A Promising mRNA Vaccine for Treating Glioblastoma Brain Cancer

Wilms tumor 1 (WT1) is identified as a GBM TAA that is related to several solid tumors and leukemias, as well as promoting carcinogenesis. Immunotherapy against WT1 has been beneficial in treating recurrent GBM, as most GBM specimens overexpress WT1 [44]. The WT1 protein has been designated by the National Cancer Institute (NCI) as the main validated target for the development of cancer vaccines [45, 46]. Peptide vaccines against WT1 have been used in several clinical trials, and the results have shown a reduction in detectable WT1 transcript levels and therapeutic effects [47]. WT1 expression can be seen at low levels in normal cells, but it is increased in certain malignancies, including GBM [48]. This suggests that responses from anti‐WT1 T cells, especially those produced by high‐avidity T cells, could eventually become tolerogenic [49]. Researchers have recently proposed that one way to help leukemia patients overcome immunological tolerance is by the production of low‐avidity CTLs. On the other hand, high‐avidity T cells are more efficient in eliminating CML than low‐avidity TCRs for WT1‐MHC. While highly immunogenic, WT1 poses a challenge because it is also expressed at low levels in healthy tissues (e.g., kidneys, hematopoietic precursors), leading to central tolerance—a process that deletes or dampens high‐affinity T cells reactive to self‐antigens like WT1 [48, 49, 50, 51, 52, 53, 54, 55].

Survivin is an apoptosis‐inhibitory protein that is associated with a poor prognosis and a low overall survival rate in CNS malignancies, including gliomas and other tumors [56]. Survivin has the ability to interfere with apoptosis, boost cancer stem cell proliferation, increase tumor cell invasion, and contribute to chemotherapy resistance in cancer cells [57]. Strong tumor antigen survivin binds to MHC class I molecules on cancer cell surfaces to provide T lymphocytes with a stimulatory ligand; that is, these modified peptides also stimulate higher‐avidity T‐cell responses, overcoming preexisting tolerance and decreasing T‐cell exhaustion by enhancing tumor clearance and synergizing with checkpoint inhibitors [58, 59]. Vaccination boosts this process by expanding low‐affinity survivin‐reactive T cells and converting them into potent, durable effectors—effectively transforming modest baseline immunity into robust antitumor activity, and given the modest immunogenicity of wild‐type survivin, the vaccination may strengthen preexisting immunity [60, 61, 62]. Compared to the matching wild‐type survivin peptide, SurVaxM (SVN53–67/M57‐KLH), a recently developed peptide vaccine made from the human survivin protein sequence (containing amino acids 53–67), elicited a stronger antitumor immune response against tumor cells [60]. In a phase IIa trial, the safety, immunologic effects, and survival of newly diagnosed GBM (nGBM) patients receiving adjuvant TMZ plus SurVaxM after surgery and chemoradiation were assessed. SurVaxM was shown to be well tolerated and safe [63]. For nGBM, the combination offers a viable treatment. When treating nGBM patients in this way, progression‐free survival (PFS) could be a suitable overall survival (OS) substitute. This is based on the strong correlation observed between prolonged PFS and improved OS in the study population. Additionally, PFS offers an earlier and less confounded measure of therapeutic efficacy in aggressive cancers like GBM, where OS can be affected by postprogression treatments and extended follow‐up times [63, 64].

