Experimental therapies

Handbook of Clinical Neurology, Vol. 134 (3rd series) Gliomas M.S. Berger and M. Weller, Editors © 2016 Elsevier B.V. All rights reserved

Chapter 11

Experimental therapies: gene therapies and oncolytic viruses 1

M. MAHER HULOU1, CHOI-FONG CHO1, E. ANTONIO CHIOCCA1*, AND ROLF BJERKVIG2,3 Harvey Cushing Neuro-Oncology Laboratories, Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

2

NorLux Neuro-Oncology Laboratory, Department of Oncology, Luxembourg Institute of Health, Luxembourg 3

Department of Biomedicine, University of Bergen, Norway

Abstract Glioblastoma is the most common and aggressive primary brain tumor in adults. Over the past three decades, the overall survival time has only improved by a few months, therefore novel alternative treatment modalities are needed to improve clinical management strategies. Such strategies should ultimately extend patient survival. At present, the extensive insight into the molecular biology of gliomas, as well as into genetic engineering techniques, has led to better decision processes when it comes to modifying the genome to accommodate suicide genes, cytokine genes, and tumor suppressor genes that may kill cancer cells, and boost the host defensive immune system against neoantigenic cytoplasmic and nuclear targets. Both nonreplicative viral vectors and replicating oncolytic viruses have been developed for brain cancer treatment. Stem cells, microRNAs, nanoparticles, and viruses have also been designed. These have been armed with transgenes or peptides, and have been used both in laboratory-based experiments as well as in clinical trials, with the aim of improving selective killing of malignant glioma cells while sparing normal brain tissue. This chapter reviews the current status of gene therapies for malignant gliomas and highlights the most promising viral and cell-based strategies under development.

BACKGROUND High-grade gliomas (HGGs) represent 75% of all gliomas and are classified by the World Health Organization (WHO) as grade III and IV tumors (Louis et al., 2007). Their infiltrative nature and eventually inexorable growth define their malignancy, and they commonly become more aggressive and resistant to current therapies over time, leading ultimately to patient death. Grade III anaplastic astrocytomas, grade III anaplastic oligoastrocytomas, grade III anaplastic oligodendrogliomas, and grade IV glioblastoma multiforme (GBM) are the most common primary brain tumors and these tumors represent the focus of this chapter. Recently, results from The Cancer Genome Atlas have led to the stratification of these tumors into proneural, classic, or

mesenchymal types based on their transcriptomic profiles (Phillips et al., 2006; Verhaak et al., 2010). The current standard of care for HGGs consists of symptom management and maximum safe surgical resection followed by concomitant radiation and chemotherapy. This, however, has proven to be inadequate with an only modest improvement in overall survival. Therefore, novel treatments are urgently needed to improve clinical management strategies that ultimately should extend patient survival. Four major features contribute to the poor prognosis of HGGs: (1) the physiologic isolation of tumor cells in the brain parenchyma where the blood–brain barrier (BBB) is intact; (2) the highly aggressive and infiltrative nature of these tumors, making complete resection impossible; (3) the immune sanctuary status of the brain,

*Correspondence to: E. Antonio Chiocca, MD, PhD, Chairman and Professor, Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston MA 02115, USA. Tel: +1-617-732-6939, Fax: +1-617-734-8342, E-mail: [email protected]

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allowing tumor cells to further expand along fiber tracts and blood vessels; and (4) the recent discovery of tumor stem-like cells that show capacities of self-renewal and resistance to conventional radio- and chemotherapy (Singh et al., 2004; Bao et al., 2006). To overcome these challenges, novel treatments targeting the very basic elements of the tumor cells and the tumor microenvironment have emerged. These targeting strategies have been clustered under the term gene therapy. In this chapter, we will discuss some recent advances in the treatment of HGGs using genes, viruses, microRNAs, stem cells, and nanotechnology. Gene therapy was first conceptualized in 1972 by Friedmann and Roblin, who proposed that defective genes can be replaced with functional genes in patients who suffer from hereditary monogenetic diseases such as recessive enzymatic deficiencies and blood coagulation factor VIII or IX deficiencies in hemophiliacs. Over time, the concept of gene therapy widened with the introduction of “therapeutic” genes to cells, where in particular treatment of cancers became a major focus. Different gene delivery strategies have been proposed, including replicating viruses, nonreplicating viruses, and stem cells. Today, the extensive gain of knowledge in molecular biology and genetic engineering has allowed for better decision processes related to the evaluation and modification of the tumor genome to accommodate suicide genes, cytokine genes, and tumor suppressor genes that can kill cells and/or boost the defensive immune system against neoantigenic cytoplasmic and nuclear targets.

VIRUSES AS THERAPEUTIC AGENTS A major limitation associated with systemic delivery of chemotherapeutics is their toxic side-effects on nontumor cells. In the context of gene therapy, a suicide gene (typically transported within a replication-deficient virus or a bacterium) is delivered to tumor cells where it expresses an enzyme that converts a systematically delivered inactive prodrug to active lethal compounds (Ezzeddine et al., 1991; Duarte et al., 2012). The best-studied “suicide” gene is the herpes simplex virus-derived enzyme thymidine kinase (HSV-Tk). Once the tumor cells are transduced with the herpesvirus, they start producing the viral thymidine kinase (Tk) that converts a systematically delivered drug such as ganciclovir into ganciclovir monophosphate. This is further converted by cellular kinases into ganciclovir triphosphate which disrupts cellular DNA replication, resulting in S-phase delay and G2M-phase arrest (Halloran and Fenton, 1998). As a result of ganciclovir-induced cell cycle arrest, apoptosis is initiated (Wei et al., 1998; Tomicic et al., 2002). The strategy described above

