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Adenoviruses are DNA viruses that contain about 36kb of double-stranded DNA. Once they have entered the nucleus, genes from the early region 1 (E1a and E1b) are transcribed. Four noncontiguous regions of the genome are expressed (E1 to E4) during the early phase of viral replication. Part of their function is to act as master transcriptional regulators that begin the process of viral gene expression leading to genome replication. Once DNA replication has begun, viral transcription is governed by the major late promoters. Viral-encoded functions can be separated into cis and trans elements. The cis elements are responsible for the origin of replication or the packaging signal that condenses DNA and need to carried by the virus itself. However, the trans genes may be replaced or complemented by the transgene that is being inserted.

Overview of adenovirus as a gene therapy vector
The ideal in oncolytic gene therapy is a vector that can be administered noninvasively, is able to target specifically, and can have regulated expression of therapeutic transgene products for defined time periods.

While there is no one vector that will be optimal for all applications, adenoviruses have features that make them attractive for gene therapy applications. Firstly, adenoviral vectors have a broad tropism and high transfection efficiencies compared with other available vectors. Besides being able to accommodate a comparatively large transgene segment (up to 7.5 kilobase pairs), adenoviruses can also transduce in non-proliferating cells. Adenoviruses are suitable as vectors because they are relatively easy to manipulate using recombinant DNA techniques. Since the adenoviral genome does not undergo rearrangement at a high rate, the inserted foreign genes are maintained through multiple rounds of viral replication. Furthermore, adenoviruses are found in a large number of different species, with most adults having had exposure to serotypes most commonly used in gene therapy. The low pathogenicity in humans of adenoviruses only result in symptoms associated with the common cold.

Oncolytic Conditionally Replicative Adenoviruses (CRAds)
Adenovirus replication may be regulated in a variety of means for the purpose of generating a CRAd. Most of these methods may be broadly classified into two major strategies. The first strategy includes completely or partially deleting the viral genes that have become expendable in tumor cells, such as genes responsible for cell cycle activation through p53 (a tumor suppressor protein) or Rb binding. The second strategy involves controlling the viral gene transcription through the replacement of native viral promoters with tumor-specific promoters (tsp). Such selective viral replication in tumor cells should ideally lead to lysis and death of the cells.

McCormick’s group at Onyx proposed an adenoviral vector that utilized the first strategy. Since p53-deficient cells were common in tumors, an E1b-55kDa-deleted adenovirus was hypothesized as being able to replicate selectively. The E1b-55k gene encodes a protein that binds and inactivates p53 in normal cells to start viral replication. Since E1b-55kDa-deleted adenovirus is not able to inactivate p53, they would not be able to replicate in normal cells. However, in cancer cells that have lost p53, an E1b-55kDa-deleted adenovirus could replicate since there is no requirement for the E1b-55kDa protein to inactivate p53. Despite the novel idea, the E1b-55kDa-deleted adenovirus, known as dl 1520 or Onyx – 015 proved to be a failure. In experiments that involved a variety of cell lines, Rothmann reported a lack of correlation between p53 status and dl 1520 replication after titering the viral progeny.

Hallenbeck from Novartis and Henderson from Calydon pursued the second strategy. They started efforts to treat hepatocellular and prostate carcinomas by using α-fetoprotein (AFP) and prostate-specific antigen (PSA) promoters to drive the adenovirus E1a gene, respectively. However, these efforts did not prove too promising as well. Even though there was some degree of specificity with these promoters, the cellular promoters were not able to keep proper fidelity in the viral genome. Specificity was prevented through even low levels of viral products such as E1a that were sufficient for replication.

Delivery and intratumoral spread of replicative adenoviral vectors
Tumor targeting should be emphasized in order to spare normal cells from infection, thus minimizing toxicity. The delivery requirements for localized or disseminated cancer may be drastically different. Tumor targeting in localized tumors may not be critical because direct injection routes offer high rates of success. However, systematic delivery is required for disseminated cancer, thus increasing the role for tumor targeting.

Adenoviruses get cleared from the blood very effectively, especially by the liver Kupffer cells. Capsid modifications done to alter charge or hydrophobicity may be performed to increase virus persistence in the blood and thus facilitate virus delivery to the tumors.

