Against the Resilience of High-Grade Gliomas: Gene Therapies (Part II)

Introduction: High-grade gliomas (HGGs) still have a high rate of recurrence and lethality. Gene therapies were projected to overcome the therapeutic resilience of HGGs, due to the intrinsic genetic heterogenicity and immune evasion pathways. The present literature review strives to provide an updated overview of the novel gene therapies for HGGs treatment, highlighting evidence from clinical trials, molecular mechanisms, and future perspectives. Methods: An extensive literature review was conducted through PubMed/Medline and ClinicalTrials.gov databases, using the keywords “high-grade glioma,” “glioblastoma,” and “malignant brain tumor”, combined with “gene therapy,” “oncolytic viruses,” “suicide gene therapies,” “tumor suppressor genes,” “immunomodulatory genes,” and “gene target therapies”. Only articles in English and published in the last 15 years were chosen, further screened based on best relevance. Data were analyzed and described according to the PRISMA guidelines. Results: Viruses were the most vehicles employed for their feasibility and transduction efficiency. Apart from liposomes, other viral vehicles remain largely still experimental. Oncolytic viruses and suicide gene therapies proved great results in phase I, II preclinical, and clinical trials. Tumor suppressor, immunomodulatory, and target genes were widely tested, showing encouraging results especially for recurrent HGGs. Conclusions: Oncolytic virotherapy and suicide genes strategies are valuable second-line treatment options for relapsing HGGs. Immunomodulatory approaches, tumor suppressor, and target genes therapies may implement and upgrade standard chemoradiotherapy. Future research aims to improve safety profile and prolonging therapeutic effectiveness. Further clinical trials are needed to assess the efficacy of gene-based therapies.


Introduction
High-grade gliomas (HGGs) are deadly brain tumors accounting for 70% of all central nervous system neoplasms [1][2][3], and the optimization of their management is among the most demanding challenging of the modern neuro-oncology. The reasons for their resilience toward treatment strategies depend on the high cell turnover, pathological neoangiogenesis, and genetic landscape heterogenicity [4][5][6][7][8][9][10][11]. Established guidelines include gross total surgical resection followed by adjuvant chemoradiotherapy [12,13]. In the effort to improve the prognosis of these tumors, characterized by a median survival of only 12-15 months [14][15][16][17], those treatment options considered as "standard of care" have been recently augmented with newer tailored and immune-based technologies. Recent advances in genetic, nanotechnologies, biotechnologies, and translational medicine provided the means for the development of more sophisticated approaches, including gene therapies which have polarized growing attention during the last few years [18][19][20][21][22][23][24][25][26].
blood-brain barrier and cross the tumor cell membrane via endocytosis. Liposomes are lipid vesicles which load electrostatically DNA and RNA plasmid and transfer genes to target cells. Polymers are macromolecules which directly bind DNA and include nucleotides into the tumor cells genome. The polyethyleneimine (PEI), a linear polymer, was widely tested, frequently combined with polyethylene glycol (PEG) or β-cyclodextrin to improve biodistribution and increasing tumor targeting [38][39][40][41]. The polymer polyamidoamine (PAMAM) was also employed in delivering therapeutic genes to glioma cells [42,43]. Moreover, iron oxide nanoparticles (SPIONs), enriched with PEG or PEI, were used as genetic carriers, allowing them to be displayed in magnetic resonance imaging [44,45]. Table 2 presents a comparison between viral and non-viral carriers.