was introduced in 1986 (Moolten, 1986), and was later used in research to demonstrate a therapeutic efficacy of HSV-TK/ganciclovir on subcutaneously implanted glioma cells (Ezzeddine et al., 1991; Martuza et al., 1991). It has later become clear that the expression of HSV-TK and the death of tumor cells can lead to a significant CD8 + T-cell-mediated vaccination response that may further improve therapeutic efficacy. There have now been several clinical trials in humans with GBM where Tk is delivered into tumors using adenoviral vectors. A phase III trial in Europe (ASPECT trial) evaluated the safety and efficacy of applying adenoviral vectors carrying HSV-TK gene (Ad-Tk) followed by intravenous ganciclovir in newly diagnosed GBM patients (Westphal et al., 2013). Despite a slight improvement in the median time to death or reintervention, the overall survival was not improved compared to standard of care. In the USA, a phase I trial for newly diagnosed GBM showed that a similar Ad-Tk vector was well tolerated when given in combination with standard chemoradiation (Chiocca et al., 2011). This trial has now progressed to a multi-institutional phase II trial, which, unlike the aforementioned ASPECT trial, has shown encouraging efficacy results (presented at the American Society of Clinical Oncology; Aguilar et al., 2015). The difference between ASPECT and this trial may be related to the synergistic effect demonstrated by the concomitant use of Ad-Tk and radiation (Nestler et al., 2004). The differences may also be related to vector design. Moreover, the results indicate that the therapy may be more effective in patients whose tumors were gross totally resected. Another strategy is adenoviral delivery of the bacterial enzyme cytosine deaminase (CDA) to glioma cells (Huber et al., 1994). The CDA converts the inactive nontoxic 5-fluorocytosine to the toxic 5-fluorouracil. In this study, the researchers also demonstrated increased cytotoxic effects in cancer cells when this system was combined with radiation therapy due to an irreversible inhibition of DNA synthesis. In comparison to the suicide gene delivery using replication-deficient virus, a replicating virus exemplified by Toca511 (vocimagene amiretrorepvec) is also being tested in multiple phase I and II clinical trials in the USA (Tai et al., 2005; Perez et al., 2012; Huang et al., 2015). Similar to what has been described for the replication-deficient adenovirus in the previous paragraph, the replicating Toca511 retrovirus, that expresses the activating enzyme CDA, has demonstrated encouraging safety and efficacy in several clinical studies (Tai et al., 2005). The advantage of using replicating retroviruses is based on their ability to infect the tumor more extensively, leading to a higher expression of the toxic CDA enzyme.

EXPERIMENTAL THERAPIES: GENE THERAPIES AND ONCOLYTIC VIRUSES

Gene delivery to modify the tumor microenvironment The tumor stroma represents the first physical barrier to viral dispersion. Recent studies have investigated the effects of the manipulation and degradation of the extracellular matrix on viral activity. One study demonstrated increased adenoviral infectivity with the use of protease enzyme (Kuriyama et al., 2000). Others have also used oncolytic herpesvirus to carry the bacterial enzyme chondroitinase ABC-1 to degrade hyaluronan and other major extracellular matrix components. This strategy led to better viral replication in the tumor cells (Dmitrieva et al., 2011). Another major challenge pertaining to tumor microenvironment is the regulation and manipulation of the GBM vasculature. Normally, tumor neovascularization is regulated by numerous pro- and antiangiogenic factors. In GBMs, the tumor cells outgrow the normal vasculature, which leads to hypoxia and an increase in proangiogenic factors. Bevacizumab or Avastin (Roche, South San Francisco, CA) is an antivascular endothelial growth factor monoclonal antibody that has recently been investigated in clinical trials in the USA and Europe (RTOG-0825 trial and AVAglio trial). The initial results from both trials were presented at the 2013 American Society of Clinical Oncology meeting; both trials demonstrated improved progression-free survival but no improvement in overall survival (Chinot et al., 2014; Gilbert et al., 2014). Alternatively, a combination therapy approach targeting multiple angiogenic pathways has shown better results (Zhang et al., 2012). Angiostatin, an inhibitor of angiogenesis, was inserted into oncolytic HSV-1 (oHSV-1) and demonstrated a significantly increased survival, especially when combined with a low dose of bevacizumab (Zhang et al., 2012).

Oncolytic virotherapy Viruses are traditionally viewed as microorganisms that infect cells, by coopting their synthetic machinery, leading to cell lysis. Oncolytic viruses have many features that make them distinct from other biologic therapies. They proliferate selectively in tumor cells and increase with time, as opposed to regular drugs which display pharmacokinetic profiles that decrease with time. In addition, oncolytic viruses are equipped with safety features, leading to a relatively high therapeutic index, allowing their safe administration in clinical trials. Multiple experiments during the last decades have shown tumor regression after intratumoral viral administration. The first laboratory-based engineered oncolytic virus was reported in 1991 by Martuza and colleagues, who demonstrated the potential use of a

