ISSN 00268933, Molecular Biology, 2012, Vol. 46, No. 6, pp. 780–789. © Pleiades Publishing, Inc., 2012. Original Russian Text © N.V. Gubanova, A.S. Gaytan, I.A. Razumov, V.A. Mordvinov, A.L. Krivoshapkin, S.V. Netesov, P.M. Chumakov, 2012, published in Molekulyarnaya Biologiya, 2012, Vol. 46, No. 6, pp. 874–886.
REVIEWS UDC 577.2:616006;61:578.7
Oncolytic Viruses in the Therapy of Gliomas N. V. Gubanovaa, e, A. S. Gaytanf, I. A. Razumova, b, e, V. A. Mordvinove, A. L. Krivoshapkine, f, S. V. Netesova, b, and P. M. Chumakova, c, d a
Novosibirsk State University, Novosibirsk, 640090 Russia; email:
[email protected] b State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk Region, 630559 Russia c Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia d Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA e Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090 Russia f Meshalkin Institute of Circulation Pathology, Novosibirsk, 630055 Russia Received May 28, 2012; in final form, June 14, 2012
Abstract—Despite the modern advances in medicine, cure for malignant glioblastomas remains an elusive goal. Both the invasive nature and the location in vital areas of the brain make this type of tumors difficult to treat surgically, while adjuvant therapy fails to bring the expected results. Frequent recurrence and invasive ness of malignant gliomas are due to the resistance of glioma stem cells to conventional radiation therapy and chemotherapy. Technological achievements in constructing recombinant viruses yielded strains with high oncolytic activity toward glial tumors. Many of these strains have passed Phase I clinical studies and proved to be highly safe. Although the approach is obviously promising, the available strains are not efficacious enough to cure the disease and need further improvement. The review summarizes the results reported for the most successful variants of oncolytic viruses that have come down to clinical trials and discusses the prospects of new approaches to viral therapy of malignant gliomas. DOI: 10.1134/S0026893312060064 Keywords: gliomas, oncolytic viruses, antitumor therapy, molecular oncology
INTRODUCTION Malignant tumors of the brain constitute only a minor portion (1–1.5%) of all cancers and originate mostly from glial cells (more than 60%) [1]. Of all malignant gliomas, 70% are multiform glioblastomas, 15% are anaplastic astrocytomas, and the remaining 15% are less aggressive gliomas [2]. Malignant gliomas have an extremely poor prognosis and are particularly difficult to treat. The mean relapsefree survival is approximately 6 months in glioblastoma patients, and their mean overall survival is no more than 9–12 months. In spite of the apparent progress in understanding the mechanisms of the origin and progression of malignant gliomas and numerous new treatments, the mean sur vival increased only by 2–3 months over the past 30 years [3, 4]. Medical care is still palliative in the vast majority of cases. Gliomas are characterized by an infiltrative growth and occur in functionally important regions of the brain, making their radical surgical removal extremely difficult. Disease progression and Abbreviations: MRI, magnetic resonance imaging; EGFR, epi dermal growth factor receptor; GST, glioblastoma stem cell; NDV, Newcastle disease virus; PFU, plaqueforming unit; Ad, adenovirus (in serotype designations); PKR, RNAdependent protein kinase R; TCID, tissue culture infectious dose; eIF2α, eukaryotic translation initiation factor 2α; RR, ribonucleotide reductase; ECM, extracellular matrix.