Passive tumor targeting may be accounted for due to the porosity of the endothelial barrier. Besides the spleen and the liver vessels, only tumor vessels allow the extravasation of particles in the adenovirus size range. Through the attachment of antibodies or other ligands, such as epidermal growth factor and basic fibroblast growth factor, to its capsid, adenovirus has been able to achieve active tumor targeting. In contrast with delivery that requires the removal of natural tropisms of adenovirus, intratumoral spread requires the tropism to be broadened to many different entry pathways. Furthermore, intratumoral spread requires capsid modifications to be genetically incorporated in order for them to be present in progeny. In order to broaden tropism, small peptides such as stretches of lysine to bind heparan sulfate and RGD-motif containing peptides to bind αv integrins have been inserted. The oncolytic potential of Onyx-015 in vitro and in vivo increased through the insertion of a polylysine at the fiber C terminus.

Immune response control of replicative adenoviruses
Virotherapy can be severly hindered by the neutralizing immune response. Patients with elevated serum neutralizing antibodies were found to have less frequent therapeutic responses when tested in early clinical trials with wild-type adenovirus. However, lack of appropriate animal models has impeded progress in the study of the immune system with replicative adenoviruses. The replication of Ad2 in murine cells have been studied to determine a suitable murine model. The cell lines B9 and SN161 were reported to produce progeny at levels 50 fold and 25 fold, respectively, lower than the human A2780Cp cell line. Such work may eventually lead to immune-competent animal models that allow for virotherapy and the study of immune system modulation.

Multimodality treatments of replicative adenoviruses
Besides being used singularly, replicative adenoviruses have also been used in combination with conventional anti-cancer approaches. The efficacy of such an approach has been studied through the configuration of a toxin gene, such as cytosine deaminase or herpes thymidine kinase, into replicative adenoviruses. Freytag developed a replicative adenovirus configured with a thymidine kinase / cytosine deaminase fusion gene and found that the toxin killed cells with administration of the prodrug and increased tumor sensitivity to radiation.

A novel combination therapy that has been attempted utilizes both conditionally replicating adenovirus and replication defective adenovirus. Lee et. al. hypothesized that E1 deleted adenoviruses can become replication-competent when co-transduced with CRAd, with the resulting increased production of E1 deleted adenoviruses helping to increase transduction efficiency. It was reported that the combination of CRAd and an E1 deleted adenovirus did indeed show increased gene transfer and therapeutic efficacy in model tumors in vivo.

The future of CRAds: Arming them
Even though CRAds such as Onyx-015 or CN706 have been used clinically, the results often indicated that although the oncolytic adenoviruses are safe, they are not able to alter significantly the progression of cancer. In order to increase the potency of CRAds, they may be used as a platform for delivery of a therapeutic transgene. This creates an armed replicating adenovirus that increases antitumor properties because the transgene dose is augmented by viral replication.

The placement of transgenes is crucial in a CRAd. Since replication is crucial to the functioning of the CRAd, an insert should not disrupt CRAd replication. Such disruption could occur either by transgene activity or by genomic alterations caused by the insert. Furthermore, since Ad5 can only stably accommodate up to 38kb, these packaging limits must be taken into account. In order to use the adenoviral genome space to its maximum, the endogenous gene expression machinery such as promoter or polyadenylation and splicing signal can be used. Furthermore, since adenoviral genes are well characterized, transgene expression may be predictably modulated.

Transgene under endogenous control
Armed CRAds may use native expression of viral genomic control. The transgene is inserted in replacement of the viral gene that is unnecessary for replication in the target cells. Direct expression of the transgene is allowed since the native gene control elements are untouched. Since the E3 transcription unit is not critical for viral replication, Doronin et. al. deleted the entire E3 region and reinserted the gene coding for adenovirus death protein (ADP), an E3 gene product leading to cell lysis and viral release. Therefore, late in the infection cycle, ADP was expressed by the major late promoter and led to high levels of expression.

Endogenous control is also utilized by linking a transgene to a viral gene by an internal ribosome entry site (IRES), thus allowing both genes to be expressed in a single transcript. The transgene is thus expressed in the same amount and timing as the gene that it is linked to. Researchers have linked a therapeutic transgene to the viral fiber gene and expressed p53, nitroreductase , and yeast cytosine deaminase , among others. All of the studies found that the inserted transgene was expressed late in the infection cycle to high levels.

Transgene under exogenous control
Armed CRAds can also be placed under the control of a non-native promoter. The E3 region, which is nonessential for viral replication, is often deleted to insert larger expression cassettes than may be accommodated by the wild-type genome and they can be divided into two broad strategies. Firstly, the expression of a single transcript that has a therapeutic transgene linked by IRES to the viral E1A gene, is driven by the exogenous promoter (usually tsp or constitutively active promoter). This strategy was utilized by Ye et. al. when they constructed a CRAd with an E1A-IRES-TRAIL cassette driven by an AFP promoter.

