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Cancer is one of the leading causes of death globally comprising 13% of all deaths (7.6 million deaths) in 2008. Although well established conventional therapies including surgery, radiotherapy and chemotherapy are existed, we still need new therapies and strategic plans. Local therapies such as surgery and radiotherapy have been limited in disseminated tumours. Likewise, chemotherapy has some intolerable adverse effects and sometimes, pre-existing chemo-sensitive tumours are resistance to chemotherapy after prolonged used. Thus, we really need certain promising therapies to handle these problems. Recent years, oncolytic virotherapy is uprising and promising for the various types of cancers. Ideology of virotherapy treating the malignancy has been introduced since early 20th century. However, since early oncolytic viruses were targeted not only tumour cells but also the normal cells, interest in the virotherapy was declined. Therefore, many trials related with such therapy were terminated during the following decades.
Late 1990s, interest of virotherapy was re-active with the advance of modern biotechnology. Today, concern of the virotherapy is high and it has the potential promises as a reasonable cancer treatment by itself or conjunction with other conventional therapies such as surgery, radio and/ or chemotherapy (synergic effect). Advance technology allows the development of oncolytic viruses which only effective on dividing cancer cells but not attack the normal dividing cells. There are generally two types of oncolytic viruses namely non-engineered (naturally occouring) and engineered agents. Both types may destroy the malignant cells without harming the normal cells. Generally, oncolytic viruses only infect and preferentially replicate within the cancer cells followed by lyses these cells. In recent years, many therapeutic virus candidates are emerging and testing their oncolytic prosperities with preclinical and clinical trials. Among them, adenovirus H101 was the first virus approved by Chinese food and drug administration (FDA) in 2004 as the adjuvant oncolytic virotherapy combined with pre-existing conventional chemo- and radiotherapy in the head and neck cancers.
2. Type of oncolytic viruses
Oncolytic viruses are principally divided into 4 types according to their mechanisms of action. There are intrinsically tumour selective viruses, virulent gene deleted viruses, promoter inserted viruses and pseudotyped viruses. Genetic modified oncolytic viruses are manipulated whether insertion of the transgenes or deletion of the virulence genes.
1) Naturally occouring tumour selective viruses are the viruses that are not genetically modified, direct targeting on the malignant cells. For instance, New castle disease virus, Vesicular stomatitis virus, Poliovirus and Reovirus are intrinsically tumour selective. However, affectivity is less due to depend on the natural strength of their lytic properties.
2) Virulent gene deleted oncolytic viruses are more popular because their selectivity on target tumours are more specific without infectivity to normal ones. For example, herpes simplex virus, adenovirus, measles virus and vaccinia virus can be modified by deletion of their virulence protein coding genes.
3) In addition, inserting of foreign genetic elements such as promoter region boost tumor specificity and selectivity of oncolytic viruses. Thus, tumour cells allow the replication of these viruses because only tumour cells can activate the promoter region of them. For example, prostate specific antigen (PSA) promoter inserted adenovirus CG7870 applies in prostate cancer and promising results were came out.
4) Pesudotyped oncolytic viruses are modified with ligands which target tumour selective cell surface receptors. Therefore, they solely have their infectivity on malignant cells. (E.g. adenovirus delta 24RGD). Moreover, these viruses may reduce toxicity and dose requirement.
3. Characteristic features of standard oncolytic virus
Since viruses can infect not only the malignant cells but also the functioning cells, oncolytic virotherapy is the critical therapy. Hence, safety and efficacy of the virotherapy are considerable issue and still challenging for further improvement. Potential oncolytic viruses are needed to confirm or compare whether they have real ideal characters of oncolytic virus or not. Standard characters of the oncolytic virus stated that (1) they only replicate within tumour with high multiplication rate, not on normal cells (2) less or no infectivity and virulence than their wild types (3) genetically stable so that mutations and recombination with other viruses are minimized for manufacturing and safety issues. DNA virus is more stable than RNA virus (4) can inactivated anytime with antiviral drugs or other mechanisms (5) considerable mass production (commercially available) can be possible with good manufacturing practices. Therefore, for development of virotherapy, all oncolytic viruses should be fulfilled above criteria. Adenovirus and Herpes simplex virus (HSV) have high selectivity and specificity on tumour cells with massive replication rate of 1000 folds in 1st cycle. Besides, they are considerably stable whereas terminate anytime with their respective antiviral therapy (e.g. adenovirus is self-limiting and HSV is treated by acyclovir).
