Oncolytic virus

Overview
An oncolytic virus is a virus used to treat cancer due to their ability to specifically infect cancer cells, while leaving normal cells unharmed. Viruses can be engineered to be replication competent only in cancer cells, leading to cell lysis and dose amplification at the site of the tumour. Replication competent viruses can efficiently deliver anticancer genes to cancer cells and also have an inherent ability to lyse tumour cells.

Requirements for an Oncolytic Virus
The virus should be able to tolerate storage, and production at high titres. A double stranded DNA genome is advantageous because it has greater stability during storage, and reduces the chances of hazardous mutations. Viruses like adenoviruses and herpes simplex virus are the most suitable, and have been the most extensively studied.

Generating Tumour Selectivity
There are two main approaches for generating tumour selectivity: transductional and non-transductional targeting. Transductional targeting involves modifying the specificity of viral coat protein, thus increasing entry into target cells while reducing entry to non-target cells. Non-transductional targeting involves altering the genome of the virus so it can only replicate in cancer cells. This can be done by either transcription targeting, where genes essential for viral replication are placed under the control of a tumour-specific promoter, or by attenuation, which involves introducing deletions into the viral genome that eliminate functions that are dispensable in cancer cells, but not in normal cells. There are also other, slightly more obscure methods.

Transductional Targeting
This approach to tumour selectivity has mainly focused on adenoviruses, although it is entirely viable with other viruses. However, it should be recognised that increasing the host tissue range of a virus has serious safety implications.

The most commonly used group of adenoviruses is serotype 5 (Ad5), whose binding to host cells is initiated by interactions between the cellular coxsackievirus and adenovirus receptor (CAR), and the knob domain of the adenovirus coat protein trimer. Li et al (1999) showed that CAR is necessary for adenovirus infection by showing that CAR-negative cells could be made adenovirus-sensitive by transfection with CAR cDNA. Virus internalisation depends on an Arginine-Glycine-Asparagine (RGD) motif at the base of adenovirus coat protein that binds to integrins, causing endocytosis. It has been suggested that CAR has a role in cell adhesion, and possibly tumour suppression. Although expressed widely in epithelial cells, CAR expression in tumours is extremely variable, leading to resistance to Ad5 infection. Retargeting of Ad5 from CAR, to another receptor that is ubiquitously expressed on cancer cells, enables reduction of Ad5 tropism, enhancing infection of CAR deficient target cells. This can be done in one of two ways:

Adapter Molecules
Bi-specific adapter molecules can be administered along with the virus to redirect viral coat protein tropism. These molecules are fusion proteins that are made up of an antibody raised against the knob domain of the adenovirus coat protein, fused to a natural ligand for a cell surface receptor. The use of adapter molecules has been shown to increase viral transduction. However, adapters add complexity to the system, and the effect of adapter molecule binding on the stability of the virus is uncertain.

Coat Protein Modification
This method involves genetically modifying the fiber knob domain of the viral coat protein to alter its specificity. Wickham et al (2003) added short peptides to the C-terminal end of the coat protein, which successfully altered viral tropism. The addition of larger peptides to the C-terminus is not viable because it reduces adenovirus integrity, possibly due to an effect on fiber trimerisation. The fiber protein also contains a HI-loop structure, which can tolerate peptide insertions of up to 100 residues without any negative effects on adenovirus integrity. Davydova et al (2004) inserted an RGD motif in the HI loop of the fiber knob protein, shifting specificity toward integrins, which are frequently over-expressed in Oesophageal Adenocarcinoma. When combined with a form of non-transductional targeting, these viruses proved to be effective and selective therapeutic agents for Oesophageal Adenocarcinoma.

Transcriptional Targeting
Transcriptional targeting places an essential viral gene under the control of a tumour-specific promoter, meaning the gene is only expressed in cell types where all the transcription factors required for promoter function are active. A suitable promoter should be active in the tumour but inactive in the majority of normal tissue, particularly the liver, which is the organ that is most exposed to blood born viruses. Many such promoters have been identified and studied for the treatment of a range of cancers.

