Current Issues and Future Directions of Oncolytic Adenoviruses
Authors: Masato Yamamoto, David T Curiel
Journal: Molecular Therapy (2009)

Oncolytic adenoviruses (Ads) constitute a promising new class of anticancer agent. They are based on the well-studied adenoviral vector system, which lends itself to concept-driven design to generate oncolytic variants. The first oncolytic Ad was approved as a drug in China in 2005, although clinical efficacy observed in human trials has failed to reach the high expectations that were based on studies in animal models. Current obstacles to the full realization of efficacy of this class of anticancer agent include (i) limited efficiency of infection and specific replication in tumor cells, (ii) limited vector spread within the tumor, (iii) imperfect animal models and methods of in vivo imaging, and (iv) an incomplete understanding of the interaction of these agents with the host. In this review, we discuss recent advances in the field of oncolytic Ads and potential ways to overcome current obstacles to their clinical application and efficacy. Oncolytic adenoviruses (Ads) constitute a promising new class of anticancer agent. They are based on the well-studied adenoviral vector system, which lends itself to concept-driven design to generate oncolytic variants. The first oncolytic Ad was approved as a drug in China in 2005, although clinical efficacy observed in human trials has failed to reach the high expectations that were based on studies in animal models. Current obstacles to the full realization of efficacy of this class of anticancer agent include (i) limited efficiency of infection and specific replication in tumor cells, (ii) limited vector spread within the tumor, (iii) imperfect animal models and methods of in vivo imaging, and (iv) an incomplete understanding of the interaction of these agents with the host. In this review, we discuss recent advances in the field of oncolytic Ads and potential ways to overcome current obstacles to their clinical application and efficacy. Oncolytic replication–selective viruses are a new class of anticancer agents with great therapeutic potential.1Alemany R Balagué C Curiel DT Replicative adenoviruses for cancer therapy.Nat Biotechnol. 2000; 18: 723-727Crossref PubMed Scopus (398) Google Scholar,2Yamamoto M Conditionally replicative adenovirus for gastrointestinal cancers.Expert Opin Biol Ther. 2004; 4: 1241-1250Crossref PubMed Scopus (10) Google Scholar,3Yamamoto M Curiel DT Cancer gene therapy.Technol Cancer Res Treat. 2005; 4: 315-330Crossref PubMed Scopus (33) Google Scholar,4Curiel DT The development of conditionally replicative adenoviruses for cancer therapy.Clin Cancer Res. 2000; 6: 3395-3399PubMed Google Scholar The selective replication of the viruses in cancer cells amplifies the initial viral inoculum, leading to destruction of the infected cells by virus-mediated cytolysis. The viral progenies are thereby released and can spread through the tumor mass to infect neighboring cancer cells, resulting in self-perpetuating cycles of infection, replication, and oncolysis. Past cancer gene therapy clinical trials have defined major limitations of replication-defective vectors for cancer gene therapy; unable to infect the majority of the cells within a clinically presented three-dimensional solid tumor mass. Replication-selective viruses are designed to overcome such clinical limitations of cancer gene therapy by virus replication/spread whereas the restriction of their replication to tumor cells embodies the safety of oncolytic viruses for clinical usage.2Yamamoto M Conditionally replicative adenovirus for gastrointestinal cancers.Expert Opin Biol Ther. 2004; 4: 1241-1250Crossref PubMed Scopus (10) Google Scholar,3Yamamoto M Curiel DT Cancer gene therapy.Technol Cancer Res Treat. 2005; 4: 315-330Crossref PubMed Scopus (33) Google Scholar Although several oncolytic viruses have been identified to date,5Russell SJ RNA viruses as virotherapy agents.Cancer Gene Ther. 2002; 9: 961-966Crossref PubMed Scopus (134) Google Scholar,6Mohr I To replicate or not to replicate: achieving selective oncolytic virus replication in cancer cells through translational control.Oncogene. 