Clinical aspects

Description

	• Several strategies for gene therapy of glioblastoma are under investigation but none have been approved yet.
	• Most gene therapies are single applications, which may not eradicate the tumor to prevent recurrence.
	• Combination of gene therapy with other techniques is being pursued to increase the chances of cure of glioblastoma.

Various strategies for gene therapy of glioblastoma are shown in Table 2.

Table 2. Strategies for Gene Therapy of Glioblastoma

Viral vector-mediated insertion of drug sensitivity genes
	• Direct intratumoral injection of genetically modified neurotrophic viruses
	• Insertion of drug sensitivity genes
	• Suicide gene therapy: herpes simplex virus-thymidine kinase
	• Use of Escherichia coli gpt gene to sensitize glioma cells to prodrug 6-thioxanthine
Use of viral vectors containing radiation-inducible promoters Regulated toxin gene therapy
	• Baculovirus as diphtheria toxin gene vector
Transfer of apoptosis-inducible FADD/MORTI gene
Gene transfer into brain tumors by using targeted adenoviral (Ad) vectors
	• Single chain antibody combined with Ad vector and targeted to EGFR receptor
Gene transfer into brain surrounding tumors by using targeted adeno-associated viral (AAV) vectors
	• Transduction of normal cells in the brain with an AAV vector encoding interferon-beta (IFN-beta)
Selective oncolysis by genetically engineered microorganisms: bacteria and viruses
Cytokine gene therapy
Immunogene therapy
Cell-based gene therapy
	• Mesenchymal stem cells engineered to produce therapeutic molecules
	• Neural stem cells engineered to produce therapeutic molecules
	• Grafting of stem cells producing therapeutic molecules such as IL-4 gene
Growth factor manipulation
	• Apoptosis induced by introduction of gene for nerve growth factor receptor TrkA
	• Inhibition of epidermal growth factor receptor-associated tyrosine kinase receptor
Antiangiogenesis approaches directed against tumor blood vessels
Oncogene antagonism: anti MYC oncogene MAD therapy
Insertion of tumor suppressor genes
	• Transfer of wild-type p53 or p27
	• Retinoblastoma gene transfer
Antisense therapy
	• Blocking of action of transforming growth factor beta 2 by triplex-forming oligonucleotides
	• Episome-based antisense cDNA transcription of insulin like growth factor 1
	• Antisense vascular endothelial growth factor
	• Oligodeoxynucleotides targeted to tumor necrosis factor
RNA interference (RNAi)-based approaches
	• MicroRNA (miRNA)-based gene therapy
	• siRNA directed against EGFR and its variants
	• siRNA directed against PI3K/Akt signaling pathways
	• Telomerase inhibition by RNAi
Gene editing
	• CRISPR-Cas9

Currently, gene therapy of brain tumors is the responsibility of neurosurgeons. Close cooperation with gene therapy laboratories and scientists working with these technologies will be required. With future advances and development of noninvasive methods, it is feasible that neuro-oncologists, interventional neuroradiologists, and neurologists may be carrying out such procedures in patients with tumors at early stages, where surgery is not required.

Indications

No gene therapy for glioblastoma has yet been approved in the United States, but several clinical trials are in progress. Selection criteria are set for each clinical trial. Neurosurgical management of these patients is still the primary approach, followed by chemotherapy or radiotherapy. Gene therapy may be used in conjunction with surgery that may be required initially for decompression of large tumors and confirmation of diagnosis. Gene therapy may be used as an alternative to chemotherapy/radiotherapy or in cases of recurrences following excision as well as chemotherapy-resistant tumors. With improvement in diagnostic imaging and methods of delivery, it feasible that small tumors detected early may be treated noninvasively or by minimally invasive gene therapy procedures.

Contraindications

None have yet been identified.

Results

The goal of gene therapy is the cessation of growth of the tumor. Prognosis of glioblastoma remains poor; current gene therapy methods have not improved patient survival significantly compared to the most aggressive non-gene therapy methods (ie, chemotherapy). Gene therapy is usually a 1-time treatment and cannot be repeated for residual and recurrent tumor.

Herpes simplex virus-thymidine kinase gene therapy reached the most advanced (phase 3) of all gene therapy methods in clinical trials for glioblastoma. The results in patients were less striking than in experimental animals. There was reduction in the size of the tumor with slight prolongation of life, but the tumors still grew and the patients died. The development of this therapy was discontinued because of lack of significant efficacy. Several modifications of viral-vector mediated gene therapy are still in clinical trials. Clinical evaluation of patients in clinical trials can be monitored by brain imaging. The expression of an exogenous gene introduced by gene therapy into patients with gliomas can be monitored noninvasively by PET. It is unlikely that any of the approaches reviewed here can eradicate glioblastoma. Unless the tumor is eradicated completely, it recurs rapidly, and eventual mortality may be unchanged. Among genetically modified microorganisms, bacteria appear to be more promising than viruses. Genetically modified bacteria that selectively destroy glioblastoma while sparing normal brain tissue can be more precise than any surgical tool and can be left in until the tumor destruction is complete; they can then be killed with an antibiotic (13).

Clinical trials of gene therapy for glioblastoma. As of August 2021, 101 clinical trials for gene therapy of this tumor are listed on the U.S. government website clinicaltrials.gov. This is a small fraction of all clinical trials of glioblastoma that number over 1660. The database lists completed, terminated, and ongoing trials.