4. Tumour selective mechanism
With the knowledge of the malignant cells' molecular biology, oncolytic virotherapy can be created to attack the tumour cells selectively. Cancer cells undergo changes ranging from subtle point mutation to chromosomal instability. Inherent tumour selective viruses specifically attack the tumour cells by targeting the specific tumour promoting pathway of the malignant cells such as activated Ras and AKT pathway, defective interferon (IFN) pathway etc.
RNA-activated protein kinase (PKR) pathway is a natural process that inhibits viral protein synthesis. Physiologically, interferon (IFN) secreted from infected cells phosphorylates the PKR which subsequent phosphorylates eIF-2α. Then, phosphorylated eIF-2α interfere the oncolytic viral protein synthesis that require for their virulence. In contrast, Ras mutation and defective IFN in malignant cells disturbs the PKR pathway and favours the oncolytic virus activity. For instance, herpes simplex virus (HSV) containing neurovirulence gene γ34.5 that binds with intracellular phosphatase and dephosphorylates eIF-2α allowing replication of HSV in both normal and tumour cells. However, deletion of this gene permits to replicate only in Ras mutated or interferon (IFN) defective cells. Controversially, recent finding suggested that γ34.5 deleted HSV can also replicate in PKR functional malignant cells. It is seen to be defects in PI-3 kinase pathway which favours translation of γ34.5 mutant HSV. Moreover, genetically modified adenovirus (dl331), VAI mutant strain, prefers to replicate only in tumour cells with Ras activation. Similarly, dl331 is also effective in Epstein-Barr virus (EBV) associated tumours such as nasopharyngeal malignancy because Epstein-Barr virus expresses viral associated RNAs (VA RNAs) that defect PKR pathway.
Many cancer cells over-express receptors for virus in high level. Thus, exploiting this mechanism, many oncolytic viruses are selectively homed in their specific malignant cells. For example, over-expression of intracellular adhesion molecule 1 (ICAM-1) and decay acceleration factor (DAF) in tumour favours to infect Coxsackie virus A21. Besides, Newcastle disease virus (NDV) binds sialic acid receptors. Alpha virus similarly uses heparin sulphate or ICAM-1 as its receptors. Both of them are highly express in tumor population. Therefore, these viruses are highly concentrated in tumour cells.
Figure : Several oncolytic viruses attacking the RNA activated protein kinase (PKR) control system
(Wang et al., 2005)
5. Immunogenicity of virotherapy
Likewise as many other viruses, oncolytic viruses also stimulate and activate the body defense mechanisms including innate as well as adopted immunity. These viruses produce the viral proteins required for their replication within tumour cells. These proteins also stimulate the MHC class I gene to present it on the cellular surface of tumour cells as well as on the normal cells. MHC class I antigen was recognized by cytotoxic T cells (CTLs) or CD8+ cells which may destroy any cells representing MHC I antigens. Therefore, nature immunity allows eliminating both tumour and normal non-dividing cells. So, oncolytic viruses may also destroy normal cells apart from abnormal ones. An immune mechanism on the oncolytic viruses is one of the major constraints for developing modern virotherapy. However, to date, genetically modified viruses can only replicate and lyses p53 mutant cells. They cannot inactivate p53 gene of the normal host cells. p53 is functional and prevent replication of these viruses in the normal host cells. So, they are allowed their functions only in mutant tumour cells.
6. Conversion of oncogenic to oncolytic
Many oncogenic viruses are potential to use as oncolytic therapy nowadays after genetically manipulation. Generally, 15-20% of the carcinogenesis is contributed by various kinds of oncogenic viruses such as herpes papilloma virus (HPV), Epstein-Barr virus etc. Reversely, these viruses can be changed to treat the malignancies by exploiting their lytic effects on the dividing cells. One of the classical examples is herpes simplex virus type 2 which is ongoing trials in many tumour cell lines by deleting its oncogenic genes coding thymidilase kinase or ribonucleotide reductase. Therefore, even tumourogenic agents can be used as oncolytic therapy by engineering their oncogenic gene components.