an inevitable relapse after therapy are most likely due to persisting glioma stem cells (GSCs), which are highly invasive and highly resistant to radiotherapy and chemotherapeutics [5, 6]. A complex multidisciplinary approach accepted in modern neurooncology involves a surgical removal of the tumor with subsequent adjuvant radiotherapy and chemotherapy. Surgery is aimed at removing tumor tissue to a maxima extent without creating new neuro logical deficiency. Hightech neuronavigation stations are used for the purpose [7], and intraoperative high field magnetic resonance imaging (MRI) instruments are finding broad use. A method that received global recognition involves a microsurgical resection of malignant gliomas on the basis of intraoperative fluo rescence of 5aminolevulinic acid (5ALA) metabo lites, which selectively accumulate in malignant glioma cells [8]. The method makes it possible to demarcate a glioma with a high precision, thus facili tating its optimal resection [9]. While studies are continued to improve the efficacy of surgical, radiation, and chemotherapeutical treat ments, new methods are also sought. Great expecta tions are associated with the socalled targeted approaches, including gene therapy, immune therapy, selective radiation exposure of malignant cells, and oncolytic viruses [10].
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The use of oncolytic viruses to treat malignant glio mas is of special interest. A profound understanding of the mechanisms of virus replication and virus–host interactions, as well as the progress in constructing recombinant viruses, made it possible to design virus strains possessing therapeutic properties. Many of such strains passes Phase I clinical trials and proved to be safe at the concentrations proposed, but the effi cacy of these strains in glioma treatment still waits for its accurate evaluation. The review discusses the prop erties of the most promising oncolytic virus strains that have come down to clinical trials and considers the possibilities of new approaches to virotherapy of malignant gliomas of the brain. PATHOMORPHOLOGICAL AND MOLECULAR GENETIC CHARACTERISTICS OF BRAIN TUMORS Glial tumors are the most abundant among pri mary tumors of the central nervous system, varying in the extents of cell differentiation and malignancy. Anaplastic astrocytoma (grade III according to a WHO classification) and glioblastoma (WHO grade IV) constitute a main group of malignant gliomas. Ana plastic astrocytoma is an infiltrative neoplasm that is characterized by focal or dispersed anaplasia and has a higher proliferation index as compared with lower grade astrocytomas (pilocytic and fibrillar astrocyto mas, which are WHO grade I and II, respectively). Histological diagnosis is based on atypical nuclei and a higher mitotic activity of glioma cells. Histologically, glioblastoma is tissue with prolifer ating blood vessels and necrotic foci [11]. Glioblasto mas arise de novo in the majority of cases or result from progression of lowergrade glial brain tumors, including fibrillar astrocytomas (malignancy grade II) and anaplastic astrocytomas (grade III). A glioblas toma is classed as secondary in the latter case. By MRI, glioblastoma has a highcontrast peripheral region with uneven outlines and a central heteroge neous region with signs of necrotic changes. Perifocal edema is usually substantial. Tumorigenesis is caused by inherited or somatic mutations of certain genes, which control important biological processes. Mutations or chromosome aber rations may activate oncogenes and/or inactivate tumor suppressor genes. Certain genetic alterations are accompanied by loss of heterozygosity, where one allele may be altered by mutations and the other is lost as a result of a deletion of an extended chromosome region. The most frequent alteratins in glioblastoma are deletions of region q22–qter (83%) [12, 13] or the total short arm (72%) [13] of chromosome 10 and supernumerary chromosome 7 fragments, including the short arm (78%) and region q11.1–q22 (83%) [13]. These genetic alterations result in amplification of region 7p21, which harbors the epidermal growth factor receptor (EGFR) gene. Autocrinous stimula tion of EGFR activates the Ras pathway and thus stimulates cell proliferation. Region q23.3 of chromo MOLECULAR BIOLOGY