2005; 24: 7697-7709Crossref PubMed Scopus (26) Google Scholar,7Barber GN Vesicular stomatitis virus as an oncolytic vector.Viral Immunol. 2004; 17: 516-527Crossref PubMed Scopus (110) Google Scholar replication-selective adenoviruses (Ads) based on human serotype 5 of species C possess a number of advantages.1Alemany R Balagué C Curiel DT Replicative adenoviruses for cancer therapy.Nat Biotechnol. 2000; 18: 723-727Crossref PubMed Scopus (398) Google Scholar,2Yamamoto M Conditionally replicative adenovirus for gastrointestinal cancers.Expert Opin Biol Ther. 2004; 4: 1241-1250Crossref PubMed Scopus (10) Google Scholar,3Yamamoto M Curiel DT Cancer gene therapy.Technol Cancer Res Treat. 2005; 4: 315-330Crossref PubMed Scopus (33) Google Scholar,4Curiel DT The development of conditionally replicative adenoviruses for cancer therapy.Clin Cancer Res. 2000; 6: 3395-3399PubMed Google Scholar Human serotype 5 Ads, which are associated with relatively mild diseases, are well characterized, their genomes can be manipulated with relative ease, and they can be purified to high titer.2Yamamoto M Conditionally replicative adenovirus for gastrointestinal cancers.Expert Opin Biol Ther. 2004; 4: 1241-1250Crossref PubMed Scopus (10) Google Scholar,8Yamamoto M Curiel DT Nonreplicating DNA viral vectors for suicide gene therapy: the adenoviral vectors.Methods Mol Med. 2004; 90: 61-70PubMed Google Scholar Moreover, the long history of usage as replication-defective adenoviral vectors for cancer gene therapy has defined the strategies for cancer targeting, including targeting based on binding and infection as well as the strategies to restrict the replication of Ads to tumor cells.1Alemany R Balagué C Curiel DT Replicative adenoviruses for cancer therapy.Nat Biotechnol. 2000; 18: 723-727Crossref PubMed Scopus (398) Google Scholar,2Yamamoto M Conditionally replicative adenovirus for gastrointestinal cancers.Expert Opin Biol Ther. 2004; 4: 1241-1250Crossref PubMed Scopus (10) Google Scholar,3Yamamoto M Curiel DT Cancer gene therapy.Technol Cancer Res Treat. 2005; 4: 315-330Crossref PubMed Scopus (33) Google Scholar,4Curiel DT The development of conditionally replicative adenoviruses for cancer therapy.Clin Cancer Res. 2000; 6: 3395-3399PubMed Google Scholar The latter can be achieved via placing the expression of viral genes, most commonly the E1A gene, under the control of tumor- or tissue-specific promoters, or via the complete or partial deletion of viral genes required for replication in normal cells, but not in tumor cells.1Alemany R Balagué C Curiel DT Replicative adenoviruses for cancer therapy.Nat Biotechnol. 2000; 18: 723-727Crossref PubMed Scopus (398) Google Scholar In this sense, Ad is one of a few systems which are capable of concept-based design of the vector structure in the context of oncolytic viruses. In 2005, State Food and Drug Administration in China approved mutation-based oncolytic Ad (H101; Sunway Biotech, Shanghai, P.R. China) as a drug for head and neck squamous cell carcinoma for local injection based on good responses observed in clinical trials.9Yu W Fang H Clinical trials with oncolytic adenovirus in China.Curr Cancer Drug Targets. 2007; 7: 141-148Crossref PubMed Scopus (211) Google Scholar This brings the hope that oncolytic Ads can be used in patients in the countries other than China. In recognition of their therapeutic potential, replication-selective Ads have been rapidly translated into human clinical trials in patients with advanced cancer,10Hecht JR Bedford R Abbruzzese JL Lahoti S Reid TR Soetikno RM et al.A phase I/II trial of intratumoral endoscopic ultrasound injection of ONYX-015 with intravenous gemcitabine in unresectable pancreatic carcinoma.Clin Cancer Res. 2003; 9: 555-561PubMed Google Scholar,11DeWeese TL van der Poel H Li S Mikhak B Drew R Goemann M et al.A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy.Cancer Res. 2001; 61: 7464-7472PubMed Google Scholar where their safety has been demonstrated. In this regard, a phase I