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HSV-1 mutant in the treatment of GBM. Since then, at least 20 clinical trials have been conducted in GBM research alone using different oncolytic viruses modified to target cancerous cells. To reduce undesired toxicity, oncolytic viruses can be engineered with selective mutations or deletions that cause them to selectively replicate in tumor cells. For example, the adenovirus Delta24-RGD has been engineered with mutations in the viral E1A gene that restricts viral replication to cells with P16 tumor suppressor defects and also with an RGD motif to allow for selective attachment of the oncolytic viruses to glioma cells expressing specific integrins (Gomez-Manzano et al., 2004; Jiang et al., 2014). oHSV-1, with a defective ICP6 gene expression, has also been shown to selectively target cells that harbor p16 tumor suppressor defects (Aghi et al., 2008). Also, poliovirus (that has been engineered to express nonpathogenic polioviral proteins) has been modified to target cells that express the CD155 receptor present on glioma cells (Merrill et al., 2004). Moreover, measles virus has been tested in clinical trials for a variety of cancers, including GBM, where the virus is used in a highly attenuated form of an existing vaccine for humans (Allen et al., 2013). The most extensively studied oncolytic virus in glioma therapy is HSV-1, with deletion of both copies of g34.5 genes. The g34.5 is widely recognized as an important neurovirulent factor in oHSV-1. It produces a protein, ICP 34.5, which activates a phosphatase enzyme that leads to the dephosphorylation of the eukaryotic initiation factor (eIF2a), which prevents a shut-off of protein production. This allows the virus to exploit the host cellular machinery for replication. The virus designated as HSV1716 lacks both copies of g34.5 and was evaluated in 33 patients in three clinical trials (Rampling et al., 2000; Papanastassiou et al., 2002; Harrow et al., 2004). Patients in all trials tolerated the viral administration into the tumor mass without any incidence of encephalitis. Moreover, patients showed an increase in viral DNA copy numbers at the injection sites, indicating some level of replication in the tumors even with the attenuated HSV1716. Furthermore, two clinical trials tested the safety and efficacy of employing one more safety feature to ICP34.5-deleted HSV. The ICP6 gene encoding for ribonucleotide reductase was inactivated in G207 virus, which resulted in a more selective replication in tumor cells compared to quiescent surrounding tumor cells. The safety of G207 was confirmed in a two-step dose escalation study in which 13% of the total 1.15  109 pfu was infused in first stage and the remaining viral dose was injected into the resection cavity 2–5 days postoperatively (Markert et al., 2009). Only minimal systemic toxicity was documented by the local use of the virus.

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Although systemic intravenous administration is possible, it is associated with many disadvantages mediated by pre-existing antioncolytic virus antibodies, the induction of anti oncolytic virus antibodies after multiple administrations, and the sequestration of the oncolytic viruses in the liver (Chiocca and Rabkin, 2014). Different approaches have been attempted to shield the virus; mesenchymal cells and neural stem cells (NSCs) have been proposed, but the efficacy among different cell lines varied depending on tumor cell permissiveness to virus and transfer of the oncolytic virus to the neoplastic cells (Chiocca and Rabkin, 2014). Oncolytic viruses can also be coated in nanoparticles (NPs: typically <100 nm in size) to ensure longer vascular circulation and less neutralization by the host immune system (Russell et al., 2012). Recently, a method to selectively “silence” an oncolytic virus in a cell and then awake it at a later stage (i.e., when tumor hypoxia is encountered) has been described (Nakashima and Chiocca, 2014).

Oncolytic virus immunotherapy The brain has traditionally been regarded as an immunoprivileged organ (Assi et al., 2012). Different immunologic models have been developed to promote effective immunotherapy against glioma. Interferons have been extensively studied for their ability to exert antiproliferative effects on viruses. They act as cytokines that activate and recruit immune effector cells such as macrophages, natural killer (NK) cells, and cytotoxic T cells (Qin et al., 2001). Interferon expression is induced by viral infection and can inhibit the cell cycle in the S phase in solid tumor cells and in the G1 phase in Daudi’s Burkitt’s lymphoma cells and other tumors (Qin et al., 2001). Moreover, they stimulate the immune system by inducing major histocompatibility complex class 1 expression, activate T-helper cells, and attract macrophages and NK cells to act against glioma cells. An excellent example is the gene transfer of interferon-a to mice tumor cells, which stimulates immunologic effector cells to infiltrate and block tumor growth (Ferrantini et al., 1993, 1994; Kaido et al., 1995). A replication-deficient adenovirus has also been used to carry immune-boosting interferon-b to mediate an effective antitumor response to subcutaneously implanted gliomas in mice (Qin et al., 1998, 2001). Tumor regression and survival were significant in many laboratory-based experiments, and thus, this strategy was taken to clinical trials (Chiocca et al., 2008). The role of host immune reactions to oncolytic virus infection is complex. On one hand, the immune system works to block efficient viral replication and spread within the tumor (Ikeda et al., 1999). On the other hand, viral infection of tumor cells acts as an in situ vaccination

that elicits a CD8+ T-lymphocyte response. Furthermore, interaction between the virus, the tumor cell, and the immune system unleashes an inflammatory cascade of signaling factors (i.e., chemokines and cytokines) and immunogenic cell death signals to induce antitumor immunity. Despite the importance of the adaptive system in antitumor immunity, different cell types in the tumor microenvironment play a role in the destruction and inhibition of viral infection and intratumoral propagation. Research has shown a rapid recruitment of the NK cells to the tumor site, causing a premature clearance of oHSV-1 in both athymic and immunocompetent mouse models (Alvarez-Breckenridge et al., 2012). This study showed that human NK cells killed GBM cells through an interaction between NK receptors and their ligands. In a similar model, the innate defense mechanism was blocked by the histone deacetylase inhibitor valproic acid through inhibition of the STAT5/T-BET signaling pathway in NK cells (Alvarez-Breckenridge et al., 2012). The interference with the NK cell action towards oHSV-infected GBM cells, with the transiently acting pharmacologic agent, resulted in an initial burst of oncolytic virus replication followed by a secondary redirection of CD8 + T cells to establish a long-term vaccination effect.