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some 10 contains the PTEN tumor suppressor gene, whose product acts as a phosphatase to prevent activity of PI3K kinase, which is stimulated by growth factors and increases cell proliferation. Loss of PTEN facili tates an increase in proliferative activity in glioblas toma [14]. The EGFR/Ras/PI3K/AKT signaling pathway is activated in 88% of glioblastomas [15]. In 87% of malignant gliomas there is an alteration of the signaling pathway involving the tumor suppres sor p53 [15], which ensures the stability of the genome and controls the induction of apoptosis. Inactivation of p53 is mediated by several mechanisms, including missense point mutations, which most often result in amino acid substitutions in positions 175, 248, and 273 [16]; deletions of the total p53 gene with loss of heterozygosity in region 17p13 (35% of cases); ampli fication of the genes for the inactive regulators MDM2 (14%) and MDM4 (7%); and mutations or deletions with loss of heterozygosity of region 9p21 with CDKN2A (49%), whose product р14ARF acts as a negative regulator of Mdm2. The signaling cascade that affects the function of the tumor suppressor protein known as the retinoblas toma protein (pRb) is the third in frequency of genetic alterations in glioma (77%) [16]. Hypophosphorylated pRb binds with transcription factors of the E2F family to render them inactive. Cyclindependent kinases (CDK4/6) phosphorylate pRb in the early S phase, and the pRb–E2F complex dissociates as a result. This stimulates transcriptional activity of E2F and triggers the genes involved in DNA synthesis, leading to a sub sequent cell division. Genes involved in this signaling pathway are often affected by mutations in glioblasto mas, as well as in many other cancers. For instance, 11% of glioblastomas display deletions of the pRb gene or amplification of the genes for pRbphosphorylating cyclindependent kinases (CDK4 in 18%, CCND2 in 2%, and CDK6 in 1% of tumors). In turn, cyclin dependent kinase activity is regulated by р14ARF and p16INK. The former prevents the assembly of the CDK4/6–cyclin D complex, suppressing its activity. The CDKN2A/CDKN2B genes (region 9p21), along with the genes involved in p53 signaling, are most fre quently affected by deletions and mutations in glio blastomas; e.g., their deletions are found in 55 and 53% of cases, respectively [16]. The high frequency of genetic alterations suggests a substantial contribution to glioblastoma development for the relevant factors and provides an opportunity to design targeted therapeutic approaches. Information about these alterations makes it possible to construct therapeutic virus strains that would exert an oncolytic effect toward tumor cells with certain genetic defects. ROLE OF GLIOMA STEM CELLS IN TUMOR MALIGNANCY GSCs seem to play an important role in tumor aggressiveness [17] because a direct correlation was observed between the GSC number and tumor malig nancy [18]. Tumor cells possessing stem properties
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were initially found in lymphomas [19] and, more recently, in breast [20], prostate [21], colon [22], and lung [23] tumors. In 2002, Ignatova et al. [24] were the first to identify and to describe stem cells in glial tumors. Representing only a minor fraction of the tumor cell population, GSCs are capable of asymmet ric divisions. Each of the divisions yields two daughter cells, of which one is a stem cell and the other lacks multipotency but is capable of producing progeny cells with different phenotypes [6]. These are neural or glial cells that express tissuespecific markers (βtubulin or GFAP, respectively) and cells with a combined pheno type, expressing both markers characteristic of neu rons and markers characteristic of neuroglial cells. Genetic lesions that are typical of glioma and cause cell malignant transformation are acquired at the GSC stage, as was initially demonstrated for p53 mutations [24]. The GSC origin is still a matter of discussion. It is possible that GSCs are neural stem cells that have undergone transformation, but have not lost the capa bility of self reproduction. Alternatively, GSCs may be dedifferentiated astrocytes that have acquired multi potency as a result of mutations [25]. GSCs display a higher resistance to most of the available antitumor drugs and radiotherapy [26], which is possibly due to their capability of efficiently repairing the DNA lesions introduced by the treat ment [27]. GSCs are characterized by elevated expres sion of the gene for ABCG2 transporter, which facilitate drug elimination from the cell and thereby sustain the resistance to