7. Novel oncolytic viruses
Adenovirus is interested in treatment of brain tumour especially in glioma multiforme. This tumour is never metastasized and contributed as single lesion. Moreover, it is almost resistance to systemic therapy because of blood-brain barrier and lack of cell mediated antigen drainage. However, fortunately, oncolytic viruses can replicate and spread within the tumour population since blood brain barrier create immune privileged site. In glioma cells, tumour suppressor gene (Rb) is inactivated and lack of expression. Taking this advantage, genetically modified adenoviruses are constructed by deletion of eight amino acids in Rb binding region of E1A protein. Therefore, they are unable to replicate in the normal cells because viruses cannot inactivate Rb gene of the functioning cells. But they can easily divide within the malignant cells owing to the lack of Rb protein. Additionally, adenoviruses induce autophagy in infected cells (malignant cells) through down-regulation of AKT/TOR pathway. Many genetically engineered adenoviruses are still on trials including in vitro and in vivo tests.
ONYX-015 (dl1520) is a simple adenovirus lack of E1B 55K protein which do not replicate in the normal cells. So, they only express their functions in p53 mutant cells. In other word, their function does not work in p53 competent cells. Onyx-015 is a first oncolytic virus that has been approved by china FDA to treat the head and neck cancers especially refractory nasopharyngeal cancer combining with standard cisplatin based chemotherapy. Onyx-015 should be given intratumoural or peritumoural injection because of their side effects (e.g. flu-like syndrome). In addition, it showed 50% response rate observed in phase I and II clinical trials of pancreatic cancer. Recent studies found that Onyx-015 replication is not solely dependent on p53 function. Onyx-015 can replicate within some p53 competent cells whereas sometimes, cannot replicate even in mutant p53 cells. It may think to be reliable on E1B 55K independent nuclear late mRNA export of the tumour cells but not in functionally normal cells. Besides, other co-founding effects should be considered. For example, increasing the temperature (fever, hyperthermia or drugs) promotes replication of E1B deficient adenovirus in the malignant cells rather than normal ones.
dl250 mutant strain is engineered by deletion of E1B 19K which is homologue of Bcl-2 and also inhibits pro-apoptotic protein Bax. Functionally, Bcl-2 is the anti-apoptosis protein. Hence, deletion of E1B 19K leads to permit cell death in Bcl-2 over-expressed tumour cells. It may also reduce expression of anti-apoptosis proteins and various growth factors. This type of virus is more potent in anti-tumour activity than dl1520.
Delta 24 (dl922-947) adenoviruses are genetically modifying agents which are deleted the gene related with virulence factors and upregulate the transcription elements sensitive to the transcription factors of tumour cells. E.g. Delta-24 is modified by deleting of 24 nucleotides (pRb binding region) in E1A gene so that it is unable to inactivate Rb gene in the normal cells. So, it can only effective in the malignant cells. Now, many studies promise that it has potent anti-tumor effect in glioma. E1A mutants are more superior in oncolysis than E1B strains in vivo and vitro. Besides, Delta-24 RGD is more efficient in treating the low CAR (coxsackie-adenovirus receptor) expression malignant cells such as glioma and bronchogenic carcinoma cells.
7.2. Herpes Simplex virus
First herpes simplex virus (dlsptk) as an oncolytic agent is developed in 1991 that is deleted in thymine kinase (TK) genes required for nucleic acid metabolism. Lacking of this gene, Herpes Simplex virus (HSV) lose its ability of replication in the normal cells. Hence, HSV only prefer to infect the tumour cells. HSV is a primarily potential treatment in several paediatric cancers including brain tumours. So far, γ34.5 deleted HSV are tracking on the clinical trials. These all vectors directly target to the tumour cells by deletion of neurovirulant gene γ34.5 (30kb) which is not essential for replication of the malignant cells. G47Δ HSV virus is derived from G207 parent virus. They are constructed by deletion of both copies of γ34.5 gene (1kb) and deletion of 312bp in ICP47 gene increasing oncolyitc efficacy. Also, they promote MHC class I expression in the tumour cells enhancing the immunogenicity of these cells. G207 variant was completed phase I study in glioblastoma multiforme resulting with no serious side-effects. Similarly, HSV 1716 is a genetically engineered variant by manipulating HSV1 stain 17 and deleting both copies of neurovirulent gene, γ34.5. Pilot study has already completed in Metastatic melanoma. NV 1020 (R7020) stain is the chimeric recombinant of HSV 1 and 2 with deleting one copy of γ34.5 gene, UL24 and 56 genes. Originally, it is developed as HSV vaccination. However, recently, it is still ongoing phase II trial on hepatic metastases of colorectal cancer by direct infusion into the hepatic artery.