Classification of Gene Therapies for High-Grade Glioma
Gene therapies can be categorized based on molecular mechanisms, carriers involved, and therapeutic gene transferred. The most promising strategies include the replicationcompetent oncolytic viruses (OVs), suicide gene therapy, tumor suppressor gene delivery, immunomodulatory strategies, and gene target therapies. Table 3 reports the classification of gene therapies for HGGs.
The oHSV1716, the first generation oHSV, was devoid of both γ34.5 copies. oHSV1716 was tested in several clinical trials, showing good results as an adjuvant agent for HGGs treatment. In 2000, Rampling et al. evaluated the toxicity of oHSV1716 after intratumoral inoculation. They treated nine patients affected by relapsing HGGs showing a good safety profile [51]. In 2002, Papanastassiou et al. administered 1 × 10 5 plaque-forming units (PFUs) of oHSV1716 to 12 patients with recurrent HGGs and, 9 days after the inoculation, tumors were surgically removed. Histological findings demonstrated the active intratumoral viral replication [52]. oHSV1716 was also employed in phase II clinical trial for HGGs treatment, combined with dexamethasone and surgery (#NCT02031965). Despite the good tolerance, the major weakness of this strategy lies in the deletion of γ34.5, which reduces viral activity and efficacy [53]. The oHSVG207, deleted γ34.5 and inactivated ICP6, was employed in a phase I clinical trial which reported radiological evidence of antitumor activity in 21 patients and an excellent safety profile (dose 3 × 10 9 PFU) [54]. Nine years later, the same group tested the injection of oHSVG207 directly in surgical cavities after surgery, as adjuvant therapy. Histology confirms viral replication activity and radiologic evidence proved the antitumor activity [55]. Several phases I, and I/II clinical trials employed the HSVG207, locally administered, as a single agent or in combination with radiation therapy. Results showed few side effects, a synergic effect with concurrent radiotherapy, but the efficacy is still limited (#NCT00028158, # NCT03911388, #NCT00157703, # NCT02457845).

CRAd
CRAds are non-enveloped DNA adenovirus engineered by the removal of E1A-B genes, which inhibit the binding to the retinoblastoma protein (pRB) and p53, respectively, and block infected cell apoptosis.
The ONYX-015, modified with deletion of E1B genes, selectively targets tumor cells with aberrant p53 pathways [56]. A phase I clinical study employed the ONYX-015 with a dose-escalation protocol. It was injected into the surgical cavity after removal of 24 HGGs (dose from 1 × 10 7 to 1 × 10 10 PFUs). No side effects were registered, but the progressionfree survival (PFS) rate was only 46 days, and overall survival (OS) of 6 months [57]. A phase I clinical trial evaluated the combination of ONYX-015 with cisplatin and fluorouracil, as adjuvant therapy after surgical removal (#NCT00006106). The study showed good tolerance to the OVs, but the treatment efficacy was still not significant. DNX-2401 (Ad5Delta24) deleted in the E1A gene, selectively target glioma cells harboring pRb pathways mutations [58]. In 2018, Lang and colleagues treated 37 patients with relapsed HGGs with intratumoral injection of DNX-2401. Patients were stratified into two groups, the first received a single dose, the second was treated by resection followed by the inoculation of DNX-2401 in the surgical cavity. The median OS was of 9.5 months and 13.0 months for the group 1 and 2, respectively (#NCT00805376) [59]. Several phase I trials tested the intratumoral inoculation of DNX-2401 with temozolomide (#NCT01956734), interferon-γ (INFγ) (#NCT02197169). In 2017, at the American Society of Clinical Oncology (ASCO) Annual Meeting I, Lang and colleagues presented the results of their clinical study (#NCT02197169) on 27 enrolled patients affected by recurrent HGGs, of which nine were treated with DNX-2401 as monotherapy and 18 with DNX-2401 and INFγ. The 12-month OS was 33% and 18-month was OS was 22% in both groups, independently from the type of treatment.
Despite encouraging results in volumetric tumor reduction with single DNX-2401/INFγ administration, no significant difference in survival was reported between the two groups [60].
An active phase II trial is studying the combination of intratumoral DNX-2401 and adjuvant systemic pembrolizumab for HGGs treatment of 49 patients with malignant brain tumors (#NCT02798406). An ongoing phase I trial is evaluating the efficacy of DNX-2401 after conventional surgery (#NCT03896568).
DNX-2440, the mutant variant of DNX-2401, was engineered with the insertion of the OX40 ligand gene. The OX40, expressed on glioma cells, boosts the antitumoral immune response. DNX-2440 is currently under evaluation for HGGs treatment (#NCT03714334).

MV
MV, an enveloped RNA virus, exhibits the mutated hemagglutinin envelope glycoprotein H, also known as Edmonston strain, which selectively targets the CD46 on glioma cells [61,62]. MV was engineered to express the circulating carcinogenic embryonic antigen (CEA), useful to assess the virus replication and oncolytic activity [63]. A phase I study tested the toxicity of MV-CEA association and no severe side effects were reported (#NCT00390299) [64]. MV was also designed to express interleukin-13 (IL-13) directed to the IL-13Rα2 receptor on glioblastoma (GBM) cells, or the single-chain antibody versus the vIII variant of epidermal growth factor receptor (EGFRvIII) [65][66][67].