chemotherapy [28–30]. Chemotherapy and radiation therapy of gliomas eliminate mostly the mature tumor cells, but not the tumor stem cells, thus alleviating the patient’s condition, but failing to pre vent a tumor relapse due to surviving GSCs. GSC surface markers, such as CD133, CD117, CD71, and CD45, are well characterized and allow GSC isolation from clinical samples [31]. According to the available estimates, these cells account for 5– 30% of the total tumor mass and have a characteristic genetic profile, which reflects both their malignancy and stemness. In view of the high GSC resistance to the available anticancer treatments, an active search is carried out to identify new therapeutics possessing cytotoxic activity toward GSCs [17, 32, 33]. Designing drugs on the basis of oncolytic viruses holds a particular promise [34]. VIRUS STRAINS WITH ONCOLYTIC PROPERTIES AND THE MECHANISMS OF THEIR ACTION In 1961, virus sensitivity of tumors of the glial ori gin was first observed in a model of the rabies virus reproducing in glioblastomas induced with methyl cholanthrene in mice [35]. Cultivation of the herpes simplex viruses (HSV), rabies, and measles viruses in glioma cells and other cells of the neural origin was described more recently, but an oncolytic effect was not observed [36–39]. In 1992, studies of virus oncol ysis of glioma were launched in Japan with the use of
the parotitis virus, which belongs to the family of paramyxoviruses [40, 41]. More recent data showed that the measles and Newcastle disease (NDV) viruses are more promising as oncolytic agents. Measles Virus Spontaneous regression of Hodgkin’s lymphomas after vaccination against measles was described in 1984 [42–44]. More recently, the measles virus was used as a basis to construct the oncolytic strain MV CEA, which expressed the carcinoembryonic antigen, a secreted protein whose level in a patient’s serum reflects virus activity in the body [45]. The virus repli cated well and caused apoptosis when used to infect a human glioblastoma cell panel. When the virus was tested in vivo in a model of U87 human glioblastoma cells grafted to athymic mice, an appreciable thera peutic effect was observed toward both subcutaneous and intracranial tumors and the time course of the process could be detected by the plasma level of the carcinoembryonic antigen [45]. MVCEA lacked tox icity upon intracranial administration in both mice [45] and rhesus monkeys [46]. A Phase I clinical trial of the MVCEA strain started in 2006, and its comple tion is expected in 2013 (http://www.clinicaltrials.gov). Newcastle Disease Virus The Newcastle disease virus (NDV) is being evalu ated in the greatest number of clinical studies among oncolytic viruses. NDV is pathogenic for the majority of birds and causes only a mild fever in humans [47]. NDV replicates well in cultured human cells, demon strating a high selectivity toward tumor cells. The mechanism of NDV tropicity to tumor cells is unclear. It was found, however, that virus replication requires activation of the Ras signaling pathway, namely, its branch associated with small GTPase Rac1 [48]. Ras activation was observed in 88% of glioblastomas, indi cating that the development of NDVbased therapeu tics is promising. As early as 1992, cytopathic activity of NDV was observed in vitro with human and rat cul tured neuroblastoma cell lines [49]. When NDV was administered to athymic mice with IMR32 human neuroblastoma cell xenografts, tumor regression was detected, and NDV was suggested as a promising onco lytic agent [50]. In 1996, two attenuated NDV strains, HuJ (attenuated by selection) and MTH68/H (natu rally attenuated), were put to clinical trials. In a study of the MTH68/H strain, a 14yearold patient with highgrade glioblastoma received daily intravenous injections with the virus, resulting in marked tumor regression and a significant neurological improvement. The patient could attend school and continued to receive intravenously NDV 2.5 × 108 PFU, thrice a day for more than three years without further disease progression [51]. MTH68/H caused tumor regression and increased the survival in four other patients with relapsing glioblastomas [52]. A combination of the strain with valproic acid induced tumor regression in a MOLECULAR BIOLOGY