Clinical outcomes of oncolytic virotherapy and gene therapy As mentioned above, there has been a number of clinical trials of oncolytic viruses for glioma and other cancers (Tables 11.1 and 11.2). Most of the glioma trials are still in phase I but some oncolytic viruses have made it to phase II and phase III, bringing closer the advent of the first US Food and Drug Administration approval of oncolytic viruses. For instance, oHSV-1-expressing granulocyte–macrophage colony-stimulating factor (GM-CSF) is in the last phase III trial for the treatment of melanoma (talimogene laherparepevec: T-Vec). Patients in the trial were randomized to receive T-Vec or GM-CSF alone, and preliminary results have shown a 16% durable response rate in the T-Vec arm compared to 2% in the control GM-CSF group.

“Arming” oncolytic virus with therapeutic transgenes Many oncolytic viruses can accommodate exogenous DNA or RNA. Oncolytic viruses armed with therapeutic genes are used in an attempt to produce proteins that have the ability to enhance antitumor efficacy. One example is the use of immunomodulatory genes in oncolytic virotherapy. In GBM patients, the immune response is compromised and treatment efficacy depends on

Table 11.1 Completed glioblastoma multiforme (GBM) gene therapy trials Median survival (months)

Year (reference)

No survival benefit compared with the control group Clear survival benefit in comparison with historic and standard care control groups No serious adverse events; evidence for tumor transduction as well as local and systemic type 1 T-helper cell cytokine elevation 16/30 patients with serious adverse events Limited gene transfer detected; responses only in small tumors No serious adverse events No serious adverse events; 1/12 CR No dose-limiting toxicity observed; CD3 + T-cell infiltration of the tumor Apoptosis induction observed at highest dose; transgene expression in tumor No treatment-associated serious adverse events: ex vivo manipulation of hematopoietic progenitor cells without influence on engraftment p53 expression and activity confirmed within 5 mm of injection site No virus shedding; no tissue toxicity Treatment well tolerated

12

2000 (Rainov, 2000)

14.4

2004 (Immonen et al., 2004)

7.5

2005 (Colombo et al., 2005)

8.4

2003 (Prados et al., 2003)

n.d.

1999 (Ram et al., 1997)

8.6 6.8 12.4

1999 (Shand et al., 1999) 1998 (Klatzmann et al., 1998) 2011 (Chiocca et al., 2011)

4.1

2008 (Chiocca et al., 2008)

14.8

2006 (Cornetta et al., 2006)

9.9

2003 (Lang et al., 2003)

12 4

2003 (Germano et al., 2003) 2003 (Smitt et al., 2003)

No serious adverse events; no virus shedding Survival not different in comparison with control group Well tolerated 2  1011 vp; no virus shedding 4/12 patients with serious adverse effects; 1/11 SD

15

2000 (Sandmair et al., 2000)

Trial phase

Disease

Virus

Results

III

Untreated GBM

IIB

Operable primary or recurrent malignant glioma

Retroviral vector expressing HSV-TK delivered Adenoviral vector expressing HSV-TK

I/II

Recurrent GBM

Retroviral vector expressing HSV-TK and IL-2

I/II

Progressive or recurrent GBM

I/II

Progressive or recurrent malignant brain tumors Recurrent GBM Recurrent GBM Primary malignant glioma

Retroviral vector expressing HSV-TK delivered Retroviral vector expressing HSV-TK

I/II I/II IB I

Retroviral vector expressing HSV-TK Retroviral vector expressing HSV-TK Adenoviral vector expressing HSV-TK

I

Recurrent or progressive malignant glioma Poor prognosis brain tumors

Adenoviral vector expressing human IFN-ß Retroviral vector expressing MGMT

I

Recurrent glioma

Adenoviral vector expressing p53

I I

Recurrent malignant glioma Recurrent malignant glioma Primary or recurrent malignant gliomas

Adenoviral vector expressing HSV-TK Adenoviral vector expressing HSV-TK

I

Adenoviral vector expressing HSV-TK Retroviral vector expressing HSV-TK

I

Recurrent malignant brain tumors

Adenoviral vector expressing HSV-TK

I

Progressive or recurrent pediatric malignant brain tumors Recurrent GBM

Retroviral vector expressing HSV-TK

I

Retroviral vector expressing HSV-TK and IL-2

Evidence for tumor transduction and transgene activity

7.4 4.5

2000 (Trask et al., 2000)

N/A

2000 (Packer et al., 2000)

8.5

1999 (Palu et al., 1999)

HSV-TK, herpes simplex virus-derived thymidine kinase; IL-2, interleukin-2; CR, complete response; IFN-b, interferon-b; MGMT, O6-methylguanine-DNA methyltransferase; SD, stable disease.

