulate the apoptosis receptor Fas/Apo-1 by inducing its internalization. Proc Natl Acad Sci USA. 1998; 95: 10072-10077.

52. Kleinberger T, Shenk T. Adenovirus E4orf4 protein binds to protein phosphatase 2A, and the complex down regulates E1A-enhanced junB transcription. J Virol. 1993; 67: 7556-7560.

53. Bondesson M, Ohman K, Mannervik M, Fan S, Akusjarvi G. Adenovirus E4 open reading 4 protein autoregulates E4 transcription by inhibiting E1A transactivation of the E4 promoter. J Virol. 1996; 70: 3844-3851.

54. Estmer Nilsson C, Petersen-Mahrt S, Durot C, Shtrichman R, Krainer AR, Kleinberger T, et al. The adenovirus E4-ORF4 splicing enhancer protein interacts with a subset of phosphorylated SR proteins. EMBO J. 2001; 20: 864-871.

55. Marcellus RC, Lavoie JN, Boivin D, Shore GC, Ketner G, Branton PE. The early region 4 orf4 protein of human adenovirus type 5 induces p53 independent cell death by apoptosis. J Virol. 1998; 72: 7144-7153.

56. Lavoie JN, Nguyen M, Marcellus RC, Branton PE, Shore GC. E4orf4, a novel adenovirus death factor that induces p53-independent apoptosis by a pathway that is not inhibited by zVAD-fmk. J Cell Biol. 1998; 140: 637-645.

57. Shtrichman R, Kleinberger T. Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells. J Virol. 1998; 72: 2975-2982.

58. Whyte P, Buchkovich KJ, Horowitz JM, Friend SH, Raybuck M. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988; 334: 124-129.

59. Fueyo J, Gomez-Manzano C, Yung WK. The functional role of tumor suppressor genes in gliomas: clues for future therapeutic strategies. Neurology. 1998; 51; 1250-1255.

60. Barnett BG, Crews CJ, Douglas JT. Targeted adenoviral vectors. Biochim Biophys Acta. 2002; 1575: 1-14.

61. Zamanian M, La Thangue NB. Adenovirus E1a prevents the retinoblastoma gene product from repressing the activity of a cellular transcription factor. EMBO J. 1992; 11: 2603-2610.

62. Jiang H, Gomez-Manzano C, Lang F, Alemany R, Fueyo J. Oncolytic adenovirus: preclinical and clinical studies in patients with human malignant gliomas. Curr Gene Ther. 2009; 9: 422-427.

63. Doronin K, Shayakhmetov DM. Construction of targeted and armed oncolytic adenoviruses. Methods Mol Biol. 2012; 797: 35-52.

64. Fueyo J, Gomez-Manzano C, Alemany R, Lee PS, McDonnell TJ, Mitlianga P, et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene. 2000; 19: 2-12.

65. Mariko W, Masato A, Rei K, Emiko S, Kuniaki F, Hideo U, et al. E1A, E1B Double-Restricted Adenovirus with RGD-Fiber Modification Exhibits Enhanced Oncolysis for CAR^Deficient Biliary Cancers. Cancer Res. 2007; 13: 2007.

66. Jiang H, Gomez-Manzano C, Rivera-Molina Y, Lang FF, Conrad CA, Fueyo J. Oncolytic adenovirus research evolution: from cell-cycle checkpoints to immune checkpoints. Curr Opin Virol. 2015; 13: 33-39.

67. Dmitriev I, Krasnykh V, Miller CR. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor independent cell entry mechanism. J Virol. 1998; 72: 9706-9713.

68. Fueyo J, Gomez-Manzano C, Alemany R, et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene. 2000; 19: 2-12.

69. Fueyo J, Alemany R, Gomez-Manzano C. Preclinical characterization of the antiglioma activity of a tropism-enhanced adenovirus targeted to the retinoblastoma pathway. J Natl Cancer Inst. 2003; 95: 652-660.

70. Nemunaitis J, Ganly I, Khuri F, Arseneau J KuhnJ, Mc Carty T. Selective Replication and Oncolysis in p53 Mutant Tumors with ONYX-015, an E1B 55kD Gene-deleted Adenovirus, in Patients with Advanced Head and Neck Cancer: A Phase II Trial. Cancer Res. 2000; 60: 6359-6366.

71. Negrini S, Gorgoulis VG, Halazonetis TD. Genomic instability-an evolving hallmark of cancer. Nat. Rev. Mol. Cell Biol. 2010; 11: 220-228.

72. Fulda S. Evasion of apoptosis as a cellular stress response in cancer. Int J Cell Biol. 2010; 2010: 370835.

73. Kurokawa M, Kornbluth S. Caspases and kinases in a death grip. Cell. 2009; 138: 838-854.

74. Mahalingam D, Szegezdi E, Keane M, Jong S, Samali A. TRAIL receptor signaling and modulation: Are we on the right TRAIL? Cancer Treat Rev. 2009; 35: 280-288.

75. Abdulghani J, El-Deiry WS. TRAIL receptor signaling and therapeutics. Expert Opin Ther Targets. 2010; 14: 1091-1108.

76. Holoch PA, Griffith TS. TNF-related apoptosis-inducing ligand (TRAIL): a new path to anti-cancer therapies. Eur J Pharmacol. 2009; 625: 63-72.