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child with anaplastic astrocytoma and caused mass apoptosis of tumor cells, although a steady growth of one of the foci has led to a fatal outcome [53]. Clinical studies of strain HuJ involved 14 patients with glio blastoma multiforme. The virus dose was gradually increased from 0.1 × 106 to 55 × 106 infectious units in the initial phase of the study, and the patients received additional three maximal doses thereafter. The patients’ survival was found to increase to 66 weeks, and a complete response to therapy was achieved in one patient and lasted 3 months. However, a disease relapse followed complete tumor regression [54]. The results indicated that the oncolytic strain has minimal toxicity and an appreciable efficacy. Reoviruses Reoviruses, which belong to one genus within a separate virus family, usually fail to cause serious dis eases in humans, although they are associated with gastrointestinal and respiratory symptoms, which go away without any substantial sequels. The reovirus genome is fragmented doublestranded RNA. When viruses find their way into normal cells, RNAdepen dent protein kinase R (PKR) is activated to stop pro tein synthesis and virus replication in the host cell. PKR activation is suppressed by the Ras oncogene in many cancer cells [55, 56]. The high frequency of Ras activation in glioblastomas [57] and a low pathogenic ity of reoviruses allowed their therapeutic use. Prelim inary results indicated that reoviruses exert a distinct cytopathic effect toward U87 and U251N cultured glioma cells and in an in vivo model [58] and are low toxic to primates [59]. In 2008, 12 patients with a recurrent glioblastoma multiforme were subject to intratumor administration of a reovirus preparation at a dose increasing from 107 to 109 TCID50 in a Phase I clinical study (Calgary, Canada). Ten patients had tumor progression, one had stabilization, and one was not evaluable for response. The overall survival was 21 weeks in the study, reaching 54 weeks in one of the patients. The doses proved to be safe, and the drug was well tolerated [60]. The oncolytic drug Reolysin was designed on the basis of reoviruses by Oncolytic Bio tech [61]. The drug was evaluated in Phase I clinical trials in solid tumors; the trials showed that the drug is safe when used at 108–1010 TCID50 and that the disease was stabilized in 45% of the cases [62]. Phase I clinical trials of Reolysin in patients with glioblastoma multi forme was completed in the United States in 2010 (http://www.clinicaltrials.gov/ct2/show/NCT00528684). Recombinant Herpes Simplex Virus Strains Owing to a better understanding of the mecha nisms of virus interactions with the cell and the progress in recombinant DNA technology, it became possible to take advantage of the tumor cellspecific genetic defects and to construct the virus strains that replicate preferentially in tumor cells. The majority of MOLECULAR BIOLOGY
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studies in the field focus on recombinant strains of herpes viruses and adenoviruses. The following approach was used to construct the oncolytic herpes virus HSV1716. Viral infection of cells initiates the homodimerization and subsequent phosphorylation and activation of kinase PKR. Acti vated PKR phosphorylates its natural substrate, the alpha subunit of eukaryotic translation initiation fac tor2 (EIF2alpha), leading to the inhibition of cellu lar protein synthesis and virus replication. Herpes viruses utilize the ICP34.5 gene to overcome this nat ural antivirus resistance mechanism. The gene codes for the γ34.5 protein, which induces protein phos phatase PP1 of the host to dephosphorylate eIF2α to restore both protein synthesis and virus replication. Strain HSV1716 has an ICP34.5 deletion, and its rep lication is consequently blocked in normal cells. In tumor cells, PRK autophosphorylation is inhibited because of Ras activation, thus allowing selective rep lication of the recombinant virus [47, 63–65]. Herpes virus strain G207 has a deletion of both ICP34.5 copies and an inactivating insertion of the LacZ reporter gene in the region of the ICP6 gene, which codes for ribonucleotide reductase (RR). The enzyme is essential for virus replication because it pro duces deoxyribonucleotides, which are utilized in virus DNA synthesis. RR is encoded by the cell genome as well. Its expression depends on activity of E2Ffamily transcription factors, which are con trolled by the pRb tumor suppressor. Replication of the virus depends on its own RR in normal nondivid ing cells. In tumor cells, eukaryotic RR compensates for the lack of viral RR in the strain G207 and thus contributes to its selectivity to