Adverse effects

Safety aspects of herpes simplex virus-thymidine kinase with ganciclovir therapy. Safety of this therapy has been established in clinical trials with direct implantation of herpes simplex virus-thymidine kinase retroviral vector. There was no associated systemic toxicity or evidence of systemic spread of retroviral vectors with this form of in vivo gene transfer. Intracerebral injection of replication-defective adenovirus-bearing herpes simplex virus-thymidine kinase gene with intravenous ganciclovir in baboons produces significant neurotoxicity. The toxicity is minimal at lower doses of the vector, corresponding to the upper range of anticipated therapeutic doses in humans.

Antivector immune response induced by adenoviral vector. This poses a problem by failure of any subsequent attempt at adenoviral mediated gene therapy by using first generation vectors. High-capacity adenovirus vectors, carrying the conditional cytotoxic gene herpes simplex virus type 1-thymidine kinase, may induce tumor regression even in the presence of systemic anti-adenovirus immunity.

Safety aspects of adenoviral vector-based gene therapy. Adenoviral vectors have the potential to induce chronic inflammation in the brain parenchyma that may have adverse effects on the function of the normal brain tissue surrounding the tumor.

Scientific basis

	• Viral vectors have been used to transfer drug-sensitivity genes into glioblastoma followed by use of prodrugs that are activated into metabolites that are toxic to dividing tumor cells but spare the normal brain.
	• Oncolytic viruses are genetically modified to lyse tumor cells.
	• Lower doses of radiation can be used for enhancement of the effects of gene therapy on glioblastoma.
	• Antisense oligodeoxynucleotides can recognize specific gene sequences and downregulate gene expression and are used for targeting oncogenes that are expressed in brain tumors.
	• CRISPR gene editing can be used to target and modify the function of genes involved in glioblastoma growth.

Viral vector-mediated insertion of drug sensitivity genes. Drug-sensitivity genes encode enzymes that activate prodrugs into their toxic metabolites. To transfer these genes into tumor cells in vivo, modified viruses and other agents can be employed as vectors. Three types of viral vectors have been found to be efficient for this purpose: (1) retrovirus, (2) herpes virus, and (3) adenovirus.

Retroviruses. The brain has several advantages of safety and efficacy for retroviral-mediated in vivo gene transfer. Retroviral vectors (that only integrate and express genes in proliferating cells) do not affect the nondividing brain cells but transfect the actively dividing tumor cells. The brain is an immunologically privileged site, and gliomas further depress local immunity due to downregulation of interleukin-2 secretion and high affinity interleukin-2 receptors on T-lymphocytes. The xenogeneic murine cells, therefore, survive for longer periods in the brain than in other tissues.

Retrovirus-mediated delivery of a reporter gene, Escherichia coli LacZ, into cerebral gliomas can be obtained by direct intratumoral injection of retroviral particles but has a low efficiency of gene delivery (only 0.1% of tumor cells). Retrovirus-mediated transfer of the drug-sensitivity gene, herpes simplex virus-thymidine kinase, into tumor cells has been reported by several laboratories. Herpes simplex virus-thymidine kinase phosphorylates nucleoside analogs such as ganciclovir and acyclovir, leading to their incorporation into DNA during its replication. The incorporated nucleoside can cause DNA breaks that lead to cell death. This has been demonstrated in animal models. Two mechanisms are operative in this effect. One is the selective targeting of replicating DNA strands by ganciclovir metabolites, and the other is selective retrovirus-mediated gene integration and expression in dividing cells. Both mechanisms spare the normal nondividing glia and neurons. The effect of ganciclovir can be enhanced by intravenous RMP-7, a bradykinin analog that increases permeability of the blood-brain tumor barrier.

Herpes simplex virus. Replication of herpes simplex virus results in cell death. This property can be utilized for the killing of tumor cells. The aim of constructing an effective herpes simplex virus vector for brain tumor therapy is to render its replication selective for neoplastic cells. This is done by deletion in the viral thymidine kinase gene. Herpes simplex virus modified this way can only replicate in dividing cells that possess large amounts of endogenous thymidine kinase. Postmitotic brain cells, such as neurons and glia, have low levels of endogenous thymidine kinase. Direct injection of such a mutant virus into intracerebral tumors in experimental animals leads to improved survival of the animals. Herpes simplex virus mutants retain the ability to confer ganciclovir sensitivity on tumor cells because the viral thymidine kinase gene is intact. Ganciclovir treatment results in potentiation of the vector's cytotoxic action against glioblastoma cells.

Adenoviruses. Efficacy of adenovirus-mediated transfer of herpes simplex virus-thymidine kinase gene and ganciclovir administration in the treatment of rat gliomas has been demonstrated.

Adenoviral vector has the following advantages for treating brain tumors:

	• It can be easily produced at high titers.
	• It can be directly injected as purified particles into brain tumors without the need for virus producing cells.
	• It does not integrate into the host genome. This avoids the risk of insertional mutagenesis.
	• Gene delivery by adenovirus is independent of the host cell cycle. This feature allows higher transduction rates than with retroviruses because only a small number of glioma cells are replicating at any given time.

Disadvantages of adenoviral vectors are as follows:

	• Actual release in vivo cannot be quantified.
	• Antigenicity of viral proteins; this might limit repeated treatments. This problem has been seen in gene therapy trials for cystic fibrosis, but it is uncertain if this problem would occur in the brain, as it is an immunologically privileged site.
	• Unlike retrovirus vectors, they infect all cells of the brain. This lack of discrimination could result in significant toxicity to the normal surrounding brain. This can be remedied by use of tissue-specific promoters. One example is glial fibrillary acidic protein, a gene that is found in glial cells. Transfer of a vector containing a cytotoxicity gene under the control of a glial fibrillary acidic protein promoter should result in expression only within the cells that normally synthesize glial fibrillary acidic protein.