Table 11.2 Completed glioblastoma multiforme (GBM) oncolytic virotherapy trials Trial phase

Disease

Virus

Results

I/II

Recurrent GBM

NDV-HUJ

IB

Recurrent GBM

I

Recurrent malignant gliomas

G207: ICP6-inactivated and ICP34.5deleted HSV Reovirus

I

Recurrent malignant glioma

ONYX-015: E1B and E3-deleted AdV

I I

High-grade glioma Malignant glioma

HSV1716: ICP34.5-deleted HSV HSV1716: ICP34.5-deleted HSV

I I

Recurrent malignant glioma Malignant glioma

HSV1716: ICP34.5-deleted HSV G207: ICP6-inactivated and ICP34.5deleted HSV

1/11 CR, not durable; virus recoverable from body fluids No CR or PR; immune cell infiltration posttreatment detectable 1/11 SD; virus shedding detectable, but not persistent Immune cell infiltration in recurrences of treated tumors No virus-associated toxicity; 3/12 SD 1/12 CR; viral DNA recoverable; indication for intratumoral viral replication No reactivation of latent HSV No virus-associated toxicity; viral DNA recoverable

Median survival (months)

Year (reference)

7.4

2006 (Freeman et al., 2006)

6.6

2009 (Markert et al., 2000)

4.8

2008 (Forsyth et al., 2008)

6.2

2004 (Chiocca et al., 2004)

n.d. n.d.

2004 (Harrow et al., 2004) 2002 (Papanastassiou et al., 2002)

n.d. 6.2

2000 (Rampling et al., 2000) 2000 (Markert et al., 2000)

NDV-HUJ, HUJ strain of Newcastle disease virus; AdV, adenovirus; HSV, herpes simplex virus; CR, complete response; PR, partial response; SD, stable disease.

EXPERIMENTAL THERAPIES: GENE THERAPIES AND ONCOLYTIC VIRUSES tumor cell lysis and the ability of the oncolytic virus to induce an in situ vaccination before the virus is cleared by the innate immune system. For example, interleukin-12 (IL-12) promotes Th1 responses and cellular immunity. One study demonstrated the efficacy of arming oHSV with IL-12 (G47△-mIL12) in the treatment of murine model of GBM. In vitro studies have shown that G47△-mIL12 and the unarmed oHSV replicate in a similar way; yet a significant increase in overall survival was particularly demonstrated when mice bearing intracerebral GBMs were treated with G47△-mIL12 (Cheema et al., 2013). Another example is the use of transgenes to produce proteins to block neoangiogenesis that is required for tumor growth. One study examined the efficacy of g34.5–, ICP6– oHSV carrying an endostatin-angiostatin fusion gene in glioma-bearing mice. The study showed a significant inhibition of the endothelial cells, which was translated to an increased overall survival in mice bearing human glioma stem cells when compared to the control groups (Zhang et al., 2014). In addition, in recent studies, proapoptotic genes have been inserted in oHSV to evaluate their therapeutic efficacy. As an example, experiments using a recombinant G47d oHSV that carried the transgene encoding for a secretable tumor necrosis factor-related apoptosisinducing ligand (TRAIL), revealed a relatively high efficacy. In these studies, the intracerebral/intratumoral inoculation of TRAIL-carrying oHSV increased overall survival of severe combined immunedeficiency (SCID) mouse models bearing oHSV-resistant GBM cells (Tamura et al., 2013).

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In summary, virotherapy has evolved as a promising field for GBM treatment. The robust bystander cytotoxic effect, the ability of the virus to mount an effective immune response, and the oncolytic effect of replicationcompetent viruses contribute to the benefits of using viral agents for treating gliomas. However, long-term therapeutic effects, limitation of dispersion by the extracellular matrix, rapid stimulation, and clearance of the viral particles by the innate immune system are still major challenges that need to be addressed for effective use in humans.

THE EMERGING ROLES OF MICRORNAS IN THE MANAGEMENT OF GBM MicroRNAs (miRNAs or miRs) are evolutionarily conserved small, noncoding, single-stranded ribonucleic acid (ssRNA) molecules that are transcribed from DNA and function as regulators of cellular protein translation. The 21–23-nucleotide-long RNA transcripts work as repressors of target gene expression through binding to complementary sequences in the 3’ untranslated region of the targeted messenger RNA (mRNA), thus inhibiting posttranscriptional gene expression (Krol et al., 2010). Each microRNA may exert its regulatory effects on many target mRNAs and many mRNAs are targeted by multiple microRNAs, orchestrating a highly complex regulatory network. MicroRNAs were first discovered in 1993 in a study on Caenorhabditis elegans and have become increasingly important in glioma research after the discovery of their role in glioma tumor growth, invasion, angiogenesis, and chemoresistance (Table 11.3)

Table 11.3 MicroRNAs for which extensive functional information is available for glioblastoma multiforme (GBM)

MicroRNA

Expression in GBM

miR-128

Low

miR-26a

High

miR-7 miR-124 miR-21

Low Low High

miR-296 miR-326

High Low

miR-451

High

Functions

References

Proliferation (in vitro and in vivo) Stem cell self-renewal Transforming proliferation (in vitro and in vivo) Apoptosis Proliferation (in vitro) Invasion differentiation Proliferation (in vitro) Invasion differentiation Proliferation (in vitro and in vivo) Apoptosis invasion Chemoresistance Angiogenesis Proliferation (in vitro and in vivo) Apoptosis invasion Switch regulating invasion, growth, and survival in response to metabolic stress Chemoresistance

Godlewski et al. (2008) Huse et al. (2009)

Kefas et al. (2008) Silber et al. (2008) Chan et al. (2005); Corsten et al. (2007); Gabriely et al. (2008); Papagiannakopoulos et al. (2008); Zhou et al. (2010) Wurdinger et al. (2008) Kefas et al. (2009) Godlewski et al. (2010)