cancer cells. Expression of reporter LacZ provides a convenient histological marker for monitoring the distribution of virus infec tion through tissues of the body [63, 65, 66]. The antitumor properties of the above recombinant oncolytic herpes viruses were evaluated both in vitro and in vivo [67–72]. Strain HSV1716 was examined in three Phase I clinical trials. In one of them, the virus (105 PFU) was injected directly into the tumor in nine glioblastoma patients. Toxicity and new latent herpes virus infection were not detected, while the treatment decelerated tumor progression and increased the sur vival to 3 years in one patient and to 4 years in two patients [73]. In another study, intratumoral adminis tration of the virus was performed to 12 patients 4– 9 days before a surgical resection of the tumor. Signs of virus replication were observed in the tumor histologi cally [74]. The third study involved 12 patients, no tox icity was observed, and the survival increased in three patients (15, 18, and 22 months) [75]. Strain G207 passed Phase I and Phase Ib clinical trials. Phase I trials involved 21 subjects, and intratu moral injections of the viruses were done at no more than 3 × 109 PFU. The treatment did not display any toxicity. A positive response was observed in eight patients, and the posttherapy survival of one patient reached 5.5 years [76]. Phase Ib studies involved six patients with relapsing glioblastoma, and the dose was
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reduced to 1.15 × 109 PFU. The virus was adminis tered directly into the tumor through a steriotactically guided catheter. The tumor was removed 2–5 days after the treatment, and the virus continued to be administered into the resulting cavity. The patient did not receive chemotherapy. The treatment improved condition in three patients, and all of the patients were alive one month after the treatment. On average, pro gression of the disease occurred 3 months later, and the overall survival was more than 6 months. There were no virusrelated toxic effects [77], but the effi cacy of the viruses needs further improvement. Recombinant Adenovirus Strains Many studies focus on the use of oncolytic variants based on adenoviruses. Design of such variants was reviewed in [78]. Only one recombinant strain dl1520, which is also known as ONYX015 and is capable of selective replication in tumor cells, came through clin ical trials for glioblastomas. The selective oncolytic effect of the strain is due to a deletion and a mutation of the gene coding for E1B55K. Natural adenovirus variants utilize this protein in complex with the prod uct of viral E4orf6 to inhibit the p53 tumor suppressor and to prevent p53 from inducing apoptosis in the infected cell before the virus cycle is complete. When E1B55k is lost, virus replication would only be suc cessful if p53 is inactive [79, 80]. Results of more recent studies suggested an additional mechanism that may determine the tumor selectivity of ONYX015 and is independent of the p53 status. It was observed that transport of virus RNA from the nucleus into the cytoplasm is more efficient in tumor cells compared with normal cells, allowing a more robust lytic replica tion [81]. Many cancer cells were reported to have altered protein composition of nuclear pores, which are directly involved in nucleocytoplasmic transport [82], but the effect of the changes on the efficiency of virus replication is still poorly understood. Phase I clinical trials of ONYX015 involved 24 glioblastoma patients, which were divided into groups of six patient, and each group received the ONYX015 at a particular dose from 107 to 1010 PFU injected into a total of 10 sites within the resected glioma cavity. The results of the trials showed that the treatment did not cause serious side effects. Tumor growth was stopped in one patient and decelerated in one other patient; three patients, who had received 109 or 1010 PFU of the virus, survived for more than 19 months [83]. As for Russian developments, Cancerolysin, which was designed at the State Research Center of Virology and Biotechnology “Vector” (Novosibirsk), should be noted. Like ONYX015, the virus strain employed is defective in the gene coding for E1B55K. Cancerolysin was effective in cultured cells [84] and in vivo [85] in preclinical studies, and Phase I clinical trials demon strated its epidemiological safety and good tolerability [86]. Phase II clinical trials in glioblastoma patients are now planned.