PVS-RIPO
PVS-RIPO is an attenuated Sabin poliovirus engineered by the replacement of the internal ribosomal entry site (IRES) with the IRES from a human rhinovirus, to reduce the viral neuropathogenicity [68][69][70]. The tropism of PVS-RIPO for tumor cells is determined by the poliovirus receptor CD155, expressed on HGGs cells [71,72].
PVS-RIPO was tested for treatment of pediatric (#NCT03043391) and adult recurrent HGGs as monotherapy, in combination with a single-cycle of lomustine (#NCT02986178), or with the anti-PDL1 antibody atezolizumab (NCT03973879). Results from the aforementioned clinical trials showed a sufficient anticancer efficacy, but a low safety profile. Table 4 reports a comprehensive summary of clinical trials on oncolytic virotherapy for HGGs.

Suicide Gene Therapies
The suicide gene strategy is grounded on the viral transfer of "suicide genes" to target cells, which encode for enzymes able to convert prodrug to active compound [73,74]. The inactive prodrug is administered systematically and activated at the tumor site by the suicide enzymes, resulting in oncolytic effect and tumor cell apoptosis [75]. For HGGs treatment, the suicide transgene evaluated in clinical and preclinical studies are as follows: herpes simplex virus thymidine kinase (HSV-TK), cytosine deaminase (CD), and E. coliderived purine nucleoside phosphorylase (PNP) (Figure 3).

HSV-TK
The HSV-TK enzyme catalyzes the monophosphorylation of the ganciclovir/valacyclovir, which is after triphosphorylates and activates intracellular kinases. The active drug blocks the S-phase and arrests the cell circle, leading to inhibition of DNA synthesis and tumor lysis [76][77][78][79].
In 2000, the phase III clinical study piloted by Rainov and colleagues tested the effect of HSV-TK gene therapy for 248 patients with newly diagnosed HGGs. Patients received an intratumoral inoculation of retroviral HSV-TK, followed by standard surgery, radiation therapy, and systemic administration of ganciclovir for 2 weeks. The study group was compared to the control one (conventional surgery and radiotherapy) and not significant differences in PFS and OS were founded [80]. Two recruiting phase I/II clinical trials tested the HSV-TK combined with replication-defective adenoviral vector (ADV/HSV-TK) and administered with valacyclovir (#NCT03603405, #NCT03596086). Results demonstrated the safety of this strategy with promising antitumoral efficacy. In 2000, Sandmair et al. conducted a phase I clinical trial intending to prove the efficacy and transinfection efficiency of replication-defective retrovirus or adenovirus-mediated HSV-TK/ganciclovir. 21 patients with newly diagnosed or recurrent HGGs were recruited, divided in two groups, and treated with retrovirus or adenoviruses. The OS of the adenovirus-mediated strategy was much higher [81]. Germano and colleagues tested the same ADV/HSV-TK strategy in a phase I trial and results showed a PFS of 112 weeks and an OS of 248 weeks [82]. In 2008, the ASPECT phase III clinical trial studied the ADV/HSV-TK for HGGs treatment. Out of 236 patients recruited who underwent surgical resection, 119 were randomized for the inoculation of ADV/HSV-TK locally at the tumor cavity, followed by systemic ganciclovir for two weeks. The PFS rates were 268 days and 308 days, and OS were 452 days and 497 days for the study group compared to the control one, respectively [83]. In 2016, Wheeler et al. enrolled 48 patients, harboring newly diagnosed HGGs, treated with ADV/HSV-TK and postoperative intravenously valacyclovir. This group was compared to the control one, treated with conventional surgery and adjuvant chemoradiotherapy. The PFS rate was 8 months for the study group and 6.5 months for the control one; the OS was 17 versus 13.5 months for the study and the control group, respectively (#NCT00589875). Results demonstrated the greatest effectiveness in the use of AD as the carrier for HSV-TK gene therapy.
An active phase I trial is evaluating a combined innovative approach, exploiting ADV/HSV-TK and the prodrug valacyclovir, associated with a checkpoint inhibitor (nivolumab), chemotherapy (temozolomide), and conventional radiation, to evaluate the safety profile and achievability of this enhanced strategy (#NCT03576612).