Replication-defective adenovirus vectors have been used to deliver reporter gene LacZ into brain tumor cells. Promising results have been obtained for delivery of herpes simplex virus-thymidine kinase gene to treat experimental rat gliomas, but the question of safety remains to be addressed, as these viruses are neurotoxic. Recombinant adenovirus vectors are used in several human clinical trials and may prove useful for the treatment of brain tumors. Although randomized, controlled trials have shown that adenoviral mediated gene therapy has significant anti-tumor effect, efficient gene delivery into the brain still presents a major problem.

Glioblastoma may be refractory to adenoviral vector mediated gene therapy because of the lack of the primary adenoviral receptor on tumor cells. Adeno-associated virus, a non-pathogenic human parvovirus, may be used as a vector for gene therapy of glioblastoma by intra-tumoral injection.

Glioblastoma expresses high levels of type 2 somatostatin receptors, which can be targeted for improving transduction efficiency in these tumors. A new adenoviral vector was designed based on the introduction of the full-length somatostatin somatotropin release-inhibiting factor sequence into it, and it was shown that low doses of this vector were sufficient to infect high-grade human glioblastoma cells with marked enhancement of gene expression (19).

In vivo application of viral vectors. Most investigators have utilized murine retroviral vectors for ex vivo gene transfer, but in vivo transfer of genes into cancer cells is preferred. In the earlier in vivo approaches, retroviral vectors were injected directly into the tumor mass, but the transduction frequency was low (1% to 3%). The direct implantation of vector-producing cells has improved the efficiency of gene transfer to more than 50%. Selective delivery of viral particles has been achieved in multifocal gliosarcoma tumors in rat brain by intra-arterial injection combined with disruption of the brain-tumor barrier. A phase I/II trial has evaluated the efficacy of repeated administration of virus-producing cells containing the herpes simplex virus thymidine-kinase gene followed by ganciclovir treatment in adults with recurrent glioblastoma. Results show this method of treatment to be feasible with some evidence of efficacy. Reported toxic effects may be related in part to the method of gene delivery. This approach has not been developed further.

Oncolytic viruses. Oncolytic viruses are genetically modified to lyse tumor cells. They are replication-selective rather than replication-defective viral vectors. Oncolytic viruses differ from viral vectors in that they increase in number in tumor cells and lyse the cells directly, not by transducing specific genes. Most oncolytic viruses currently investigated in clinical trials are derived from adenovirus or herpes simplex virus (HSV) type I. An adenovirus ONYX-015 was shown to be effective in delaying the growth and partial destruction of subcutaneous human malignant glioma xenografts deriving from primary tumors. It was not approved in the United States or Europe but was licensed by a Chinese company and is approved for use as an anticancer agent. Reolysin, a proprietary formulation of reovirus, can freely replicate and kill tumor cells with an activated Ras pathway. It is in phase II clinical trials for brain cancer. A better understanding of the interactions of the host's immune system with the virus is required before oncolytic virus therapy of malignant brain tumors can be introduced into clinical practice. Studies both in a mouse glioma model as well as on glioma stem-like cells from patients suggest that the efficacy of viral oncolysis HSV type 1 may be enhanced when used in combination with inhibitors of histone deacetylases or other proteins that modulate cellular trafficking of these therapeutic viruses (23).

Drug sensitivity genes. Genes that have been shown to possess antitumor effect are as follows:

	• Drug-enhancing genes that code enzymes that catalyze the conversion of a prodrug into active anticancer metabolites.
	• Immune-response enhancer genes that encode factors (such as interleukin-2) that increase immune response against tumors.
	• Tumor suppressor genes such as p53.
	• Antisense RNA.

Herpes simplex virus-thymidine kinase. This gene encodes for the enzyme thymidine kinase that catalyses phosphorylation of the anti-herpes drugs acyclovir and ganciclovir. Cellular kinases then convert the monophosphate form of the drug to a triphosphate form that inhibits the association of deoxynucleoside triphosphate with DNA polymerase. Finally, the triphosphate forms incorporate into DNA and act as a chain terminator.

Cytosine deaminase. The normal function of this bacterial gene is to deaminate cytosine into uracil. 5-fluorocytosine was developed as a prodrug, and can be converted into the highly toxic 5-fluorouracil that causes nucleotide strand breaks. Transfer of cytosine deaminase genes into mammalian cells renders them selectively sensitive to 5-fluorocytosine. Stereotactic injection of cytosine deaminase gene into established rat gliomas, followed by systemic administration of FC-5 in vivo results in prolongation of survival. This strategy is considered likely to be useful for the treatment of brain tumors.

Deoxycytidine kinase. This enzyme phosphorylates cytosine arabinoside to ara-CMP. Cytosine arabinoside is the most effective agent for treatment of acute myeloid leukemia. It incorporates into replicating DNA and terminates DNA chain elongation. It penetrates the blood-brain barrier and has a relative lack of toxicity against postmitotic cells. This agent normally has low effect on solid tumors such as glioblastoma because deamination by cytidine deaminase is more rapid than phosphorylation by deoxycytidine kinase. Transduction of glioma cells by retroviral and adenoviral vectors expressing deoxycytidine kinase gene increases the sensitivity of these cells to the cytotoxic effect of cytosine arabinoside both in vitro and in vivo.

Cytochrome P450 2BI. Cyclophosphamide is an inactive prodrug that must be metabolized by a liver-specific enzyme cytochrome P450 2BI into its active metabolite 4-hydroxy cyclophosphamide, which exerts an anticancer effect by disrupting strand links in DNA during all phases of the cell cycle. When transfected into glioma cells, retroviral vectors bearing cytochrome P450 2BI induce chemosensitivity to prodrug cyclophosphamide.