Cyclooxygenase-2 enzyme (Cox-2) expression is elevated in a range of cancers, and has low liver expression, making it a suitable tumour-specific promoter. Davydova et al (2004) targeted AdCox2Lluc, a conditionally replicating adenovirus (CRAd), against Oesophageal Adenocarcinoma by placing the early genes under the control of a Cox-2 promoter (adenoviruses have two early genes, E1A and E1B, that are essential for replication). When combined with transductional targeting, AdCox2Lluc showed potential for treatment of Oesophageal Adenocarcinoma. Cox-2 is also a possible tumour-specific promoter candidate for other cancer types, including ovarian cancer.

A suitable tumour-specific promoter for Prostate Cancer is prostate specific antigen (PSA), whose expression is greatly elevated in prostate cancer. CN706 is a CRAd with a PSA tumour-specific promoter driving expression of the adenoviral E1A gene, required for viral replication. Rodriguez et al (1997) showed that the CN706 titre is significantly greater in PSA-positive cells.

Attenuation
Cancer cells and virus-infected cells have similar alterations in their cell signalling pathways, particularly those that govern progression through the cell cycle. A viral gene whose function is to alter a pathway is dispensable in cells where the pathway is defective, but not in cells where the pathway is active. Attenuation involves deleting viral genes, or gene regions, to eliminate viral functions that are expendable in tumour cell, but not in normal cells.

For adenovirus replication to occur, the host cell must be induced into S-phase by viral proteins interfering with cell cycle proteins. The adenoviral E1A gene is responsible for inactivation of several proteins, including Retinoblastoma, allowing entry into S-phase. The adenovirus E1B gene inactivates p53, preventing apoptosis. Adenoviruses with mutant E1B have been shown to replicate selectively in p53 deficient cells. One of these viruses, ONYX-015, has a deletion in the E1B coding region, preventing E1B expression, has been shown to be capable of tumour-selective ‘tissue destruction’ in head and neck cancer. ONYX-015 combined with chemotherapy proved effective in a high proportion of cases, and has progressed to phase III clinical trials and even marketing approval in China (Onyx-015/H101). However, research into ONYX-015 has been discontinued indefinitely for financial reasons.

Carette et al (2004) used Ad5- Δ24E3, a CRAd with a 24 base pair deletion in the retinoblastoma-binding domain of the E1A protein, making it unable to silence retinoblastoma, and therefore unable to induce S-phase in host cells. This means Ad5-Δ24E3 is only able to replicate in proliferating cells, such as tumour cells. The adenovirus was used to deliver short hairpin RNA, which was able to reduce expression of the luciferase target gene in target cells to 30%, relative to the control, by RNA interference.

The herpes simplex virus genome contains the enzymes thymidine kinase and ribonucleotide reductase, whose cellular forms are responsible for the production of dNTP’s required for DNA synthesis and are only expressed during the G1 and S phases of the cell cycle. These enzymes allow herpes simplex virus replication in quiescent cells, so if they are inactivated by mutation the herpes simplex virus will only be able to replicate in proliferating cells, such as cancer cells. The G207 herpes simplex virus mutant contains a LacZ insertion, inactivating ribonucleotide reductase, as well as deletion of a virulence gene for safeties sake, has progressed to clinical trials for the treatment in brain cancer.

Vesicular Stomatitis Virus
Vesicular stomatitis virus (VSV) is a rhabdovirus, consisting of 5 genes encoded by a negative sense, single-stranded RNA genome. In nature, VSV infects insects as well as livestock, where it causes a relatively localized and non-fatal illness. The low pathogenicity of this virus is due in large part to its sensitivity to interferons, a class of proteins that are released into the tissues and bloodstream during infection. These molecules activate genetic anti-viral defence programs that protect cells from infection and prevent spread of the virus. However in 2000, Stojdl and Lichty et al. demonstrated that defects in these pathways render cancer cells unresponsive to the protective effects of interferons and therefore highly sensitive to infection with VSV. Since VSV undergoes a rapid cytolytic replication cycle, infection leads to death of the malignant cell and roughly a 1000-fold amplification of virus within 24h. VSV is therefore highly suitable for therapeutic application, and several groups (Stojdl et al., 2003, Ahmed et al., 2004, Ebert et al. 2005, Porosnicu et al., 2003) have gone on to show that systemically-administered VSV can be delivered to a tumor site, where it replicates and induces disease regression, often leading to durable cures. Attenuation of the virus by engineering a deletion of Met-51 of the matrix protein ablates virtually all infection of normal tissues, while replication in tumor cells in unaffected (Stojdl et al., 2003).