CD
The bacterial enzyme CD catalyzes the activation of the prodrug 5-fluorocytosine (5-FC) in oncolytic 5-fluorouracil (5-FU), selectively in glioma cells [28,84,85]. Toca 511, a replication-competent retrovirus, loads the CD and transinfect tumor cells. It promotes the expression of CD, which active the 5-FU, able to irreversibly blocks DNA synthesis and leads to cell apoptosis [86].
From 2016, Cloughesy and his group employed the Toca 511/5-FC in two clinical trials. The first one (#NCT01156584) tested the Toca 511 via stereotactic transcranial injection or intravenously injection [87]. In the subsequent study (#NCT01470794), they administered Toca 511 in the surgical cavity with a subsequent administration of Toca FC antifungal agent. Patients affected by recurrent or progressive HGGs were enrolled, and results showed a good safety profile and a median OS of 12-14 months [86]. Despite these initial encouraging results, Cloughesy et al. 2019 designed a phase III study which reported the therapeutic failure of Toca 511/5-FC, compared to the standard of care, in 271 patients with recurrent HGGs (#NCT02414165).
Moreover, the co-administration of antibiotic therapy, which suppresses the intestinal flora, could overactivated the PNP gene therapy intensifying the prodrug conversion [95,96]. Table 5 reports a comprehensive summary of clinical trials on suicide gene therapies for HGGs.

Tumor Suppressor Gene Therapies
Oncogenesis predicts the loss of the physiological regulatory function of some tumor suppressor genes which control the cell cycle and death. HGGs harbor deletions and mutations of specific tumor suppressors, more frequently the p53, p16, and phosphatase and tensin homologue (PTEN) [97]. The goal of tumor suppressor gene strategies is to transfer antitumoral functional genes to glioma cells, in order to restore normal function ( Figure 4).

p53
The TP53 is the most common suppressor gene which codifies for p53 protein, fundamental in cell replication and apoptosis, found mutated in more than 50% of HGGs, 30% newly diagnosed, and 70% relapsed [98,99]. P53 is involved in angiogenesis inhibition and DNA repairing mechanisms. The most accredited strategy includes the replication-deficient adenovirus in which the E1 gene is replaced by the wild-type p53 and conveyed by a cytomegalovirus promoter (Ad5CMV-p53). E1 deletion makes the virus unable to activate the infectious process, while the CMV promoter increases p53 gene expression [30,100,101]. Ad5CMV-p53 proved to block the glioma cell cycle, inhibit angiogenesis, and induce tumor apoptosis in many preclinical trials. [100,[102][103][104][105][106]. In 1998, Badie et al. tested the efficacy of Ad-mediated p53 gene therapy, combined with radiation, in p53-mutant rat glioma models. Results showed 85% of tumor cell apoptosis in 24 h [107]. Later, Cirielli and his group studied the transfer of AdCMVp53 in human intracranial HGG cells in mice. 100 days after treatment all rats survived [103]. Another preclinical experimental study was conducted, in 2014, by Kim and colleagues. They designed a nanodelivery system able to carry the p53 gene into glioma cells through the blood-brain barrier. They reported a high rate of tumor suppression in GBM xenograft mice [108].
As regards clinical studies, two completed phase I trials employed the Ad5CMV-p53 as neo-and adjuvant therapy for relapsing HGGs. In both studies, patients received a preoperative intratumoral stereotactic inoculation of Ad5CMV-p53, followed by conventional surgery. Afterward, Ad5CMV-p53 was directly injected several times into the tumor cavity walls. Results showed a PFS of 13 weeks and OS of 44 weeks (#NCT00004041, #NCT00004080). A phase I clinical trial tested the efficacy and safety profile of Ad-p53 for HGGs treatment. 15 patients were enrolled and preoperatively treated with a stereotactic injection of Ad-p53 through an implanted catheter. After surgical gross total removal, Ad-p53 was injected several times in the surgical cavity. Treatment demonstrated low toxicity, but still limited efficacy [105]. p16 p16 controls the cell cycle arrest at the G1-S transition, avoiding uncontrolled replication and oncogenesis [109]. Restoration of p16 function, via adenoviral carrier, shown to inhibit glioma growth and locoregional diffusion, also blocking the activity of matrix metalloproteases in the glioma microenvironment [110]. In 1997 Chintala et al. tried to restore in vitro the p16 activity in HGGs cells, through Matrigel-coated transwell inserts and fetal rat-brain aggregates, by recombinant replication-deficient adenovirus. All tests showed a substantial reduction in glioma cell replication activity and a decrease in the expression of tumor microenvironment enzymes [110]. In 2000, Hung et al. tested the injection of a retrovirus, encoding the human p16 gene, in 10 rat HGG models. Results demonstrated the inhibition of glioma cell growth [111]. In 2003, Hama and colleagues examined the interaction between p16 and radiation-induced cell death. p16-null human glioma cell lines were induced to phase G1 of the cell cycle, by means of the adenovirus-mediated p16 gene. Data suggested that p16 expression is related to tumor radiosensitive via mechanisms of abnormal nucleation in HGG cells [112]. It is relevant that the effectiveness of the p16 gene strategy is only possible if the pRB activity is preserved [113]. PTEN PTEN was found mutated in about 45% of HGGs and is involved in tumor microenvironment maintenance and proangiogenetic pathways [114,115]. Adenoviral delivery of the PTEN gene has been demonstrated to inhibit glioma proliferation and promote oncolysis [116][117][118][119]. In 1998, Cheney and colleagues designed a replication-defective adenovirus to transfer the PTEN gene in nude mice tumors. Results supported the tumor suppression activity of PTEN expression in HGGs [117]. Furthermore, as demonstrated by Davies and his group, PTEN inhibits Akt kinase activity, resulting in glioma cell death [116]. In vivo experiments, conducted by Abe and Lu, proved the adenoviral expression of PTEN able to block the angiogenetic processes and tumor proliferation in glioma cells [118,119]. In 2011, Inaba et al. demonstrated that the transmission of the PTEN gene into glioma cells, by an adenoviral vector, increased the tumor sensitivity to temozolomide and radiotherapy [120]. Table 6 presents the clinical trials on tumor suppressor gene therapies for HGGs.