Gpt. Escherichia coli gpt gene encodes xanthine-guanine phosphoribosyl transferase, a bacterial enzyme responsible for transfer of ribose phosphate to xanthine and certain xanthine analogs. This gene has been shown to sensitize rat glioma cells to killing by 6-thioxanthine or 6-thioguanine. These findings provide a basis for exploring further gene therapy strategies, based on in vivo transfer of the gpt gene to provide chemosensitivity against 6-thioguanine or 6-thioxanthine. Unlike other genes tested to date in brain tumor models, gpt gene is unique in that it does not only sensitize the cells to the prodrug, but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus, providing a means for selecting gpt positive cells.

Suicide gene therapy: herpes simplex virus-thymidine kinase. In vivo herpes simplex virus-thymidine kinase gene (suicide gene) transfer forms the basis of gene therapy for malignant gliomas. The tumor cells are sensitized to the antiviral drug ganciclovir given systemically.

Experimental studies. The tumors are eliminated in animal models with the use of herpes simplex virus-thymidine kinase strategy despite less than 100% gene transfer efficiency. This phenomenon is referred to as the "bystander effect," and allows the destruction of neighboring cells. Within a certain level of thymidine kinase enzyme threshold, the higher levels of herpes simplex virus-thymidine kinase expression correlate with better bystander effect in mediating tumor killing. The presence of this bystander effect is important in achieving complete regression of brain tumors in experimental animals; none of the viral vectors used for therapy of brain tumors can transduce 100% of the tumor cells in the brain.

Mechanisms of "bystander effect." Various possible mechanisms of this effect are listed below, and it is possible that all of these participate in this phenomenon in vivo.

	• It is possible that a transfer of phosphorylated ganciclovir moieties from herpes simplex virus-thymidine kinase-positive cells to herpes simplex virus-thymidine kinase-negative cells occurs through gap junctions. These junctions are comprised of a family of homologous proteins called connexins that are expressed in a tissue-specific manner. Astrocytes in the brain express connexin43. Transfection of a rat astrocytoma cell line with a connexin43 complementary cDNA has been reported to increase intercellular communication and to suppress growth. These results anticipate that glioblastomas will have a varied "bystander effect" during herpes simplex virus-thymidine kinase therapy, depending on the level of connexin43 expression. Normal brain tissue is protected because of obliteration of gap junctions at the interface of tumor and normal tissues. Concern has also been expressed about possible development of resistance to ganciclovir by this approach.
	• Cell fragments from cells destroyed by herpes simplex virus-thymidine kinase with ganciclovir are taken up by neighboring cells by phagocytosis and cause death of these cells.
	• Direct cell-to-cell transfer of phosphorylated ganciclovir moieties through apoptotic vesicles.
	• An immune response to ganciclovir metabolites.
	• Thymidine kinase expression in nearly vascular endothelial cells made mitotically active by tumor angiogenesis factors with consequent killing of these cells by acquisition of ganciclovir susceptibility leading to ischemia of the tumor mass.
	• Development of antitumor immunity.

Adenovirus-mediated herpes simplex virus-thymidine kinase gene transfer. A replication-deficient recombinant adenovirus bearing the Escherichia coli b-galactosidase gene has been used in a rat C6 glioma tumor model by stereotactic technique and shown to be capable of transferring genes into the central nervous system. The same technique followed by ganciclovir administration can cause reduction in tumor size in experimental. Furthermore, compared with retrovirus-mediated herpes simplex virus-thymidine kinase transfer, adenovirus-mediated transduction increased viral kinase activity by more than 600-fold and reduced by as much as 12-fold the concentration of ganciclovir required to elicit cytotoxicity. A phase I clinical trial of patients with recurrent glioblastoma treated with adenovirus/herpes simplex-thymidine kinase/ganciclovir shows that this procedure is safe. Results of an open-label, randomized, phase III trial of locally applied adenovirus-mediated gene therapy with herpes-simplex-virus thymidine kinase (sitimagene ceradenovec) followed by intravenous ganciclovir in patients with newly diagnosed glioblastoma after resection can increase time to death or reintervention, but not overall survival (31). Further clinical trials are in progress, and it will be the large randomized phase III controlled clinical trials that will provide evaluation of the success of gene therapy for the treatment of glioblastoma (08).

Activation of early growth response-1 promoter to induce sensitivity to herpes simplex virus-thymidine kinase. Early growth response-1 gene expression can be useful in enhancing the treatment of brain tumors with radiation by combining this gene therapy approach with ganciclovir administration. The drawbacks of this strategy are as follows:

	• Local transduction of normal slowly proliferating brain tissue such as endothelial and glial cells.
	• Escape of the vector into the systemic circulation may lead to the integration of the herpes simplex virus-thymidine kinase gene into normal proliferating tissues such as bone marrow.

Lentiviral vectors for gene therapy of glioblastoma. The latest generation of lentiviral vectors have improved safety profile and gene transfer capacity. Initially used in suicide gene therapy of glioblastoma, they have been modified to either express small noncoding RNAs or to overcome the resistance to both chemo- and radiotherapy in glioblastoma. Lentiviral vectors have now been adopted for engineering T cells to express chimeric antigen receptors that recognize specific glioblastoma antigens, or for transducing specific cell types that can carry therapeutic molecules to the tumor (10).

Liposome-mediated gene delivery into tumor cells. Nonviral-mediated gene transfer of herpes simplex virus-thymidine kinase using liposomes and particle bombardment has been tried. Injection of cationic liposomes containing the herpes simplex virus-thymidine kinase gene into a rat brain tumor model followed by ganciclovir treatment, leads to marked tumor regression without any histological damage in the neighboring tissue.