OncoVEXTM is recombinant vector with deletion of γ34.5 gene as well as ICP47. Deletion of γ34.5 reduces intrinsic neuro-pathogenecity of HSV whereas ICP47 deletion restores MHC class I presentation. Additionally, insertion of GM-CSF gene stimulates immune response of the host to the tumour cells. Releasing of GM-CSF promotes recruitment of dendritic cells for tumour specific response. It promotes tumour specific antigen (TSA) expression as well. Thus, OncoVEXTM affects not only on local tumour but on metastases or distant tumours. Currently, OncoVEXTM improved loco-regional control of head and neck cancers combining with chemo-radiotherapy. Intralesional injection of OncoVEX GM-CSF is ongoing phase I trials on cutaneous metastases and melanomas although it has dose related limitation such as injection site inflammation. Another advantage is that it is able to carry large transgenes up to 150kb. It is the main advantage of these viruses using for oncolytic agent although they may produce neurotoxity at high doses, difficult cloning and reactivate latent herpes infection which are hidden in the nervous systems (Ganglions).
7.3. Newcastle disease virus
Newcastle disease virus (NDV) is paramyxovirus containing single stranded RNA which causes Newcastle disease in avian (birds). Earliest NDV (73-T strain) has been started to use as a clinical trial oncolytic virus for cervical cancer in 1965. Based on their oncolytic properties, NDV is divided into lytic and non-lytic stains. Lytic strains direct lyses the targeted cells. Currently, 2 lytic strains of NDV are ongoing trials which are NDV-HUJ and PV701. Both are naturally occouring live attenuated viruses. As NDV-HUJ strain is a neurotropic virus, it applies in glioblastoma multiforme (GBM). Likewise, NDV-PV701 strain has effect on many types of tumours. Results of their trials have potential promising. One of the advantages of NDV is that it selectively replicates in the tumour cells, not on normal ones. When NDV has also studied in breast cancer patients neoadjuvant with chemotherapy, outcome was desirable with minimal adverse effects including fever, flu-like syndrome, hypotension etc. Occourance and severity of side effects is reduced in subsequent therapy due to development of NDV antibodies in patients' serum. On the other hand, non-lytic strains disturb the malignant cell metabolisms leading to allow regression of the tumours. Common non-lytic strains include Ulster stain. NDV damages the malignant cells by either direct lysis of the cells, induction of cytokine production (Interferon, Tumour necrotic factor) or enhance apoptosis including both intrinsic and extrinsic pathways. 72-T stain induces cytokine release while Ulster stain over-expresses the TRAIL receptors on tumour cells surface which may lead to apoptosis.
7.4. Mump virus and Simian virus
Mump virus is the first paramyxovirus trying to treat in variety of human malignant cells. Vaccine strain 79 (S79) has potential promising oncolytic virus because S79 can only be infected to the cancer cells but not in normal ones. Studying in nude mice, mump virus demonstrated its tumour inhibition effect significantly.
Simian virus is also a rubulavirus and among them, strain 5 can be genetically engineered as an oncolytic virus. This modified strain is able to attack several different cancer cell types significantly.