clinical trial of an intraperitoneally administered replication-selective Ad has been conducted in patients with recurrent/refractory ovarian cancer.12Alvarez RD Curiel DT A phase I study of recombinant adenovirus vector-mediated intraperitoneal delivery of herpes simplex virus thymidine kinase (HSV-TK) gene and intravenous ganciclovir for previously treated ovarian and extraovarian cancer patients.Hum Gene Ther. 1997; 8: 597-613Crossref PubMed Scopus (65) Google Scholar In this trial, the maximum tolerated dose was not reached at 1011 plaque-forming units, and patients did not experience significant toxicity with this dose of administration.12Alvarez RD Curiel DT A phase I study of recombinant adenovirus vector-mediated intraperitoneal delivery of herpes simplex virus thymidine kinase (HSV-TK) gene and intravenous ganciclovir for previously treated ovarian and extraovarian cancer patients.Hum Gene Ther. 1997; 8: 597-613Crossref PubMed Scopus (65) Google Scholar Hence, there is a precedent for the safe use of replication-selective Ads in ovarian cancer patients. However, in spite of their promise as selective cancer therapeutics, replicating Ads have shown limited efficacy in the clinical setting.3Yamamoto M Curiel DT Cancer gene therapy.Technol Cancer Res Treat. 2005; 4: 315-330Crossref PubMed Scopus (33) Google Scholar In this regard, there was no clear-cut evidence of clinical or radiologic response in any of the 16 patients with recurrent/refractory ovarian cancer who received an intraperitoneal replication–selective Ad in the phase I trial.12Alvarez RD Curiel DT A phase I study of recombinant adenovirus vector-mediated intraperitoneal delivery of herpes simplex virus thymidine kinase (HSV-TK) gene and intravenous ganciclovir for previously treated ovarian and extraovarian cancer patients.Hum Gene Ther. 1997; 8: 597-613Crossref PubMed Scopus (65) Google Scholar Similarly, phase I and II clinical trials in which patients with recurrent squamous cell carcinoma of the head and neck received direct intratumoral injection of a replicating Ad, ONYX-015, resulted in clinical benefit in <15% of cases.13Ganly I Kirn D Eckhardt G Rodriguez GI Soutar DS Otto R et al.A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer.Clin Cancer Res. 2000; 6: 798-806PubMed Google Scholar,14Nemunaitis J Khuri F Ganly I Arseneau J Posner M Vokes E et al.Phase II trial of intratumoral administration of ONYX-015, a replication-selective adenovirus, in patients with refractory head and neck cancer.J Clin Oncol. 2001; 19: 289-298Crossref PubMed Scopus (445) Google Scholar Only when combined with standard chemotherapy did this oncolytic Ad cause an objective response (at least a 50% reduction in tumor size) in 19 of 30 cases, with 8 complete responses.15Lamont JP Nemunaitis J Kuhn JA Landers SA McCarty TM A prospective phase II trial of ONYX-015 adenovirus and chemotherapy in recurrent squamous cell carcinoma of the head and neck (the Baylor experience).Ann Surg Oncol. 2000; 7: 588-592Crossref PubMed Scopus (118) Google Scholar,16Khuri FR Nemunaitis J Ganly I Arseneau J Tannock IF Romel L et al.A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer.Nat Med. 2000; 6: 879-885Crossref PubMed Scopus (1012) Google Scholar This indicates a need for implementation of novel strategies to improve the efficacy of replicating Ads while assuring safety in normal cells for clinical application in the treatment of patients with cancer. As noted, there is no conditionally replicative adenovirus (CRAd) which shows clinical efficacy as a single therapy agent so far. This highlights the recognition that further augmentation of antitumor effects is the most crucial issue in CRAd development. Although Ad has been used for cancer gene therapy due to its exceptionally high in vivo transduction efficiency, many cancers (including gastrointestinal cancers, pancreatic cancer, ovarian cancer, and hormone-refractory prostate cancer) do not express Ad primary receptor (coxsackie adenovirus receptor, CAR).2Yamamoto M Conditionally replicative adenovirus for gastrointestinal cancers.Expert Opin Biol Ther. 2004; 4: 1241-1250Crossref PubMed Scopus (10) Google Scholar,3Yamamoto M Curiel DT Cancer gene therapy.Technol Cancer Res Treat. 