The advantages of these methods are simplicity, less possibility of adverse reactions, and less cost as compared to methods using recombinant viral vectors. The disadvantages are a low level of gene transfer, lack of stable integration of injected DNA, and a lack of specificity. There are no approved trials using these techniques for treatment of malignant brain tumors.

Herpes simplex virus-thymidine kinase as radiosensitizer. 9L rat glioma cells, transduced with herpes simplex virus-thymidine kinase and treated with acyclovir, are highly sensitive to radiation, as compared with nontransduced glioma cells. This suggests that the addition of herpes simplex virus-thymidine kinase gene therapy to standard radiation therapy might improve the effectiveness of brain tumor treatment.

Gene therapy for reducing adverse effects of chemotherapy. Chemotherapy has several adverse effects, particularly myelosuppression, which significantly limit dose escalation and potential clinical efficacy. Gene therapy using mutant methylguanine methyltransferase (P140K) gene-modified hematopoietic stem cells may reduce the toxic effects of chemotherapy on hematopoietic cells. Engraftment of autologous P140K-modified hematopoietic stem cells was used in 3 glioblastoma patients with poor prognosis before they were subjected to multiple cycles of chemotherapy, resulting in increase of survival in all the 3 without adverse effects (01).

Glioblastoma frequently develops resistance to temozolomide, which can be overcome by adding O6-benzylguanine (O6BG), but the combination produces myelosuppression. Results of a prospective clinical trial have shown that gene therapy P140K-modified hematopoietic stem cells to confer O6BG resistance improves chemotherapy tolerance and outcome in these patients (02).

Combination of radiation and gene therapy. Radiation can be combined with gene therapy either for activation of gene therapy vectors or for enhancement of the effects of gene therapy so that lower doses of radiation are required for therapeutic effect. Gene therapy offers the possibility of significantly improving the efficacy of radiotherapy for glioblastoma without the need for ionizing radiation dose escalation or undue increases in normal tissue morbidity.

Combination of gene therapy with chemotherapy. An example of combination of gene therapy with chemotherapy is clinical trial NCT02414165 titled “Toca 511 and Toca FC versus standard of care in patients with recurrent high-grade glioma.” This is a randomized, open-label phase II/III trial of combination treatment using retroviral vector Vocimagene amiretrorepvec (Toca 511), a replicating virus that only infects actively dividing tumor cells to deliver the gene for enzyme, cytosine deaminase, and sustained release 5-fluorocytosine (Toca FC), the prodrug of the chemotherapy 5-fluorouracil. Once inside tumor cells, cytosine deaminase converts the prodrug to 5-fluorouracil, which destroys them as well as immunosuppressive myeloid cells, enhancing the patient’s immune system to recognize and attack the cancer cells. As of August 2018, the trial is still ongoing, but analysis of a subset of phase I (selected to proceed to phase III) showed that durable response rate of 21.7% in patients with recurrent glioblastoma who were treated with a gene therapy combination were alive 33.9+ to 52.2+ months after treatment (09).

Regulated toxin gene therapy. Experimental studies have shown that diphtheria toxin A fragment expression in human glioma cell lines using the tetracycline system derived from Escherichia coli could regulate tumor cell killing several-fold. Tetracycline system is of potential use in gene therapy applications wherein regulated expression of a therapeutic gene is an important issue.

Targeted disruptions of factors that promote growth of glioblastoma. An experimental study has demonstrated that silencing of BCL6, a prognostic biomarker and a targetable glioblastoma-promoting factor, inhibits growth of malignant cells and triggers cellular senescence (33).

Immunogene therapy. Preclinical models and clinical trials have demonstrated that despite their location within the brain, usually considered as “immune privileged” organ, brain tumors can be effectively targeted by the activated immune system following various immunotherapeutic strategies that can also be combined with cytotoxic therapies. These involve mainly the transfer of cytokine genes. There are abnormalities of cytokine-mediated cell proliferation in gliomas. Amplification of the epidermal growth factor receptor correlates inversely with the length of survival for patients with malignant gliomas. Further understanding of this phenomenon will facilitate the development of strategies for limiting glioma cell locomotion and invasion. A cytokine vaccine strategy that has been applied successfully against metastatic tumors is being extended against malignant gliomas; initial observations are encouraging.

Cytokine gene therapy. Cytokines have been administered to patients with gliomas by various methods. Some of these methods are described here briefly.

Interleukin-2. Attempts to enhance the immune response by in vivo transfer of human interleukin-2 gene for eradication of brain tumors have not been successful in patients. The long-term success of this strategy can be improved by measures to augment the host's antitumor immune response.

Granulocyte macrophage colony-stimulating factor. Glioma cells induced by this cytokine have been used as a vaccine against malignant gliomas. Preclinical studies have shown the potential of immunogene therapy of human glioblastoma.

Immunovirotherapy. In preclinical studies, pediatric brain tumors are highly sensitive to oncolytic virotherapy with genetically engineered herpes simplex virus type 1 (HSV-1) G207, which lacks genes essential for replication in normal brain tissue. A phase 1 trial (NCT02457845) has shown that intratumoral G207 alone and with radiation had an acceptable adverse-event profile with evidence of responses in patients with high-grade glioma (11). G207 markedly increased the number of tumor-infiltrating lymphocytes and increased median overall survival to 12.2 months.

Growth factor manipulation. Several growth factors are involved in the progression of malignant gliomas of the brain. Nerve growth factor can stimulate clonal growth of cells of glioblastoma. Efforts have been made to induce apoptosis by the introduction of the gene for nerve growth factor receptor TrkA. Tumor necrosis factor also plays a part in tumorigenesis, but the gene for tumor necrosis factor can be better inhibited by antisense approach.