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(Lee et al., 1993; Chiocca and Lawler, 2010; Ebert and Sharp, 2012). An emerging theme in microRNA oncology research today is its role in regulating GBM stem cell maintenance and epigenetic pathways. The cancer stem cell theory was hypothesized more than 100 years ago (Boveri, 1914) and was later conceptualized in breast cancer and leukemia, referring to a subpopulation of cancer cells that possess the ability to regenerate tumors. Cancer stem-like cells have been isolated based on their expression of the putative stem cell marker CD133 (Singh et al., 2004). However, later studies have shown an extensive cellular heterogeneity within cancer stem cell populations, which makes a clear identification of these cells difficult (Shackleton et al., 2009).Yet, cancer cells that display stem-like properties have been found to have an increased resistance to radioand chemotherapy. Furthermore, recent research has demonstrated a link between NSCs and a subpopulation of GBM cells that can be grown readily under the same conditions that are normally used to grow NSCs. This group of cells also has the capacity to proliferate aggressively, and differentiate along astrocytic, neural, and oligodendroglial lineages. Moreover, they express Nestin or Bmi-1 proteins, which are normally associated with stem and progenitor cells. MicroRNAs normally regulate NSC growth and differentiation, and their dysfunction may lead to an undifferentiated proliferative phenotype. It is tempting to speculate that miRNAs may function in cancer by subverting normal NSC functions. It has been suggested that stem cells use miRNAs to properly override the G1/S checkpoint to sustain continuous division, and that cancer cells may exploit this mechanism to bypass the G1/S checkpoint to multiply in an uncontrolled pattern. This has been described for the weakly expressed miRNA-124 and miRNA-137 in GBM cells (Silber et al., 2008). Moreover, microRNAs are emerging as promising diagnostic tools, in particular as biomarkers to assess treatment responses based to their presence in body fluids and their relatively easy detection in serum (Skog et al., 2008).

MicroRNAs in GBM The expression of microRNA in GBM cells differs from the surrounding normal brain parenchyma. Recent studies have shown that miR-21 has the highest expression levels in GBM cells, whereas several other microRNAs, namely miR-124, miR-7, and miR-128, are weakly expressed in tumor cells compared to normal brain tissues. Little is known about the mechanisms by which the GBM alters microRNA levels. It has been suggested that all genomic regulatory systems, including mutations, gene deletions, methylations, and amplifications,

play a role by altering the processing and reformation of putative target-binding sites.

EXAMPLES OF MICRORNA FUNCTION AND GBM miR-21 is significantly elevated in HGGs, as confirmed by reverse transcriptase polymerase chain reaction. Recent studies suggest that miR-21 acts as an oncogene that correlates with glioma grade (Gabriely et al., 2008). In addition, miR-21 has been shown to suppress apoptosis and promote tumor cell proliferation and invasion (Chan et al., 2005; Gabriely et al., 2008). In vivo studies have shown that miR-21 downregulation reduces tumor cell motility and growth, indicating that miR-21 may represent a potential target candidate for HGGs (Corsten et al., 2007; Gabriely et al., 2008). Unlike miR-21, miR-124 expression in GBM is relatively low (Silber et al., 2008). It is normally upregulated during adult NSC differentiation as a consequence of growth factor withdrawal. However, when GBM develops, miR-124 is downregulated. This leads to an inhibition of neuronal differentiation and an increase in tumorigenicity (Godlewski et al., 2008). Recent data suggest that miR-124 promotes G0/G1 cell cycle arrest in GBM, which makes it a potential candidate for clinical trials (Godlewski et al., 2008; Silber et al., 2008). miR-128 is also downregulated in GBMs, and it has been shown that overexpression of miR-128 is associated with a downregulation of Bmi-1, which is an epigenetic silencer that can cause severe impairment of stem cell renewal mechanisms (Godlewski et al., 2008).

Targeting microRNAs Several approaches have been proposed for therapeutic targeting of microRNAs. These include the use of expression vectors, small-nucleotide inhibitors, and antisense oligonucleotides. Vector-based approaches exploit the overexpression of inhibitors that target miRNA binding sites on specific mRNAs. This will selectively sequester endogenous miRNA and increase the expression of target mRNA. By using small nucleotide inhibitors, such as azobenzene, an inhibition of miRNA expression may be obtained through transcriptional regulation of miRNA rather than the inhibition of target recognition sites of the miRNAs. A more complex approach may be the use of antisense oligonucleotides, where encapsulated naked oligonucleotides, or modified RNA or DNA oligonucleotides, are used to bind endogenous mRNA targets. This approach will directly antagonize microRNA binding to the receptors. Despite considerable advances in microRNA field, challenges related to systemic delivery, receptor affinity, and the identification of anti-miR oligonucleotides necessitate a thorough evaluation of these therapeutic applications in vivo.