2005; 4: 315-330Crossref PubMed Scopus (33) Google Scholar,17Wesseling JG Bosma PJ Krasnykh V Kashentseva EA Blackwell JL Reynolds PN et al.Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin-targeted adenoviral vectors.Gene Ther. 2001; 8: 969-976Crossref PubMed Scopus (93) Google Scholar,18Yamamoto M Davydova J Wang M Siegal GP Krasnykh V Vickers SM et al.Infectivity enhanced, cyclooxygenase-2 promoter-based conditionally replicative adenovirus for pancreatic cancer.Gastroenterology. 2003; 125: 1203-1218Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar,19Davydova J Le LP Gavrikova T Wang M Krasnykh V Yamamoto M Infectivity-enhanced cyclooxygenase-2-based conditionally replicative adenoviruses for esophageal adenocarcinoma treatment.Cancer Res. 2004; 64: 4319-4327Crossref PubMed Scopus (59) Google Scholar Without a strategy for infecting target cells via CAR-independent pathways, achieving sufficient antitumor efficiency is difficult with the initial generation CRAd systems (Figure 1a). On this basis, various strategies have been developed to address CRAd infectivity. Specific strategies for Ad infectivity enhancement are: The Ad fiber region contains the “knob” domain which binds to the primary adenoviral receptor (CAR) on the surface of target cells. Because this position is the natural binding locale, a number of fiber-knob modifications have been endeavored to increase viral infectivity for CAR negative cancer cells. One of the most effective fiber modification is based upon infectivity enhancement via incorporation of an arginine–glycine–aspartic acid-4C (RGD-4C) motif into the HI-loop of the fiber-knob region20Krasnykh V Dmitriev I Mikheeva G Miller CR Belousova N Curiel DT Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob.J Virol. 1998; 72: 1844-1852Crossref PubMed Google Scholar,21Dmitriev I Kashentseva E Rogers BE Krasnykh V Curiel DT Ectodomain of coxsackievirus and adenovirus receptor genetically fused to epidermal growth factor mediates adenovirus targeting to epidermal growth factor receptor-positive cells.J Virol. 2000; 74: 6875-6884Crossref PubMed Scopus (170) Google Scholar (Figure 1b). The RGD-4C motif is a partial peptide sequence of fibronectin identified by phage library screening. When it was incorporated into the surface exposed HI-loop of the fiber-knob region, the Ad vector with this motif showed CAR-independent infection of the target cells. Also, oncolytic Ads with this motif showed an augmented cytocidal effect in CAR negative cancer cell lines in vitro and in vivo.18Yamamoto M Davydova J Wang M Siegal GP Krasnykh V Vickers SM et al.Infectivity enhanced, cyclooxygenase-2 promoter-based conditionally replicative adenovirus for pancreatic cancer.Gastroenterology. 2003; 125: 1203-1218Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar However, identification of new peptide motifs for Ad modification is nontrivial. Most attempts to incorporate preidentified peptide coding sequence have failed because of a lack of production of fully assembled virus, or the incorporated motif did not show sufficient affinity to the binding counterpart on the surface of the target cells. Screening of the ligand in the form presented in the fiber-knob region is logical direction but there remains the issue of adequate library diversity. Historically, Ad vectors have been derived based on subtype 2 or 5. This is the reason that CAR deficiency on the target cell is a major issue for Ad-based cancer gene therapy. Interestingly, other Ad serotype vectors do not necessarily use CAR as the primary receptor. For example, Ad35 uses CD46 for initial binding, and thus the infection is CAR independent.22Gaggar A Shayakhmetov DM Liszewski MK Atkinson JP Lieber A Localization of regions in CD46 that interact with adenovirus.J Virol. 2005; 79: 7503-7513Crossref PubMed Scopus (53) Google Scholar,23Sirena D Lilienfeld B Eisenhut M Kälin S Boucke K Beerli RR et al.The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.J Virol. 