The metabolic enzymes known as Isocitrate dehydrogenase 1/2 (IDH1/2) are encoded by the IDH1 and IDH2 genes and located on chromosomes 2 and 15, respectively [65]. IDH has been considered a potential TSA since mutation in it mainly occurs in tumor cells, not in normal human cells [66]. Mutations in IDH1, particularly the R132H variant, are found in approximately 80% of low‐grade gliomas (LGG) and are a hallmark of secondary GBM, but are rare in primary GBM. An IDH1 gene mutation suggests that the GBM is a low‐grade secondary glioma [67]. Numerous human malignancies, including gliomas, have been found to have mutations in the genes encoding these enzymes [68]. These mutations cause oncometabolite 2‐hydroxyglutarate (2HG) synthesis, genomic instability, and neoplastic transformation, which are consistently distributed in the catalytically active areas of these enzymes [69]. Schumacher et al. initially attempted to create vaccinations against mutant IDH. Mice were injected with a peptide consisting of twenty amino acids, which covered a portion of the altered catalytic pocket of the IDH enzyme. Mice were injected with a 20‐amino‐acid peptide derived from the mutated catalytic pocket of IDH (e.g., R132H), which drives cancer via D‐2HG production. Antigen‐presenting cells (APCs) internalized and processed the peptide, presenting it on MHC molecules to activate tumor‐specific CD8+/CD4+ T cells. By targeting the mutation‐induced neoepitope—absent in wild‐type IDH—these T cells bypassed immune tolerance and selectively killed IDH‐mutant tumor cells, reducing D‐2HG levels. The peptide's length likely required in vivo trimming to MHC‐compatible epitopes (8–12‐mers), with adjuvants potentially enhancing APC priming. This approach mirrors clinical vaccine strategies against IDH‐mutant gliomas, where mutant‐IDH peptides induce cytotoxic responses without attacking normal tissues. T‐helper cells specific to this mutation demonstrated a robust immunological response, according to the findings [70]. Pellegatta et al. developed an immunologically viable glioma model of the R132H mutation. Their results revealed that peptide vaccination may delay otherwise fatal intracranial glioma by about a month and, in a fraction of cases, cure it [71]. The initial phase 1 trial in humans of the IDH1 peptide vaccine was NOA‐16 (NCT02454634). Patients with grade 3 and 4 IDH1R132H astrocytoma who had just been diagnosed were enrolled [72, 73].

A distinct subset of tumor cells (about 33%) expresses the permanently active wild‐type tyrosine kinase mutant known as the epidermal growth factor receptor III variant (EGFRvIII). It is a viable target for the creation of customized immunotherapies due to its level of specificity [74]. Mechanistically, EGFRvIII promotes glioma invasion by activating HIF‐1a in a STAT3‐dependent manner and by overexpressing the antiapoptotic protein Bcl‐xL [75]. EGFRvIII exhibits persistent ligand‐independent activity even in the absence of a functioning ectodomain, which would otherwise confer ligand specificity. In addition to increased carcinogenesis, dysregulated EGFR signaling has been linked to metastasis, resistance to chemotherapy and radiotherapy, and metastasis [76].

This oncogenic protein, EGFRvIII, is a very appealing tumor‐specific target for the development of a GBM vaccination since it promotes proliferation [77]. The shortened extracellular domain produces a unique tumor neoantigen that is specific to GBM cells in both mice and people [78]. This prompted the creation of the peptide‐based vaccination rindopepimut (CDX‐110). Rindopepimut is a peptide vaccination that kills GBM cells that express EGFRvIII by including 14 amino acids from the EGFRvIII fusion site [77, 78, 79]. Three discrete reasons emerged: first, strong immunogenicity; second, low toxicity that is, KLH has an excellent safety profile in humans, with only mild side effects (e.g., transient fever or injection‐site reactions); and third, wide distribution, that is, KLH is commercially available, standardized, and compatible with clinical‐grade manufacturing, facilitating scalable vaccine production of keyhole limpet hemocyanin (KLH). This vaccine is frequently used in conjunction with it. In vivo, KLH stimulates immune responses that are dependent on T and B cells, hence fostering antigenic immune responses [80]. In fact, KLH enhances the immunogenicity of EGFRvIII‐targeted vaccines (e.g., rindopepimut) by acting as a potent carrier protein. As a foreign T‐cell‐dependent antigen, KLH stimulates robust CD4+ T‐cell help, which in turn drives both antibody production against EGFRvIII and cytotoxic CD8+ T‐cell responses to eliminate EGFRvIII‐expressing tumor cells. Its low toxicity, commercial availability, and ability to break immune tolerance make KLH an ideal partner for overcoming the weak intrinsic immunogenicity of tumor‐specific peptides like EGFRvIII. However, while KLH boosts antigen‐specific immunity, its efficacy in clinical settings may still require combination with other therapies to counteract tumor immune evasion mechanisms.