EXPERIMENTAL THERAPIES: GENE THERAPIES AND ONCOLYTIC VIRUSES

STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMAS As mentioned above, viral vectors have been extensively investigated as delivery vehicles in gene therapy. Several therapy approaches have been implemented in clinical trials based on promising results from animal experiments (Lawler et al., 2006). However, success in the clinic has been relatively modest (Pulkkanen and YlaHerttuala, 2005). This may be attributed to the failure of the viruses to distribute to the invasive glioma cell compartments as well as to an inefficient gene transfer into the GBM cells (Pulkkanen and Yla-Herttuala, 2005). To overcome this problem, carrier cells such as stem cells and progenitor cells have been tested for their migratory ability towards brain tumors in vivo. It was early shown in experimental animal models that NSCs possess the ability to migrate towards and within intracranial neoplasms where they were able to deliver cytotoxic substances that reduced tumor growth (Aboody et al., 2000). Based on these observations, several studies have been conducted to determine tumor cell tropism using different stem cell lines. At present, it has been shown that NSCs, multipotent mesenchymal stromal cells (MSCs), and hematopoietic progenitor cells all show high migratory abilities towards infiltrating tumor extensions, where they are able to deliver a therapeutic payload. In contrast, differentiated cells, such as fibroblasts, do not exhibit such a migratory tropism (Nakamura et al., 2004; Nakamizo et al., 2005; Tabatabai et al., 2005).

Neural stem cells NSCs are multipotent progenitors of the neural lineage that give rise to neurons and glial cells. They are located mainly in three regions in the adult brain: (1) the subependymal zone (Sanai et al., 2004) adjacent to the lateral ventricles; (2) the dentate gyrus of the hippocampus (Eriksson et al., 1998); and (3) in the subcortical white matter (Nunes et al., 2003). NSCs were first used to deliver cytokines to improve immune responses against gliomas (Benedetti et al., 2000). Recently, they have been employed to deliver therapeutic genes as well as oncolytic viruses. However, there are two major clinical problems (Bexell et al., 2013): immunogenicity and tumorigenicity. Some NSCs can provoke an extensive immune response capable of neutralizing the grafted nonautologous cells. Other cell lines that carry protooncogenes may develop into a secondary malignancy after implantation into cancerous tissue. To avoid such occurrences, NSCs have been engineered to carry oncolytic viruses or genes such as HSV-Tk to eliminate the administered NSCs at any given time. Herrlinger and colleagues (2000) demonstrated the feasibility of using

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NSCs carrying oncolytic viruses to target gliomas more efficiently. This technique has effectively protected the virus from host immunosurveillance and showed virus delivery to distant tumor regions. This approach also eliminated the risk of tumor transformation of the lysed stem cells.

Multipotent mesenchymal stromal cells Multipotent mesenchymal stromal cells, also called MSCs, are nonhematopoietic stem cells that can be isolated from the bone marrow stroma and peripheral blood as well as muscle and adipose tissue. They normally repair injured tissue by causing inflammation. Since tumors show similarities to nonhealing wounds, MSCs exhibit tropism for gliomas and other brain tumors (Bexell et al., 2013). In rats, MSCs showed a lack of long-distance migratory capability when they were injected a few millimeters from the tumor (Bexell et al., 2012). However, intratumoral implantation of MSCs has shown a migration to tumor extensions as well as to some distant tumor microsatellites (Bexell et al., 2009). MSCs are not without risks; they can potentially promote tumor growth (McLean et al., 2011) and increase metastatic capacity (Karnoub et al., 2007). Interestingly, independent groups have reported that nonmodified MSCs reduce tumor vascularization and suppress tumor growth without promoting tumorigenicity. Finally, similarly to NSCs, MSCs can be modified to carry conditionally replicating oncolytic viruses or suicide genes with variable efficiency (Ahmed et al., 2010).

NANOTECHNOLOGY FOR DRUG/GENE DELIVERY TO TREAT GBM Nanotechnology focuses on the design and application of multivalent, multifunctional NPs (Ferrari, 2005). Over the last few decades, NPs have received considerable attention based on their ability to deliver therapeutics or genes to the diseased site while minimizing toxicity to healthy tissues. This may lead to a better therapeutic efficacy compared to conventional chemotherapy (Tiwari and Amiji, 2006; Jin and Ye, 2007). Several nanoplatforms have been developed ranging from synthetic NPs (i.e., liposomes (Malam et al., 2009), iron oxide (Chertok et al., 2008), and gold (Huang et al., 2007)) to biologic NPs (i.e., virus). The NPs are generally administered intravenously and are designed to be stable, nontoxic, biocompatible, and biodegradable. They are also designed to have a prolonged blood circulation time with a sustained drug release profile (Olivier et al., 1999; Tiwari and Amiji, 2006). Although NPs offer many advantages, their size (typically ranging from 50 to 300 nm) does not allow them to freely diffuse across the BBB. Most NPs that have been investigated require

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receptor-mediated transport through the brain endothelium for proper delivery of the therapeutic payload into the brain parenchyma, as discussed below.