2004; 78: 4454-4462Crossref PubMed Scopus (220) Google Scholar There are basically two approaches for incorporating other subtypes' tropism into adenoviral vectors. One approach is to make a vector fully based on alternate subtype vectors (Figure 1c); the other is to design an Ad2/5-based vector with an alternate subtype's binding domain incorporated (chimeric) (Figure 1d). The first method has the advantage that all parts of the capsid consist of alternate subtype Ad proteins, thus the distribution is assumed to be completely the same as the parental virus. However, in this approach there is no guarantee that the CRAd replication processes after viral entry are as efficient as those of Ad2/5. In recognition of this fact, chimeric approaches are more frequently adaptable to oncolytic Ads, which require efficient post-transduction processes for efficient replication.19Davydova J Le LP Gavrikova T Wang M Krasnykh V Yamamoto M Infectivity-enhanced cyclooxygenase-2-based conditionally replicative adenoviruses for esophageal adenocarcinoma treatment.Cancer Res. 2004; 64: 4319-4327Crossref PubMed Scopus (59) Google Scholar,24Liu Y Wang H Yumul R Gao W Gambotto A Morita T et al.Transduction of liver metastases after intravenous injection of Ad5/35 or Ad35 vectors with and without factor X-binding protein pretreatment.Hum Gene Ther. 2009; 20: 621-629Crossref PubMed Scopus (23) Google Scholar,25Hoffmann D Heim A Nettelbeck DM Steinstraesser L Wildner O Evaluation of twenty human adenoviral types and one infectivity-enhanced adenovirus for the therapy of soft tissue sarcoma.Hum Gene Ther. 2007; 18: 51-62Crossref PubMed Scopus (23) Google Scholar Mosaic vectors are vectors with multiple binding moieties derived from different parental viruses and/or targeting peptides (Figure 1e). Such vectors not only embody a wider infectious range, due to multiple binding motifs, but also show higher infectivity compared to singly targeted vectors. Because enhanced infection is a crucial aspect of CRAd functionality and efficacy, such modifications have been incorporated into CRAds, which thereby show an improved cytocidal effect.26Takayama K Reynolds PN Short JJ Kawakami Y Adachi Y Glasgow JN et al.A mosaic adenovirus possessing serotype Ad5 and serotype Ad3 knobs exhibits expanded tropism.Virology. 2003; 309: 282-293Crossref PubMed Scopus (50) Google Scholar Such mosaic vectors can be derived by either pseudotyping, with 293 cells expressing different binding motifs, or by incorporating more than one fiber into the vector genome.26Takayama K Reynolds PN Short JJ Kawakami Y Adachi Y Glasgow JN et al.A mosaic adenovirus possessing serotype Ad5 and serotype Ad3 knobs exhibits expanded tropism.Virology. 2003; 309: 282-293Crossref PubMed Scopus (50) Google Scholar,27Murakami M Ugai H Everts M Belousova N Yamamoto M Curiel D A simplified method for the generation of a fiber mosaic adenoviral vector.Mol Ther. 2008; 16: S307Google Scholar Because the progeny virus needs the same efficient binding, genetic incorporation is preferred in CRAds. This method can achieve the precise selectivity embodied by employing a high affinity/specificity antibody (Ab), or a specific binding motif for the target moiety expressed on the surface of the cells28Curiel DT Strategies to adapt adenoviral vectors for targeted delivery.Ann NY Acad Sci. 1999; 886: 158-171Crossref PubMed Scopus (129) Google Scholar (Figure 1f). Such bridging can be realized by anti-knob Ab fused with targeting ligands/Abs28Curiel DT Strategies to adapt adenoviral vectors for targeted delivery.Ann NY Acad Sci. 1999; 886: 158-171Crossref PubMed Scopus (129) Google Scholar,29Douglas JT Rogers BE Rosenfeld ME Michael SI Feng M Curiel DT Targeted gene delivery by tropism-modified adenoviral vectors.Nat Biotechnol. 