Immunomodulatory Gene Therapies
HGGs resistance to standard treatments resides in the immune-escape tumor mechanisms and immunosuppressor tumor microenvironment. Immunomodulatory gene strategies are designed to implement the immune response against glioma by means of delivery of genes which encode for immunostimulatory cytokines and IFNβ/γ [48,85,121,122]. ( Figure 5).

IFN-β/γ
Adenoviral-IFN-β gene delivery was tested in many preclinical and clinical trials [123][124][125][126][127][128][129]. In 2001, Qin et al. employed adenovirus expressing IFN-β in both in vivo and ex vivo human glioma xenografts in mouses. Results showed a potential antitumoral activity with the activation of NK cells and macrophages [123]. In phase I clinical study the drug was stereotactically inoculated into glioma before surgery. Results supported the activation of immune cascade and T and NK cells recruitment in the tumor microenvironment (#NCT00031083).
Nanoparticles and liposomes were also employed for INF-β transfer. From 1999, Natsume et al. conducted in vivo experiments using murine INF-β gene directly injected via liposomes in brain gliomas in mice. Results showed in 40% of cases the total inhibition of glioma growth with a strong antitumoral T lymphocyte infiltration [124]. Moreover, the same group carried on another study with the aim to deepen the role of tumor-specific lymphocytes. Mice were re-treated with a subcutaneous or intracranial injection of glioma cells and no tumor evidence was found 50 days later. This data proved that, in addition to the anticancer effects of INF-β, the local immune response has a role in long-term antitumor efficacy [125]. In 2004, Yoshida and colleagues tested the treatment with liposome/INF-β in a clinical trial, involving five patients with HGG. Four of these experienced a total or partial response to treatment with radiological evidence of volumetric glioma reduction by 50% and concomitant low toxicity [126]. Histological findings reported a high level of immune activation, also [127]. IFN-γ has the role of reducing cancer cell proliferation and interaction with the extracellular matrix [128]. IFN-γ as monotherapy was proved to be less effective, so combination protocols are under evaluation [129]. In 2002, Ehtesham and colleagues tested the efficacy of adenoviral-mediated IFN-γ and TNF-α gene transfer in HGGs cells. They proved the antitumoral efficacy of this treatment in mice models, also highlighting local increased recruitment of lymphocytes [130].
Earlier phase studies employed non-replicating adenoviruses and HSV for delivery of IL12 to malignant glioma cells [135][136][137]. In 2012, Chiu et al. tested the intracranial injection of recombinant adeno-associated virus expressing IL12 gene (rAAV2/IL12) in the glioma mice model [135]. Later that year, Markert et al. studied the efficacy and security of γ34.5-deleted HSV1, encoding the IL12 gene, for malignant glioma treatment in rats [137]. Results of both preclinical studies showed tumor cell apoptosis, infiltration of active microglia cells, good safety profile, and strong local immune reaction.
Two recruiting phase I clinical trials tested the inducible adenoviral vector engineered to express IL12 (Ad-RTS-hIL12) with the oral vedelimex (an IL12 immunotherapeutic activator) for adult and pediatric gliomas (#NCT02026271, #NCT03330197). These studies revealed an intense upregulation of antitumor infiltrating lymphocytes.
IL4, secreted by lymphocytes, upregulates the immune cascade and B and T cells enrollment [138][139][140]. In clinical and preclinical models, the IL4 gene was virally transduced as an immunomodulatory agent for HGGs treatment. Yu and colleagues experienced the antitumoral activity of IL4, which was administered to 12 nude mice affected by gliomas. The treatment resulted in significant inhibition of glioma cell growth [138]. Okada and his group conducted clinical studies to test a vaccine constituted of IL-4-HSV-TK gene-modified autologous glioma cells, followed by systemic ganciclovir administration. Patients enrolled harbored recurrent/refractory supratentorial malignant glioma. The aim was to evaluate the safety profile, clinical efficacy, and immune response. Results reported good antitumoral activity and a strong antitumoral peripheral immunization [140].
Moreover, in 2005, Colombo et al. tested the intratumoral injection of retroviruses expressing both HSV-TK and IL2 genes, followed by intravenous ganciclovir, for treatment of 12 patients with recurrent HGGs. Few side effects were reported, and the 12 months PFS and OS was of 14% and 35%, respectively [141]. Table 7 summarizes the clinical trials on immunomodulatory gene therapies for HGGs.