Prospects of Virus Therapy of Gliomas While promising results were obtained in preclini cal studies in tumor cell lines and laboratory animals, and acceptable safety was observed for oncolytic strains, oncolytic activity in clinical trials was not high enough to justify the clinical use of the strains. To effi ciently infect all tumor cells in the body, it is necessary to provide the conditions that allow the virus to enter the tumor as easily as possible, to infect tumor cells with maximal specificity, and to induce their mass death. What factors hinder the process and prevent this promising approach from clinical application? The problem was considered in many reviews, which com prehensively discussed the completed and planned studies. To summarize the data, solution of several problems may substantially increase the efficacy of oncolytic virotherapy. Unlike in experiments with cultured cells and immunocompromised laboratory animals, an onco lytic virus has to overcome several natural barriers before entering the tumor in the patient’s brain. For instance, when a virus is injected intravenously, its major fraction is almost immediately removed from circulation by the reticuloendothelial system of the liver and spleen [87]. Huge virus doses are required to overcome this barrier, making the treatment more expensive and creating additional safety concerns. There are also adaptive barriers in an immunocompe tent organism. Neutralizing antibodies are rapidly produced in response to the virus administered, which is then rapidly removed from circulation, especially after repeated administration [88]. Indeed, antibodies to the therapeutic virus were usually found in the blood of clinical study subjects [60, 73–77, 83]. Experiments with immunocompetent mice showed that circulating antivirus antibodies can almost totally bind the injected virus [88]. The barrier may be overcome in part by injecting the virus directly into the tumor. An alternative strategy is provided by the socalled Trojan Horse technique, where viruses contained in infected susceptible carrier cells are introduced in circulation. The viruses located within cells are not recognized by the host immune system; this allows the infected cells to enter the tumor and to serve as a factory for local propagation of the virus, which then penetrates into tumor cells [89]. Infected neural stem cells were recently used to deliver viruses into brain tumors in preclinical experiments and proved more efficient than a virus suspension [90]; it is also possible to use infected mesenchymal stem cells [91]. Activation of antitumor immunity may play an important role in the efficacy of oncolytic viruses. Malignant gliomas are highly immunogenic because they express specific antigens that facilitate migration of macrophages and other immunocytes infiltrating the tumor and activate microglia [47, 92, 93]. The resulting inflammatory process is extremely complex and involves many components, which may promote immunosuppression or even stimulate the tumor growth [47, 94]. Oncolytic virotherapy should enhance the cytotoxic immune response to the tumor MOLECULAR BIOLOGY
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[77, 95, 96], and recent clinical trials support this idea [77]. To increase the immune response against the tumor, HSV1 with an ICP34.5 deletion was used to construct the socalled armed strains, which con tained the genes for the antiinflammatory cytokine interleukin 4 [97] or 12 [98, 99]. The strains displayed higher antitumor activity in the model of mouse syn genic brain tumor, and their clinical trials are now planned [47]. However, further studies are required to establish whether this approach is safe, as an addi tional risk could be associated with cerebral inflamma tion and edema. Studies of oncolytic viruses in combination with radiotherapy and chemotherapy in animal models suggest their synergistic effects [66, 100–103]. Syn ergy may be explained in part by immunosuppression, which prevents virus neutralization. To achieve good oncolytic effect, maximal virus replication is required, while antitumor immunity would stay intact or even increase. To achieve both of these goals with one virus, oncolytic herpes viruses of the next generation were designed on the basis of the strain G207. The viruses have an additional deletion of ICP47, whose product is involved in suppressing the presentation of main histo compatibility complex antigens, and early expression of their US11 is ensured to block the effect of interfer ons and thereby to increase virus replication and tumor cell lysis [104–106]. The studies discussed above demonstrate the com plexity of interactions in the system tumor–virus– immune