The second strategy involves CRAds where a TSP or constitutively active promoter only drives the transgene expression. In efforts to increase the efficacy of Onyx-015 and improve selectively of replication, numerous researchers have inserted transgenes in place of the E1B-55k gene.

Transgenes that enhance cell killing
The purpose of this class of transgenes is to bring about death of the cancer cell, independent of any viral oncolytic effect. Interfering nucleic acids aimed towards cell-cycle regulators are able to stimulate cell death. Researchers have constructed viruses where antisense cDNAs directed against gene encoding checkpoint kinase 1 and 2 have been inserted in place of another gene. As compared to unarmed controls for orthotopic models of hepatocellular cacinoma, intravenous administration of the virus along with cisplatin (chemotherapy drug) led to metastatic reductions and survival improvements. Stimulating apoptosis of the cancer cell has also been attempted to kill the cell. In comparison with CRAds expressing a reporter gene in a subcutaneous model of hepatocellular carcinoma, CRAds armed with a cytokine signaling 3 suppressor were more effective at tumor suppression

Transgenes that modulate the tumor micro-environment
Since both tumors and metastasis require extracellular matrix remodeling and new vascularization, CRAds targeting the tumor stroma (supportive framework of cancer call) usually try to modulate the extracellular matrix or inhibit angiogenesis. Researchers have successfully armed CRAds by inserting a gene in place of the E1B-55k gene that encodes for a strong antiangiogenic factor known as murine endostatin that inhibits endothelial cell proliferation. In subcutaneous models of hepatocellular carcinoma, such armed CRAds showed improved tumor suppression capabilities.

Transgenes that stimulate immune response towards tumor
The purpose of such transgenes is to recruit immune cells to the site of cancer and induce activation and proliferation. Such transgenes have an advantage in that recruitment of the immune system not only has the potential to destroy the primary tumor, but may also facilitate clearance of metastasis and long term benefits in suppressing recurrence. Monocyte chemotactic protein 3 (MCP-3) has been successfully expressed in a CRAd as part of a multiple transgene expression.

Telomerase-dependent oncolytic adenovirus for cancer treatment
Telomerase reverse transcriptase (TERT) is only expressed by tumor cells and is absent is post-mitotic cells. Huang et. al. constructed a TERT promoter regulated CRAd by replacing the adenovirus E1A promoter with that of human TERT to achieve tumor specific oncolysis. TERT promoter regulated CRAd replicated almost as efficiently as wild type adenovirus in TERT-positive cells but was drastically attenuated in TERT-negative cells. Tumor-selective replication and oncolysis in vivo was also demonstrated by an increase in adenovirus titers in tumor extracts by several orders of magnitude between 6 hour and 3 days post-vector injection into a mouse model of human hepatocellular carcinoma.

Hypoxia-inducible factor (HIF) activated oncolytic adenovirus for cancer therapy
The hypoxic (deprived of oxygen supply) fraction of tumors is the population of cells that are most resistant to radio- and chemotherapies. In order to develop new therapies targeting this hypoxic fraction, researchers have examined the hypoxia-inducible factor (HIF) that mediates transcriptional responses to hypoxia by binding to hypoxia-responsive elements (HRE) in target genes. Post and Meir developed a hypoxia / HIF dependent replicative adenovirus (HYPR-Ad) to target hypoxic cells and it was found to display hypoxia-dependent E1A expression. Furthermore, HYPR-Ad was reported to show conditional cytolysis of hypoxic but not normoxic (adequate oxygen supply) cells, demonstrating that an attenuated oncolytic adenovirus can selectively lyse cells under hypoxic conditions.

Adenovirus that targets loss of insulin-like growth factor 2 imprinting system
Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality that is found commonly in human colorectal neoplasms (abnormal mass due to abnormal growth of cells). In order to investigate use of the IGF2 imprinting system for targeted gene therapy of colorectal cancer, Nie et. al. constructed a novel replication-selective oncolytic adenovirus, Ad315-E1A. It was reported that Ad315-E1A had a significant effect against tumor growth both in vitro and in vivo. Since an oncolytic adenovirus regulated by the IGF2 LOI system possesses a strong anti-tumor effect through induction of apoptoss in vitro and in vivo in human colorectal cancer cells, they could also possibly be extended for therapeutic use in a wide variety of tumors in which LOI exists.