77. Wang SB, Tan Y, Lei W, Wang YG, Zhou XM, Jia XYZ. Complete eradication of xenograft hepatoma by oncolytic adenovirus ZD55 harboring TRAIL-IETD Smac gene with broad antitumor effect. Hum Gene Ther. 2012; 23: 992-1002.

78. Bagnoli M, Canevari S, Mezzanzanica D. Cellular FLICE-inhibitory protein (c-FLIP) signalling: a key regulator of receptor-mediated apoptosis in physiologic context and in cancer. Int J Biochem Cell Biol. 2010; 42: 210-213.

79. Fulda S. Caspase-8 in cancer biology and therapy. Cancer Lett. 2009; 281: 128-133.

80. Chawla-Sarkar M, Bae SI, Reu FJ, Jacobs BS, Lindner DJ, Borden EC. Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis. Cell Death Differ. 2004; 11:915-923.

81. Foloppe J, Kintz J, Futin N, Findeli A, Cordier P, Schlesinger Y, et al. Targeted delivery of a suicide gene to human colorectal tumors by a conditionally replicating vaccinia virus. Gene Ther. 2008; 15: 1361-1371.

82. Leveille S, Samuel S, Goulet ML, Hiscott J. Enhancing VSV oncolytic activity with an improved cytosine deaminase suicide gene strategy. Cancer Gene Ther. 2011; 18: 435-443.

83. Takagi-Kimura M, Yamano T, Tagawa M, Kubo S. Oncolytic virotherapy for osteosarcoma using midkine promoter-regulated adenoviruses. Cancer Gene Ther. 2014; 21:126-132.

84. Carette JE, Overmeer RM, Schagen FH, Alemany R, Barski OA, Gerritsen WR, et al. Conditionally Replicating Adenoviruses Expressing Short Hairpin RNAs Silence the Expression of a Target Gene in Cancer Cells”. Cancer Res. 2004; 64: 2663-2667.

85. Russell SJ, Peng KW, Bell JC. Oncolytic virotherapy. Nat Biotechnol. 2012; 30: 658-670.

86. Atherton MJ, Lichty BD. Evolution of oncolytic viruses: novel strategies for cancer treatment. Immunotherapy. 2013; 5: 1191-1206.

87. Thomas MA, Song R, Demberg T, Vargas-Inchaustegui DA, Venzon D. Effects of the Deletion of Early Region 4 (E4) Open Reading Frame 1(orf1), orf1-2, orf1-3 and orf1-4 on Virus-Host Cell Interaction, Transgene Expression, and Immunogenicity of Replicating Adenovirus HIV Vaccine Vectors. PLoS ONE. 2013; 8: 76344.

88. Zhu W, Zhang H, Shi Y, Song M, Zhu B, Wei L. Oncolytic adenovirus encoding tumor necrosis factor-related apoptosis inducing ligand (TRAIL) inhibits the growth and metastasis of triple-negative breast cancer. Cancer Biol Ther. 2013; 14: 1016-1023.

89. Norian LA, Kresowik TP, Rosevear HM, James BR, Rosean TR, Lightfoot AJ, et al. Eradication of metastatic renal cell carcinoma after adenovirus-encoded TNF-related apoptosis-inducing ligand (TRAIL)/ CpG immunotherapy. PLoS One. 2012; 7: 31085

90. Wei RC, Cao X, Gui J, Zhou XM, Zhong D, Yan QL, et al. Augmenting the antitumor effect of TRAIL by SOCS3 with double-regulated replicating oncolytic adenovirus in hepatocellular carcinoma. Hum Gene Ther. 2011; 22: 1109-1119.

91. Zhang ZL, Zou WG, Luo CX, Li BH, Wang JH, Sun LY, et al. An armed oncolytic adenovirus system, ZD55-gene, demonstrating potent antitumoral efficacy. Cell Res. 2003; 13: 481-489.

92. Takahashi M, Sato T, Sagawa T, Lu Y, Sato Y, Iyama S, et al. E1B-55K deleted adenovirus expressing E1A-13S by AFP-enhancer/promoter is capable of highly specific replication in AFP-producing hepatocellular carcinoma and eradication of established tumor. Mol Ther. 2002; 5: 627-634.

93. Yu MC, Yuan JM. The enigmatic epidemiology of nasopharyngeal carcinoma. Semin Cancer Biol. 2002; 12: 421-429.

94. Sagawa T, Takahashi M, Sato T, Sato Y, Lu Y, Sumiyoshi T, et al. Prolonged survival of mice with multiple liver metastases of human colon cancer by intravenous administration of replicable E1B-55K-deleted adenovirus with E1A expressed by CEA promoter. Mol Ther. 2004; 10: 1043-1050

95. Wirth T, Zender L, Schulte B, Mundt B, Plentz R, Rudolph KL, et al. A telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer. Cancer Res. 2003; 63: 3181-3188.

96. Kim E, Kim JH, Shin HY, Lee H, Yang JM, Kim J, et al. Ad-mTERT-delta19, a conditional replication competent adenovirus driven by the human telomerase promoter, selectively replicates in and elicits cytopathic effect in a cancer cell specific manner. Hum Gene Ther. 2003; 14: 1415-1428.

97. Jakubczak JL, Ryan P, Gorziglia M, Clarke L, Hawkins LK, Hay C, et al. An oncolytic adenovirus selective for retinoblastoma tumor suppressor protein pathway-defective tumors: dependence on E1A, the E2F-1 promoter, and viral replication for selectivity and efficacy. Cancer Res. 2003; 63: 1490-1449