system. This will certainly limit the use of immunocompromised animal models in the future [47]. Immunocompetent animals, be they transgenic or carrying syngenic brain tumors [107, 108], will pro vide better models of clinically observed cases. Another important problem is to increase the viru lence of oncolytic viruses toward tumor cells without impairing their safety and lack of effect on normal cells. To improve the specificity of delivery, viruses are modified with the proteins that increase their tropism to surface proteins or proteoglycans of the tumor cell. Natural oncolytic virus strains, such as reoviruses, the measles virus, and NDV, have surface hemagglutinins, which ensure their higher neurotropism, and the F pro tein, which determines the fusion of the infected cell with its neighbors to facilitate virus spreading through the tumor. The approach was employed in constructing the adenovirus strain Δ24RGD, which has fibrils with the ArgGlyAsp motif introduced to increase the tro pism to integrins ανβ3 and ανβ5 [109]. However, a mere increase in tropism may render the strain more toxic to healthy cells of the body, and modified promot ers may be used in such constructs to ensure the selec tive expression of viral products in tumor cells [110]. Another approach to improve the oncolytic effect of virus strains is to overcome the physical barriers pro vided by the extracellular matrix (ECM) of the tumor. The ECM contains proteoglycans (heparin sulfate and chondroitin), hyaluronic acid, collagen (the most abundant ECM component), elastin, fibronectin, and laminin. ECM components bind with various growth MOLECULAR BIOLOGY
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factors, such as the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), to pro mote angiogenesis and tumor growth [111]. In addi tion, the ECM may absorb virus particles to prevent infection of tumor cells [112]. To overcome this bar rier, it was suggested that tissues be initially treated with proteolytic enzymes, such as collagenase and hyaluronidase [113], though this procedure is feasible only during surgery. The gene for the ECMmodifying relaxin was introduced in a virus for an improved virus spreading through the tumor and a longer survival in the mouse model was observed [114]. GSCs are probably the most promising target for virothreapy of gliomas [34] because their high resis tance to therapeutic treatments is responsible for the inevitable recurrence of the disease. Many oncolytic strains were tested in a GSC panel in recent years. Adenovirus strains Ad5, Ad11p, Ad16p, CV23 [115], and Δ24RGD [80, 116] demonstrated a high efficacy against glioma stem cells in vitro. Promising results were obtained for the herpes virusbased strain G47Delta (deletions of ICP6, ICP34.5, and ICP47) [117] and especially Delta68H6, which was designed to propagate in stem cells of tumors [118]. When administered to immunodeficient mice with human GSC xenografts, the strains caused tumor reduction and suppressed selfreproduction of stem cells. To develop a chemotherapeutic, it is necessary to identify the target, to find and optimize an active com pound, and to test its pharmacological and toxicolog ical properties in preclinical and subsequent clinical studies. Only few molecules examined are introduced in medicine. The process takes years to perform, and the total cycle cost counts in billions of dollars. It is not surprising that, having completed all of these stages, pharmaceutical companies are not interested in improving their marketed products. Another situation is possible for the development of oncolytic viruses, where the results of clinical trials may provide a basis for designing an optimized virus variant, which then enters the developmental cycle again [119]. This strat egy is especially suitable for the viruses targeted to malignant gliomas because their laboratory testing in cell and animal models are low informative. Efficacy testing in animals is hardly suitable in the case of human viruses, while mouse viruses, which are not pathogenic to humans, may present a problem in pre clinical studies with mouse models [3]. At the same time, a profound understanding of the mechanisms of virus–host interaction would allow creation of new improved variants of oncolytic viruses in a directional manner in order to find cure for gliomas, which still remain an incurable disease. ACKNOWLEDGMENTS This work was supported by the Program for Support of Scientific Schools (project nos. NSh65387.2010.4 and NSh2996.2012.4) of the President of the Russian Federation, Agreement no. 11.634.31.0034, Analytical Departmental Program no. 2.1.1/9888, Integration
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