Antiangiogenic gene therapy. Gene therapy strategies, developed to interfere with the normal function of vascular endothelial growth factor receptors, have been successfully used in different experimental models to block tumor angiogenesis and to inhibit tumor growth. Different antiangiogenesis strategies may be combined. Some of the antiangiogenic gene therapies are:

Matrix metalloprotease inhibitor (MMI) gene therapy. Several studies have been conducted to show that in vivo gene therapy with matrix metalloprotease inhibitors leads to excellent prevention as well as treatment of established primary tumors and metastases in animal models. Various methods of gene transfer have been used including adenoviral vectors and liposomes. For optimal effect a high concentration of the matrix metalloprotease inhibitor was required at the tumor site. None of the studies were conducted in brain tumor models but this is a promising method for adjuvant treatment of residual tumor in combination with chemotherapy. Further research is required to determine how and which matrix metalloprotease inhibitor can exert optimal effects on malignant brain tumors.

MMAC/PTEN based gene therapy for gliomas. The tumor suppressor gene mutated in multiple advanced cancers/phosphatase and tensin homologue (MMAC/PTEN) has been shown to inhibit cell migration, spreading, and focal adhesion. Reintroduction of the MMAC/PTEN gene into human glioma cells induces significant inhibition of in vitro invasion. Further characterization of this regulation will facilitate the development of MMAC/PTEN-based gene therapy for gliomas.

Angiostatin gene delivery. Recombinant adeno-associated viral vectors carrying the angiostatin gene have been used as antiangiogenesis strategies to treat malignant gliomas in rat models, demonstrating the potential of adeno-associated virus as a safe and effective vector for antiangiogenic gene therapy of brain tumors. Intra-arterial injection of plasmids, encoding the antiangiogenic endostatin, selectively target therapeutic genes to malignant gliomas with considerable reduction in the volume of the tumors.

Targeting endothelial vasculature in brain tumors. VB-111, currently in phase II clinical trials, consists of a non-replicating adenovirus 5 (El deleted) carrying a proapoptotic human Fas-chimera (transgene) under the control of a modified murine promoter, which specifically targets endothelial cells within the tumor vasculature. VB-111 inhibits vascular density in mouse models bearing glioma xenografts, which justifies its clinical development as a treatment for glioblastoma (12).

Stem cell gene therapy. Several studies have demonstrated that neural stem cells, when implanted into experimental intracranial gliomas in vivo in adult rodents, distribute themselves quickly and extensively throughout the tumor bed and migrate uniquely in juxtaposition to widely expanding and aggressively advancing tumor cells, at the same time continuing to stably express a foreign gene. These findings suggest the adjunctive use of inherently migratory neural stem cells as a delivery vehicle for targeting therapeutic genes and vectors to refractory, invasive brain tumors.

Another approach for gene therapy of brain tumors is based on the grafting of neural progenitor cells producing interleukins. Intratumoral injection of IL-12 secreting neural stem cells in mice bearing intracranial gliomas significantly prolongs survival and leads to long-term antitumor immunity. Combining the tumoricidal potency of IL-12 with the extensive tumor tracking capability of neural stem cells results in a synergistic therapeutic benefit. Genetically engineered neural stem cells have been shown to specifically target glioblastoma grafts after traveling through brain parenchyma and hinder tumor growth through local activation of cyclophosphamide-activating enzyme cytochrome p450 2B6 (22). Expression of vascular endothelial growth factor, a motility factor for neural stem cells, correlates with their migration in the brain.

Mesenchymal and neural stem cells are suitable for therapy of glioblastoma and their use is mainly based on the homing capacity toward the tumor microenvironment (21). Availability, targeted delivery to the tumor, potential oncogenicity, and ethical issues are the main challenges for translation of stem cell technologies into clinical applications for glioblastoma.

Human embryonic stem cell-derived engineered mesenchymal stem cells have been shown to inhibit tumor growth and prolong survival in the presence of the prodrug ganciclovir after they were injected either directly into the xenografts or into the opposite hemisphere of glioblastoma in the brains of mice (04).

Results obtained in a preclinical study using mesenchymal stem cells engineered to express cytosine deaminase provided evidence that stem cell-based gene therapy might also attack glioblastoma stem cells and, therefore, be curative (03). Encouraging results of preclinical studies of stem cell-based gene therapy for glioblastoma support the argument to begin clinical studies.

Cancer stem cells (CSCs), which are a subset of tumor cells with self-renewal ability and remarkable tumorigenicity, account for the high rates of recurrence after treatment as well as development of resistance to treatment in glioblastoma patients. Strategies for development of therapies include inhibition of CSC-specific pathways and receptors; agents that increase sensitivity of CSCs to chemotherapy and radiotherapy; virotherapy; and gene therapy (17). A subset of glioblastoma stem cells is marked by cell surface expression of CD133, a glycosylated pentaspan transmembrane protein. CD133-LV (lentiviral) represents a novel tool for the selective genetic manipulation of CD133-expressing glioblastoma stem cells and can be used to answer important questions about how these cells contribute to tumor biology and therapy resistance (05).

CAR-T cell therapy for glioblastoma. Chimeric antigen receptors (CAR)-T cells combine the antigen binding site of a monoclonal antibody (MAb) with the signal activating machinery of a T cell, freeing antigen recognition from MHC restriction, and thus, breaking one of the barriers to more widespread application of cell therapy. CAR-T technology uses retroviral or lentiviral vectors to engineer CARSs, which graft an arbitrary specificity onto an immune effector cell such as a T cell. These modified T cells are then transferred to the patient. T cells expressing CARs are highly targeted like MAbs, but they also offer the potential benefits of active trafficking to tumor sites, in vivo expansion, and long-term persistence. Furthermore, gene transfer in T cells enables the introduction of countermeasures to tumor immune evasion and of safety mechanisms.