Poliovirus
Poliovirus is a natural neuropathogen, making it the obvious choice for selective replication in tumours derived from neuronal cells. Poliovirus has a plus-strand RNA genome, the translation of which depends on a tissue specific internal ribosomal entry site (IRES) within the 5' untranslated region of the viral genome, which is active in cells of neuronal origin and allows translation of the viral genome without a 5’ cap. Gromeier et al (2000) replaced the normal poliovirus IRES with a rhinovirus IRES, altering tissue specificity. The resulting PV1(RIPO) virus was able to selectively destroy malignant glioma cells, while leaving normal neuronal cells untouched.

Double Targeting
It is unlikely to be possible to make a virus entirely specific toward any tissue type by using just one form of targeting. Infection of normal tissue can result in adverse side effects. Double targeting with both transductional and non-transductional targeting methods is more effective than any one form of targeting alone. Davydova et al (2004) combined transductional targeting with a tumour-specific promoter to successfully target an adenovirus against Oesophageal Adenocarcinoma.

Bystander effect
Tumour-selective replication-competent viruses can infect and lyse cancer cells, rapidly destroying them without the need for the expression of foreign genes. Viral replication also leads to local dose amplification and improved tumour penetration. However, their intrinsic ability to lyse tumour cells can be complimented by other mechanisms that increase the anti-tumour toxicity if the virus, so it is not necessary for all cells in the tumour to be infected and lysed.

Suicide Genes
Viruses can be used as vectors for delivery of suicide genes, encoding enzymes that can metabolise a separately administered non-toxic pro-drug into a potent cytotoxin, which can diffuse to and kill neighbouring cells. One herpes simplex virus, encoding a thymidine kinase suicide gene, has progressed to phase III clinical trials. The herpes simplex virus thymidine kinase phosphorylates the pro-drug, ganciclovir, which is then incorporated into DNA, blocking DNA synthesis. The tumour selectivity of oncolytic viruses ensures that the suicide genes are only expressed in cancer cells.

Suppression of Angiogenesis
Angiogenesis (blood vessel formation) is an essential part of the formation of large tumour masses. Angiogenesis can be inhibited by the expression of several genes, which can be delivered to cancer cells in viral vectors, resulting in suppression of angiogenesis, and oxygen starvation in the tumour. The infection of cells with viruses containing the genes for angiostatin and endostatin synthesis inhibited tumour growth in mice.

Immune Response
In a number of cases, cancer cells exposed to viruses have experienced widespread necrosis, which cannot be entirely accounted for by viral replication alone. Cytotoxic T-cell responses directed against virus-infected cells have been identified as an important factor in tumour necrosis. However, since viruses are normal human pathogens, they induce an immune response, which reduces the effectiveness of viruses. For example, increased antibody titers could deactivate viruses before the tumour has been destroyed. This can be overcome by using parental viruses that are not normal human pathogens, thereby avoiding any pre-existing immunity. However, this does not avoid subsequent antibody generation. Alternatively, the viral vector can be coated with a polymer such as polyethylene glycol, shielding it from antibodies, but this also prevents viral coat proteins adhering to host cells. Deactivation of the immune system is not desirable, since it has a positive effect on tumour necrosis.

Oncolytic Viruses in conjunction with existing cancer therapies
Chen et al (2001) used CV706, a prostate-specific adenovirus, in conjunction with radiotherapy on prostate cancer in mice. The combined treatment resulted in a synergistic increase in cell death, as well as a significant increase in viral burst size (the number of virus particles released from each cell lysis). No alteration in viral specificity was observed.

ONYX-015 has undergone trials in conjunction with chemotherapy. The combined treatment gave a greater response than either treatment alone, but the results have not been entirely conclusive. ONYX-015 has shown promise in conjunction with radiotherapy.

Possible Applications
Virus gene therapy has never been used successfully against cancer, mainly due to poor transduction of cells. This problem is solved by oncolytic viruses. The use of viral agents to treat cancer is now a real possibility, and several very promising advances have been made e.g. ONYX-015.