Gene Target Therapies
The identification of specific molecular markers of HGGs allowed the development of gene target therapies, designed to directly bind specific tumor antigens, with the aim to irreversibly block oncogenic pathways. Most of these strategies are still experimental and no active clinical trials are underway ( Figure 6). EGFRvIII EGFRvIII variant, found in 30% of HGGs, is involved in mechanisms of oncogenesis and tumor progression [142,143]. Viral vectors and nanoparticles were engineered to transfer antisense or small interfering RNA directed specifically against the TK domain of glioma EGFRvIII. Several studies demonstrated a significant tumor volume reduction after treatment [144][145][146][147]. In 2006, Kang and colleagues projected antisense-RNA and small interference RNA (siRNA) expressing antisense EGFR genes which selectively bind the TK domain of EGFRvIII. After inoculation, glioma cell growth was amply reduced in vitro and in vivo models [145]. Shir and Levitzki tested antisense-RNA, transduced via viral and non-viral carriers, able to activate dependent protein kinase PKR. PKR induces cancer cell death targeting EGFRvIII in intracranial glioma xenografts [144]. In 2005, Padfield et al. confirmed the role of adjuvant miRNA therapies as promising strategies in glioma treatment. In fact, miR-7 showed high efficacy in blocking directly the EGFR pathways and downregulate MAPK/PI3K/Akt signaling, resulting in tumor cell apoptosis [148]. The cyclodextrin-modified dendritic polyamine complexes (DexAMs) were employed in the delivery of EGFRvIII siRNA and showed promising results in malignant glioma cells, also in combination with erlotinib [149].

VEGF/VEGFR
The vascular endothelial growth factor (VEGF) was found overexpressed in many malignant tumors. Adenoviral vector, loaded with anti-sense cDNA VEGF (Ad5CMV-αVEGF), was subcutaneously injected in nude mice previously infected with human glioma cells, resulting in inhibition of tumor spreading [150]. In xenografts, the direct intratumoral inoculation of PEI/VEGF siRNA showed an antiangiogenetic strong effect [151]. In 2007, Yoo and colleagues tested an oncolytic adenovirus (Ad)-based short hairpin RNA (shRNA) expression system (Ad-DeltaB7-shVEGF) directed versus the VEGF. Ad-DeltaB7-shVEGF showed high antiangiogenetic activity in the matrigel plug assay, and greater bioavailability compared to replication-incompetent adenoviruses [152]. The oncolytic adenovirus Ad-DeltaB7, was also employed in a preclinical study by Kang et al. in 2008. They designed an adenovirus able to express the transcriptional repressor Cys2-His2 zinc-finger proteins, F435-KOX, directed versus the VEGF promoter (Ad-DeltaB7-KOX). Ad-DeltaB7-KOX demonstrated high antitumor activity in a human xenografted glioma model [153].
In addition, the strategy of antagonizing the VEGF receptor (VEGFR) has proven to be effective. In 2004, Heidenreich et al. tried to inhibit the VEGFR-2 signaling pathway through transfers of a mutant-VEGFR via a retrovirus. The lack of intracellular tyrosine kinase domain in the engineered mutant-VEGFR resulted in inhibition of angiogenesis and progression in the xenografted glioma model [154]. A further study tested the coinfection of HGGs with adenovirus expressing VEGFR and an oncolytic virus dl922/947. This combined treatment resulted in more effectiveness than monotherapy [155].