Multiple infusions of CAR-T cells targeting the tumor-associated antigen IL-13 receptor alpha 2 (IL13Rα2) were administered to a patient with recurrent multifocal glioblastoma through 2 intracranial delivery routes: infusions into the resected tumor cavity followed by infusions into the ventricular system (07). There were no serious toxic effects, and regression of glioblastoma was observed, along with corresponding increases in levels of cytokines and immune cells in the cerebrospinal fluid. Intravenous delivery of a single dose of autologous T cells redirected to the epidermal growth factor receptor variant III (EGFRvIII) mutation by CAR-T in patients with recurrent glioblastoma was reported to be feasible and safe, without evidence of off-tumor toxicity or cytokine release syndrome (25).

Immunosuppressive cells, ie, regulatory T cells, tumor associated macrophages and myeloid derived suppressor cells constitute the tumor microenvironment in glioblastoma. These cells inhibit antitumor specific cytotoxic T cells functions, ultimately leading to decreased efficacy of immunotherapeutic approaches. In addition, glioblastoma is heterogeneous, with tumor cells acquiring new mutations after treatment, leading to therapy resistant disease. This supports the combination of 2 approaches against glioblastoma: attacking the tumor with gene therapy and enhancing the immune system's ability to fight it (15). Immune checkpoint blockade combined with gene therapy stops the cancer cells from hijacking the host immune system.

Aptamers for selective targeting of tumor-initiating cells. A study has adopted Cell-Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX) to identify aptamers that specifically bind to tumor-initiating cells in glioblastoma but not to human neural stem cells (18). These aptamers select and internalize into cells that self-renew, proliferate, and initiate tumors. Because they can be modified to deliver payloads, aptamers could selectively target or facilitate imaging of tumor-initiating cells to improve therapeutic outcomes in individual patients.

Insertion of tumor suppressor genes. A class of cellular genes that regulate cell growth by counteracting the action of proto-oncogenes are called "anti-oncogenes" or tumor suppressor genes. Their exact role has not been defined. Potential sites where these genes might inhibit the development of cancer include cell proliferation, differentiation and senescence, cell-to-cell communication, and chromosomal stability. Tumor suppressor genes are implicated in the genesis of many tumors. Efficacy in tumor suppression of 3 tumor suppressor genes (p53, p21, and p16) has been demonstrated in glioblastoma cell lines after retrovirus-mediated gene delivery in vitro and in vivo.

Experimental evidence indicates that adenoviral mediated gene therapy with p27, another tumor suppressor gene, has the potential to become a novel and powerful therapy for glioblastoma. Suppression of human glioma growth has been demonstrated by adenovirus-mediated retinoblastoma protein gene transfer.

Antisense therapy. Antisense approach involves inhibition of DNA transcription or mRNA translation by blocking or inserting special nucleotide sequences. This can be achieved by using antisense oligodeoxynucleotides. Other approaches include triple helix oligonucleotides and ribozymes. Various antisense strategies for brain tumors are shown in Table 3.

Table 3. Antisense Therapies in Development for Glioblastoma

Triple helix approach to inhibit insulin-like growth factor expression
	• Rat C6 glioma model • Inhibition of tumor growth
Direct in vivo administration of antisense oligonucleotides to insulin-like growth factor receptor
	• Rat C6 model • Reduced tumor burden. No need for a viral vector
Protein kinase C-alpha modified ribozyme
	• Animal tumor models • A single injection inhibits tumor growth
Recombinant adenoviral vector Ad4CMV-alphavascular endothelial growth factor carrying the coding sequence of wild-type vascular endothelial growth factor165 cDNA in an antisense orientation
	• Subcutaneous implant of human glioma in nude mice • Inhibition of tumor growth
Triple helix-forming oligonucleotides targeted to the human tumor necrosis factor gene
	• Inhibition of growth of glioblastoma cell lines • Blocks the tumor necrosis factor-dependent growth of human glioblastoma
Antisense 2-5A molecules
	• Animal tumor models • The compound works by limiting the action of telomerase
c-myc antisense oligonucleotides
	• Glioma cell lines in culture • Oligonucleotides suppress proliferation of glioma cells
Cancer vaccine: injecting malignant glioma patients with irradiated transforming growth factor beta 2 antisense gene-modified autologous tumor cells
	• Phase 1 study of the safety • Experimental studies showed prolongation of survival C6 tumor-bearing animals.
RNA interference: short inhibitory RNA targeting epidermal growth factor receptor
	• Human malignant glioma cells cultured in vitro • Specifically suppression of the epidermal growth factor receptor expression, gene silencing, and inhibition of cell growth and invasion.
Ribozyme embedded in adenoviral VAI sequence and targeted to vascular endothelial growth factor
	• U87 human glioblastoma cells • Inhibition of angiogenesis in vitro with potential of application in antiangiogenic gene therapy.

Antisense oncogene. The ability of antisense oligodeoxynucleotides to recognize specific gene sequences and to downregulate gene expression make them ideal agents for use in targeting oncogenes (such as c-myb) that are expressed in brain tumors. Oncogene targets for antisense oligonucleotide therapy in malignant glioma cells in vitro have included c-myc, c-myb, c-cis, and c-erb B.

Antisense growth factor. Malignant glial tumors express basic fibroblast growth factor. Transfection of rat C6 astrocytoma cells with antisense basic fibroblast growth factor cDNA leads to reduced levels of immunologically detectable basic fibroblast growth factor and diminished growth in vivo. This approach deserves further investigation.

Antisense protein kinase C. The protein kinase C family represents several closely related genes that are involved in signal transduction for processes such as cell proliferation, angiogenesis, and immune response. Protein kinase C has been proposed as a target for anticancer drug development. Antisense protein kinase C-a phosphorothioate oligonucleotides are in clinical development for treatment of cancer.