7.5. Vesicular stomatitis virus
Vesicular stomatitis virus (VSV) is only rhabdovirus potentially using in cancer therapy. VSV is a single stranded RNA virus considering for oncolytic therapy. Developing of the recombinant VSV virus in 1995, the role of VSV is amounting in virotherapy. In recent studies, genetically modified replication competent VSV prolonged survival of hapatocellular carcinoma, breast cancer and malignant melanoma. Oncolytic properties of VSV is more effective in type I interferon (IFN) resistance malignant cells. Tumour cells are defect in interferon (IFN) signaling pathways but activated in Ras -ERK pathway. However, VSV can also impact on the normal cells especially in high doses. Thus, early (prophylactic) interferon therapy is required concomitant with VSV virotherapy because interferon appears to prevent the viral replication within the normal cells. Using the advantage of replication within the interferon defect cells, recombinant VSV deltaM51 which is defective in M (matrix) protein (point mutation) was constructed. Matrix protein is the regulator protein that increases replication and transcription of the virus but blocks the host cells anti-viral mechanism. Studies showed that VSV deltaM51 strain has beneficial role in glioma cells xenografted nude mice. Furthermore, VSV shutdown the blood supply to the tumour leading to deprivation of oxygen and nutrients which may require for tumour growth.
Measles as oncolytic therapy is more interesting since there was significant regression of Hodgkin's lymphoma after infecting with measles virus. Resent study suggested that recombinant measles virus (Edmonston B strain) showed significant inhibition on xenograft SCID mice with human lymphoma cells. Next, Edmonston B stain specifically attracts CD 46 cell surface receptors that are highly expressed in human mesothelioma cells. Thus, this strain has highly attractive role in treatment of mesothelioma. In addition, engineered measles virus with interferon (IFN) β gene inhibits tumour angiogenesis rather than parental strain. Despite most of the people previously encountered with measles infection or vaccination in their early life which may cause therapeutic failure, the evidence highlighted that replication of measles virus was taken place even in the immune individuals. It seems to be immunosuppression due to cancer itself or concurrent use of other anti-cancer therapies such as radiotherapy, and (or) chemotherapy.
Vaccinia virus (VV) is the most potential candidate poxvirus utilized as virotherapy recently. This virus is genetically engineered by deletion of thymidine kinase genes like herpes simplex virus (HSV). For instance, JX-594 strain which is transfected with GM-CSF gene, displayed oncolytic activity in animal models. However, it may rarely affective in the normal cells. Most Vaccinia viruses kill the targeted malignant cells by apoptosis as well as traditional mechanisms. Myxoma virus, another poxvirus, is significantly effective on human glioma cancer cell lines. In addition, rapamycin (immunosuppressant) reinforced its oncolytic efficacy when using combination.
Togaviruses (Sindbis and Semliki Forest Virus) also show their potential roles in the oncolytic therapy. Sindbis virus (SIN) is an RNA virus that naturally infects human by mosquito bites. This virus binds with its receptors of 65kD (Laminin receptors) which are highly express on the tumor cells (tumour homing property). To take the advantage, Sindbis virus promotes considerably regression of the several tumor cell lines in vitro testing and xenograft SCID mice. In human study, it has promising effect on cervical and ovarian malignancies with minimal or no remarkable adverse effect on normal cells. Next, Semliki Forest Virus (SFV) may inoculate repeatedly without prominent immune response. Togaviruses favour as the oncolytic virotherapy agents due to their high replication rate, broad spectrum of host ranges, increase transgene expression and stable in blood stream. Apart from these viruses, Venezuelan equine encephalitis virus (VEE) is also a replication competent virus which is interesting in certain circumstance of oncolytic therapy.
Gamma retrovirus (moloney murine leukemia virus (MoMLV)) may have effect on the tumour cells not in the non-dividing cells. So, it may safe as oncolysis. For instance, when U87 glioma xenografted nude mice were administrated with MoMLV, significant oncolytic result has been reported. These viruses are less effect on normal cells due to lack of nuclear transport of viral genome. Certainly, they cannot replicate well within non-dividing cells. In addition, modified MoMLV viruses expressing HSV thymidine kinase (TK) have synergistic effect on glioblastoma cells combining with ganciclovir (anti-viral agent for HSV). Another retrovirus such as fomy virus has also intrinsic oncolytic property. It is researched recently on glioma implanted nude mice. However, the result is still controversial.
Table 1: Specific oncolytic viruses with their ongoing treatment regimes