Viral agents administered intravenously can be particularly effective against metastatic cancers, which are especially difficult to treat conventionally. However, blood born viruses can be deactivated by antibodies and cleared from the blood stream quickly e.g. by Kupffer cells (extremely active phagocytic cells in the liver, which are responsible for adenovirus clearance). Avoidance of the immune system until the tumour is destroyed could be the biggest obstacle to the success of oncolytic virus therapy. To date, no technique used to evade the immune system is entirely satisfactory. It is in conjunction with conventional cancer therapies that oncolytic viruses show the most promise, since combined therapies operate synergistically with no apparent negative effects.

The specificity and flexibility of oncolytic viruses means they have the potential to treat a wide range of cancers with minimal side effects. Oncolytic viruses have the potential to solve the problem of selectively killing cancer cells. However, altering the host range or tissue specificity of any virus has extremely serious safety implications. Nonetheless, the increasing understanding of viral replication mechanisms and the immune system means the effective treatment of cancer with virotherapy could be just around the corner.

Referenced Papers

 * Li Y, Pong R, Bergelson JM, Hall MC, Sagalowsky AI, Tseng C, Wang Z & Hsieh J; Loss of Adenoviral Receptor Expression in Human Bladder Cancer Cells: A Potential Impact on the Efficacy of Gene Therapy, Cancer Research, 59, 325-330 (1999)
 * Wickham TJ; Ligand-directed targeting of genes to the site of disease, Nature Medical, 9, 135-139 (2003)
 * Davydova J, Long PL, Gavrikova T, Wang M, Krasnykh V & Yamamoto M; Infectivity-Enhanced Cyclooxygenase-2-Based Conditionally Replicative Adenoviruses for Esophageal Adenocarcinoma Treatment, Cancer Research, 64, 4319-4327 (2004)
 * Rodriguez 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 Research, 57, 2559-2560 (1997)
 * Carette JE, Overmeer RM, Schagen FHE, Alemany R, Barski OA, Gerritsen WR & Van Beusechem VW; Conditionally Replicating Adenoviruses Expressing Short Hairpin RNAs Silence the Expression of a Target Gene in Cancer Cells, Cancer Research, 64, 2663-2667 (2004)
 * Stojdl DF, Lichty B, Knowles S, Marius R, Atkins H, Sonenberg N, Bell JC; Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus, Nature Medicine, 6, 821 (2000)
 * Stojdl DF, Lichty BD, tenOever BR, Paterson JM, Power AT, Knowles S, Marius R, Reynard J, Poliquin L, Atkins H, Brown EG, Durbin RK, Durbin JE, Hiscott J, Bell JC; VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents, Cancer Cell, 4, 263 (2003)
 * Ahmed M, Cramer SD, Lyles DS; Sensitivity of prostate tumors to wild type and M protein mutant vesicular stomatitis viruses, Virology, 330, 34 (2004)
 * Ebert O, Harbaran S, Shinozaki K, Woo SL; Systemic therapy of experimental breast cancer metastases by mutant vesicular stomatitis virus in immune-competent mice, Cancer Gene Therapy, 12, 350 (2005)
 * Porosnicu M, Mian A, Barber GN; The oncolytic effect of recombinant vesicular stomatitis virus is enhanced by expression of the fusion cytosine deaminase/uracil phosphoribosyltransferase suicide gene, Cancer Research, 63, 8366 (2003)
 * Gromeier M, Lachmann S, Rosenfeld MR, Gutin PH, & Wimmer E; Intergeneric poliovirus recombinants for the treatment of malignant glioma, Proc. Natl. Acad. Sci. USA, 97, 6803-6808 (2000)
 * Chen Y, De Weese T, Dilley J, Zhang Y, Li Y, Ramesh N, Lee J, Pennathur-Das R, Radzyminski J, Wypych J, Brignetti D, Scott S, Stephens J, Karpf DB, Henderson DR & Yu D. CV706, a Prostate Cancer-specific Adenovirus Variant, in Combination with Radiotherapy Produces Synergistic Antitumor Efficacy without Increasing Toxicity, Cancer Research, 61, 5453-5460 (2001)

Onkolytische Viren