1996; 14: 1574-1578Crossref PubMed Scopus (397) Google Scholar or incorporation of immunoglobulin binding domains into the viral fiber.30Tanaka T Huang J Hirai S Kuroki M Kuroki M Watanabe N et al.Carcinoembryonic antigen-targeted selective gene therapy for gastric cancer through FZ33 fiber-modified adenovirus vectors.Clin Cancer Res. 2006; 12: 3803-3813Crossref PubMed Scopus (44) Google Scholar Although this kind of targeting is functional only for initial entry, the targeting ability is high. Thus, there is a good possibility that such a bridging moiety can be applied for CRAds, especially for systemic administration. Although transductional targeting by these modifications is very useful for improving the efficacy of CRAd therapy, it does not provide sufficient levels of selectivity to the progeny viruses. This is because the transduction-based CRAd selectivity is currently imprecise and because efficient incorporation of the extrinsic targeting moiety into the intratumorally produced progeny virus is uneasy. At this time, bridging moiety-based targeting is used as a way to increase initial infectivity in CAR negative cancer cells,29Douglas JT Rogers BE Rosenfeld ME Michael SI Feng M Curiel DT Targeted gene delivery by tropism-modified adenoviral vectors.Nat Biotechnol. 1996; 14: 1574-1578Crossref PubMed Scopus (397) Google Scholar or a way to deliver the systemically administered virus to the tumor locale.31Li HJ Everts M Yamamoto M Curiel DT Herschman HR Combined transductional untargeting/retargeting and transcriptional restriction enhances adenovirus gene targeting and therapy for hepatic colorectal cancer tumors.Cancer Res. 2009; 69: 554-564Crossref PubMed Scopus (22) Google Scholar The second key issue in CRAd development is a replication control mechanism for selectivity. Aforementioned various methods have been utilized for controlling viral replication in a tumor-specific manner. However, some replication control systems may not function as originally designed. For example, ONYX-015 was originally introduced as a virus that specifically replicates in cancer cells with mutation of the p53 tumor suppressor gene.32Heise C Sampson-Johannes A Williams A McCormick F Von Hoff DD 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: 639-645Crossref PubMed Scopus (839) Google Scholar Although this virus was shown to be safe in early phase clinical trials, there are studies suggesting that the selectivity of this agent is based on p14ARF status in p53 functional cells.33Yang CT You L Uematsu K Yeh CC McCormick F Jablons DM p14(ARF) modulates the cytolytic effect of ONYX-015 in mesothelioma cells with wild-type p53.Cancer Res. 2001; 61: 5959-5963PubMed Google Scholar Another report states that replication of the same virus does not depend on both p53 or p14ARF status.34Edwards SJ Dix BR Myers CJ Dobson-Le D Huschtscha L Hibma M et al.Evidence that replication of the antitumor adenovirus ONYX-015 is not controlled by the p53 and p14(ARF) tumor suppressor genes.J Virol. 2002; 76: 12483-12490Crossref PubMed Scopus (58) Google Scholar Likewise, the CRAd agent Adδ24 has also shown incomplete specificity, allowing replication in pRb intact cells in some experimental systems.35Gomez-Manzano C Balague C Alemany R Lemoine MG Mitlianga P Jiang H et al.A novel E1A-E1B mutant adenovirus induces glioma regression in vivo.Oncogene. 2004; 23: 1821-1828Crossref PubMed Scopus (50) Google Scholar,36Balagué C Noya F Alemany R Chow LT Curiel DT Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes.J Virol. 2001; 75: 7602-7611Crossref PubMed Scopus (55) Google Scholar Thus, additional deletions have been performed to confer more stringent selectivity.35Gomez-Manzano C Balague C Alemany R Lemoine MG Mitlianga P Jiang H et al.A novel E1A-E1B mutant adenovirus induces glioma regression in vivo.Oncogene. 2004; 23: 1821-1828Crossref PubMed Scopus (50) Google Scholar,36Balagué C Noya F Alemany R Chow LT Curiel DT Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes.J Virol. 