Antisense antiangiogenic. Vascular endothelial growth factor is an endothelial cell-specific mitogen that promotes angiogenesis in solid tumors including brain tumors. Antisense vascular endothelial growth factor may provide the basis for the development of antiangiogenic gene therapy for brain tumors.

Antisense brain tumor vaccine. Transforming growth factor-beta antisense RNA expressing glioma cells have been used as a vaccine. The postulated mechanism of this effect is knocking out transforming growth factor-beta expression with antisense RNA and producing interleukin-2 in tumor cells. This would have a combined effect on stimulating immune response against the tumor.

The Recombinant DNA Advisory Committee has approved a protocol for a phase 1 study of the safety of injecting malignant glioma patients with transforming growth factor-beta antisense gene modified autologous tumor cells. Patients with glioblastoma tumors that secrete transforming growth factor-beta will have the tumor removed surgically and receive the usual radiation therapy. A sample of the tumor cells will then be cultured and transfected by electroporation with a vector that expresses transforming growth factor-beta antisense RNA. After the cells have been demonstrated to express the RNA, they will be irradiated to prevent further proliferation and injected subcutaneously into patients.

Ribozyme. Ribozymes are enzymes comprised of RNA that can act as a catalyst as well as a genetic molecule. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. A nuclease-resistant protein kinase C alpha ribozyme has been shown to block glioma cell growth.

RNA interference. The overexpression of epithelial growth factor receptor (EGFR) is a feature of glioblastoma and is present in 40% to 50% of tumors, resulting in an uncontrolled multiplication of both normal EGFR and a mutant form called EGFRvIII. RNA interference would be an ideal approach to target EGFRvIII to destroy brain cancer cells and spare healthy cells. Because of the delivery problems, the ideal physical strategy appears to be direct application of RNA interference to the tumor or resected tumor cavity. Small-interfering RNA (siRNA)-based downregulation of DNA repair protein O(6)-methylguanine-DNA methyltransferase in tumor cells can enhance the chemosensitivity of malignant gliomas against temozolomide (16).

siRNA applied to glioblastoma cells in vitro was shown to reduce gene expression of EGFR and beat-catenin at the mRNA level and significantly inhibit their migratory as well as invasive ability (30). This is potentially an effective therapy for human glioblastoma and warrants further study in vivo.

Some miRNAs (eg, miR-21, miR-26a, and miR-10b) are overexpressed and oncogenic in glioblastoma, and suppression of their gene targets may facilitate tumor cell proliferation, migration, and survival. In animal glioblastoma models, intravenous or direct intracranial injection of antitumor miRNAs suppresses tumor growth. Clinical translation of miRNAs is limited due to their rapid degradation in serum, poor blood-brain barrier penetrance, lack of tumor selectivity, and insufficient intracellular uptake. However, the use of nanoparticles, peptide carriers, and extracellular vesicles to deliver anticancer miRNAs is being explored.

One approach to miRNA-based gene therapy for glioblastoma uses 3 miRNAs with synergistic antitumor activity and extracellular vesicles transferred from genetically modified glioma stem cells to unmodified glioma stem cells to effectively deliver a therapeutic dose of their miRNA cluster (06). The results of this study are promising, but currently have limited clinical applicability as they are nonspecific. Moreover, obtaining approval for intracranial intratumoral injection of this gene therapy in humans may be difficult because the long-term consequences and genetic cross-reactivity of such a therapy is unpredictable (29).

Gene editing. A gene editing technique, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 adaptive immune system, can be used for precisely altering the genomes of living cells by adding or deleting genes. In experimental studies, deletion of SOX2 regulatory region 2 (SRR2) by CRISPR/Cas9 technology leads to a reduction in expression of SOX2 (a major driver of cancer stem cells) in glioma cells, reducing proliferation of these cells, whereas self-renewal capacity is impaired in glioma stem cells (27). Furthermore, the results indicate that prevention of SRR2 activity leads to a reduction of oncogenic properties promoted by SOX2, suggesting that SRR2 may represent a novel therapeutic target in the glioblastoma to prevent relapse of these tumors.

Small molecules designed to target FGFR3-TACC3 gene fusions, chromosomal aberrations found in cancer, are 1 form of potential treatment of glioblastoma but cause off target effects and toxicity. Experimental studies on orthotopic tumor models in mice have shown that the personalized design of a CRISPR-Cas13a-based tool against FGFR3-TACC3 fusion genes induces collateral damage in cancer cells and provides a viable strategy for precision tumor therapy (32).

Vaccines. Autologous tumor-pulsed dendritic cell vaccination in conjunction with toll-like receptor agonists was shown to be safe as adjuvant therapy in newly diagnosed and recurrent glioblastoma patients. Results of a clinical study suggest that the mesenchymal gene expression profile may identify an immunogenic subgroup of glioblastoma that may be more responsive to immune-based therapy (26).

A phase II, multicenter trial for assessing the immunogenicity of an epidermal growth factor receptor variant III (EGFRvIII)-targeted peptide vaccine showed that progression-free 6-month survival rate after vaccination was 67%, but at recurrence 82% of patients had lost EGFRvIII expression (28).

Nanobiotechnology for improving delivery of gene therapy. Nanobiotechnology, particularly by use of nanoparticles, is making a significant contribution to the improvement of drug delivery in cancer, and some of these technologies can be applied to gene therapy of glioblastoma (14). A nanoparticle preparation using low molecular weight polyethylenimine, modified with myristic acid and complexed with DNA, has been used successfully for targeted delivery of gene therapy for glioblastoma (20).