Synthetic nanocarriers SURFACTANT-COATED POLYMERS FOR BRAIN TARGETING

Since the mid-1990s, it has been shown that polybutylcyanoacrylate (PBCA) NPs coated with the surfactant polysorbate 80 can successfully deliver drugs with poor brain diffusion properties into the brain (Olivier et al., 1999; Kreuter, 2001). However, the working doses for brain delivery in mice were approaching the lethal dose 50% (LD50) of PBCA NPs, with an observation of a dramatic reduction in mice locomotor activity and with concomitant signs of distress (Kante et al., 1982; Olivier et al., 1999). In addition to the general toxicity observed, major damage to the BBB induced by polysorbate 80 and the short duration of pharmacologic effects (Olivier et al., 1999) may limit their potential clinical applications. NPs made from polylactide (PLA) or poly (lactide-coglycolide) (PLGA) polymers and methoxypoly (ethylene glycol) (mPEG) may serve as a promising alternative due to their brain biocompatibility, biodegradability, and resorbability (Menei et al., 1993; Shive and Anderson, 1997; Emerich et al., 1999). For example, mPEG2000PLA30000 NPs demonstrated no signs of toxicity in rats at the highest dose upon intravenous administration. To date, many conventional drugs have been formulated as PLA or PLGA NPs (i.e., doxorubicin (Yoo and Park, 2001), irinotecan (Onishi et al., 2003), paclitaxel (Feng et al., 2004), lidocaine (Holgado et al., 2008), and heparin (Hoffart et al., 2002)). Each formulation possesses different drug release characteristics, ranging from a few hours to several weeks (reviewed in Olivier et al., 1999). In addition to drug delivery, plasmid DNA-loaded PLA or PLGA NPs have been described, allowing sustained gene expressions in vivo for several weeks. To overcome the BBB, the surface of the NPs has to be modified with targeted ligands specific to receptors that are highly expressed on the brain capillary endothelium (i.e., transferrin) to facilitate receptor-mediated transcytosis from the blood to the brain parenchyma (Gan and Feng, 2010). While these NP formulations offer great promises for therapeutic delivery into the brain, further optimization and evaluation are necessary in order to bring them forward for clinical applications.

Solid lipid nanoparticles (SLNPs) SLNPs are attractive drug carrier systems for brain targeting and may have several advantages compared to polymeric NPs and other nanoplatform systems

(i.e., liposomes), including extended controlled drug release, prolonged NP stability, higher drug payload, cost-effectiveness, avoidance of use of organic solvents, and feasibility of large-scale production and sterilization. The use of SLNPs can alleviate the issue of solubility, permeability, and toxicity associated with several therapeutic drugs. Several preclinical studies have demonstrated the capability of SLNP drug delivery into the brain (Yang et al., 1999; Zara et al., 2002; Goppert and Muller, 2005; Manjunath and Venkateswarlu, 2006). New formulations of SLNP therapeutics are continuously being explored to improve their pharmacokinetic profiles and to obtain higher payload delivery into the brain. In this context, drug-loaded polymeric NP-stabilized gas bubbles together with low-intensity focused ultrasound exposure has been shown to transiently and selectively open the BBB in small animals (Burgess et al., 2011). Nonviral NPs for gene delivery have gained vast attention recently because they are less immunogenic and oncogenic, and generally safer than viral gene transfer (Jin and Ye, 2007). Genetic material such as DNA plasmids or RNA can be encapsulated or conjugated on to NPs, such as liposomes, silica, and other polymeric NPs (Kneuer et al., 2000; Kaul and Amiji, 2005). Nonetheless, NP gene delivery vectors suffer disadvantages such as lower efficiency compared to viral vectors (Jin and Ye, 2007). To improve gene delivery efficiency, efforts have been made to develop ligand-conjugated NPs for tumor targeting. A biodegradable NP, polyaminoethyl propylene phosphate, utilizes a controlled-release system to achieve gene transfer in the brain (Li et al., 2004). This NP does not cause detectable neurotoxicity and is biodegradable, providing sustained release of DNA at various rates.

Magnetic nanoparticles (MNPs) for thermotherapy of glioma MNPs can generate heat when exposed to alternating magnetic fields. The mechanism of hyperthermia induction and magnetic energy conversion can be explained in two ways: Neel relaxation is the internal rotation caused by rapidly occurring changes in the direction of the magnetic fields. Brownian relaxation is the external rotation of the NPs within the medium. High temperature causes cells to undergo protein denaturation, aggregation, and DNA cross-linking (Wust et al., 2002; Goldstein et al., 2003). This process can induce apoptosis and change the pH of the microenvironment. Recent studies have shown that these effects, combined with radiotherapy and chemotherapy, can have a synergistic effect. MNPs can be manufactured from various metals, including manganese, iron, cobalt, nickel, zinc, and magnesium (Pradhan et al., 2007; Atsarkin et al., 2009; Kaman

EXPERIMENTAL THERAPIES: GENE THERAPIES AND ONCOLYTIC VIRUSES et al., 2009). Cobalt ferrites-based NP, for example, produces greater heat at much lower concentration than iron oxide-based NP. However, owing to their lack of toxicity and high biocompatibility, iron oxide-based MNPs are still preferred for thermotherapy of malignant brain tumors (Huber, 2005). MNP-based hyperthermia has recently been evaluated for feasibility in several preclinical animal models and in patients with GBMs. In this context, Jordan et al. (2006) evaluated the utility of dextran-coated and aminosilane-coated iron oxidebased NPs in a rodent GBM model. Maier-Hauff et al. (2007) have since then tested this model in humans with recurrent GBM before and after administration of adjuvant fractionated radiation therapy. With a magnetic field of 100 kHz and a high concentration of iron oxide-based NPs >100 mg/mL, the temperature within the tumor was elevated to 51.2 °C and was demonstrated to be safe in the phase II clinical trial. Although the authors showed significant increase in overall survival with comparison to a reference population, further randomized studies are necessary for validation of this treatment modality. In conclusion, nanotechnology may be important for the treatment of HGGs. The ideal characteristics of NPs for drug/gene delivery include biocompatibility, low immunogenicity, long circulation time, and the ability to efficiently bypass barriers such as the vascular endothelium and the BBB. Yet, far more extensive investigations are required to study the efficacy and safety of NPs in human use. In addition, design of new NP structures to improve gene transfection efficiency is warranted. Moreover, modifications of NPs for enhanced targeting and retention in desired tissues, delivery of agents (i.e., drugs/genes), and sustained release of cargo must be further improved to promote the medical application of nanotechnology in the near future.

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