2001; 75: 7602-7611Crossref PubMed Scopus (55) Google Scholar,37Howe JA Demers GW Johnson DE Neugebauer SE Perry ST Vaillancourt MT et al.Evaluation of E1-mutant adenoviruses as conditionally replicating agents for cancer therapy.Mol Ther. 2000; 2: 485-495Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar These approaches are designed because selectivity becomes particularly important in CRAds displaying an augmented antitumor effect, whereby sufficient clinical safety must be maintained. In addition, the fact that each tumor context may present different requirements for selectivity highlights the need to develop more variety of precise control mechanisms for future CRAd design. In the process of the development of CRAds, strict control of selectivity has historically been considered the key issue relevant to developmental realization. However, wild-type Ad5 had been injected into cervical cancers in the 1950s, causing no severe toxicity.38Huebner RJ Rowe WP Schatten WE Smith RR Thomas LB Studies on the use of viruses in the treatment of carcinoma of the cervix.Cancer. 1956; 9: 1211-1218Crossref PubMed Scopus (258) Google Scholar More recently, agents without additional control mechanisms (ING007) have been approved for phase I clinical trial.39Lichtenstein DL Spencer JF Doronin K Patra D Meyer JM Shashkova EV et al.An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector.Cancer Gene Ther. 2009; 16: 644-654Crossref PubMed Scopus (40) Google Scholar,40Ying B Toth K Spencer JF Meyer J Tollefson AE Patra D et al.INGN 007, an oncolytic adenovirus vector, replicates in Syrian hamsters but not mice: comparison of biodistribution studies.Cancer Gene Ther. 2009; 16: 625-637Crossref PubMed Scopus (47) Google Scholar These are based on the data of intrinsic cancer selectivity of wild-type Ad5. However, in the context of tropism-modified Ads, the US Food and Drug Administration still has concerns vis-à-vis replicative selectivity due to possibility of infection of a wider range of normal cells as a consequence of infectivity enhancement. With respect to the achievement of replicative selectivity, two general strategies have been endeavored. The first type is based upon mutations in the viral genome which are essential for viral replication in normal cells but can be selectively compensated by particular defects in cancer cells (Figure 2a). In this regard, the dl1520 (or ONYX-015) Ad lacks the E1B region and was originally designed to achieve selective replication in cancer cells with mutated p53.32Heise C Sampson-Johannes A Williams A McCormick F Von Hoff DD 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: 639-645Crossref PubMed Scopus (839) Google Scholar Adδ24 has a mutation in the E1A region which theoretically restricts replication to cancer cells with mutated pRb.41Fueyo 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: 2-12Crossref PubMed Scopus (636) Google Scholar The second type of CRAds relies on cancer-specific, promoter-controlled transcription of the E1 region (Figure 2b). Because the E1A protein is necessary for the replication of Ad, these viruses can replicate only in cells where the controlling promoter is active. In this regard, CN706 has a prostate-specific antigen promoter–driven E1 expression cassette which enables selective replication in prostate cancer cells in an androgen-dependent manner.42Rodriguez R Schuur ER Lim HY Henderson GA Simons JW Henderson DR Prostate attenuated replication competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-specific antigen-positive prostate cancer cells.Cancer Res. 1997; 57: 2559-2563PubMed Google Scholar As an emerging strategy for CRAd replication control, post-transcriptional control can be also used. Micro-RNAs are short RNAs which are expressed in the cells and determine many aspects of the cell characteristics. By placing micro-RNA binding sequence into the context of Ad E1A, replication can be restricted to the target cells (e.g., cancer cells).43Ylösmäki E Hakkarainen T Hemminki A Visakorpi T Andino R Saksela K Generation of a conditionally replicating adenovirus based on targeted destruction of E1A mRNA by a cell type-specific MicroRNA.J Virol. 2008; 82: 11009-11015Crossref PubMed Scopus (113) Google Scholar Also, the 5′- or 3′-untranslated region placed on E1A can embody cancer specificity.44Stoff-Khalili MA Rivera AA Nedeljkovic-Kurepa A DeBenedetti A Li XL Odaka Y et al.Cancer-specific targeting of a conditionally replicative adenovirus using mRNA translational control.Bre