Discussion
The present literature review aims to outline the up-to-date gene therapies for HGGs treatment, focusing especially on the molecular mechanisms, vectors, and therapeutic genes employed.
The rationale of gene strategies lies in the reprogramming of the glioma genome, intending to induce oncolysis or the expression of the antitumoral mediators. Manufactured genes are transferred to target cells through specific carriers, engineered to selectively bind cancer cells. Viral carriers were the first vehicle used, because of their specific neurotropism, and proved their gene delivery efficacy [156]. The main limitations are the short bioavailability of viral carrier and the negligible permanence of the virus at the tumor side [157].
Combined complex of viral vehicles with immunomodulatory agents is currently under investigation to enhance the duration of the therapeutic effect [158]. Among nonviral vehicles, only the liposomes were approved to be tested in clinical trials showing low toxicity and a high biodistribution level. All other nanoparticles are still in earlier phase studies.
If viral carriers are the most suitable vehicles for gene therapy, also the oncolytic virotherapy proved to be a valuable option. OVs act as a genetic payload which directly lyses tumor cells. The apoptosis of cancer cells promotes the release of tumor-associated antigens (TAA) in the tumor microenvironment. TAA are recognized by immune cell, resulting in the burst of immune cascade [159][160][161].
Suicide gene therapy is also an excellent potential resource for HGGs treatment. This approach is based on the assumption that suicide enzymes are not expressed in healthy cells. Therefore, the intravenous administration of prodrug and the intratumoral inoculation of virus-mediated suicide genes allow restricting the therapeutic effect only to glioma cells, while reducing systemic side effects [76,82,162]. Another considerable advantage of suicide gene therapy lies in the "bystander effect", namely the ability to share transduced genes and death signals to the neighboring cells through gap junctions [163][164][165].
In the era of translational medicine, the identification of specific tumor markers and genes involved in oncogenesis, above all EGFR, VEGF, TP53, and pRB pathways, offers new insights to design the target gene strategies and tumor suppressor gene therapies [22]. These lasts are based on the rearrangement of the glioma genome with the aim of restoring lost oncosuppressive functions. Delivery of tumor suppressor genes can be exploited as a combined approach, resulting in sensibilization of glioma cells to chemoradiotherapy [106,166].
Despite good assumptions, the intrinsic heterogeneity of HGGs, the multitude of mutations, and immune evasion mechanisms constitute the major limits of all these strategies. The immunomodulatory gene therapies, including INF e cytokines delivery, were projected precisely to modulate the immunosuppressive tumor microenvironment, meanwhile increasing the oncolytic gene therapy efficacy [134].
In accordance with the data outlined in the present review, the most accredited strategies are oncolytic virotherapy (26 trials), suicide gene strategies (18 trials), and immunomodulatory gene therapies (14 trials). Results reported overall excellent effectiveness, especially as adjuvant therapies with local injection after surgery. No significant toxicity was reported and, when preoperatively administered, a role in reducing tumor volume was also demonstrated.
The future perspectives of the HGGs treatment are directed toward the progressive integration of standard chemoradiotherapy with immune-boosting strategies and new tailored gene therapies.

Conclusions
Gene therapies are projected with the aim to edit the glioma genome and overcome the therapeutic resilience of HGGs. The oncolytic virotherapy, suicide genes and immunomodulatory strategies, tumor suppressor, and target genes therapies were widely tested in clinical trials, remaining mostly still experimental approaches.
Oncolytic viruses oHSVs and CRAds were proven to be safe and feasible. HSV-TK and CD suicide genes revealed a promising potential in several preclinical studies. Although they are not included in the first-line treatment protocol for newly diagnosed HGGs, gene therapies represent a valuable option as second-line adjuvant therapy for refractory GBM.
Future perspectives provide for the development of new administration vehicles, optimize biodistribution and selectivity. Further clinical trials are essential to implement standard protocols with gene innovative strategies in therapeutic synergy.