Another study by Sampson et al. explored the mechanisms underlying disease progression after prolonged survival with EGFRvIII‐targeted vaccination. EGFRvIII, a tumor‐specific mutation, is expressed in a subset of GBM and offers a target for vaccine‐based therapies. Patients achieving extended PFS following EGFRvIII peptide vaccination were analyzed for EGFRvIII expression and immune profiles. Tumor biopsies and blood samples were evaluated to identify resistance mechanisms. The outcome illustrated that tumor recurrence was associated with the loss of EGFRvIII expression and immune evasion strategies, such as T‐cell exhaustion. These findings emphasize the need for combinatorial approaches to overcome immune escape [81]. In the same vein, the ACT IV trial investigated the efficacy of rindopepimut, an EGFRvIII‐targeting peptide vaccine, combined with TMZ. This randomized, double‐blind, phase III trial enrolled newly diagnosed EGFRvIII‐expressing GBM patients. Participants received rindopepimut or placebo alongside standard chemoradiotherapy. Outcomes included PFS and OS. The most important clinically relevant finding was the vaccine generated robust immune responses but did not improve OS or PFS compared to the control group [82]. Although the immunotherapeutic targeting of EGFRvIII may effectively eradicate neoplastic cells it might be impeded by concurrent myelosuppressive chemotherapy, such as TMZ, that offers a survival advantage in GBM. A phase II, multicenter study was conducted to evaluate the immunogenicity of an investigational EGFRvIII‐targeted peptide vaccine in patients with GBM receiving therapy with numerous cycles of standard‐dose or dose‐intensified TMZ. The results indicated that EGFRvIII‐specific immune responses emerged in all patients receiving either treatment; however, the DI TMZ regimen elicited a more pronounced amplitude of humoral and delayed‐type hypersensitivity reactions. Vaccination targeting EGFRvIII elicits immune responses in patients despite lymphopenia generated by TMZ therapy and eradicates EGFRvIII‐expressing tumor cells without causing autoimmunity [83, 84, 85, 86].

The telomerase reverse transcriptase (TERT) protein is an important enzyme complex in eukaryotic species that helps preserve and lengthen telomeres, enhancing the possibility of cell division [87]. Because TERT mutations reactivate the telomerase enzyme and immortalize malignant cells, they have been associated with the development of cancer, especially in the promoter region of the gene [88, 89]. TERT promoter mutations are common in GBM and are linked to varying prognoses based on additional genetic variables. For instance, in GBM with IDH mutations, the presence of a TERT promoter mutation is particularly linked to a better prognosis. Nonetheless, patients with mutations in the TERT promoter and unmethylated MGMT promoters typically have the worst prognoses [90, 91, 92]. To potentially treat TERT‐related tumors, researchers are presently investigating therapeutics that target TERT activity, such as vaccinations and small‐molecule inhibitors [93]. Amen et al. observed that cancer‐cell‐specific TERT inhibition via GABPB1L decrease leads to short‐term growth inhibitory effects and a compromised DNA damage response, which significantly raises the susceptibility of GBM tumors to frontline chemotherapy. Additionally, their findings support the use of TMZ chemotherapy in conjunction with GABPB1L peptide suppression as a potentially effective treatment for GBM [93]. In another study, researchers paired this strategy with the use of survivin‐mRNA and hTERT‐transfected DCs to aid in monitoring induced immunity and may serve as therapeutic targets. Their findings imply that it is possible to establish autologous cancer stem cell cultures under good manufacturing procedures (GMP). It is clear that immunization against cancer stem cells can increase the length of time without recurrence and is safe and well‐tolerated [94, 95, 96, 97, 98].

A novel vaccination strategy called the heat Shock Protein (HSP) vaccine works by using proteins with molecular chaperone activity to stop biological macromolecules that are impacted by ions, oxygen, and temperature from denaturing [99, 100]. HSPs may be useful in tumor tissues where aberrant proteins are abundant because they can reassemble misfolded proteins and direct the breakdown of aberrant ones. Studies have revealed that HSPs such as HSP96 can trigger potent immune responses and are strongly linked to gliomas. Before brain tumor‐derived HSP96 is internalized and presented as an HSP96‐chaperoned tumor antigen on class I and class II MHC, the HSP96 complex is first attached to CD91 on antigen‐presenting cells (APCs), resulting in robust immunogenicity. The advantage of the HSP vaccine over other tumor vaccines is that it better induces CD4⁺ and CD8⁺ T‐cell immune responses due to its highly precise interaction between HSP96 and APCs [101].

The HSP vaccine has the benefit of being selective in how it interacts with antigen‐presenting cells, which stimulates strong T‐cell immune responses [101]. The vaccination did not cause any major adverse effects, and the most common adverse event was minor injection site erythema. Similar to other studies, the single‐arm phase II trial that followed included 41 patients with recurrent GBM and revealed that the mOS of the HSP96 group was 42.6 weeks following vaccination without experiencing any significant side effects (Table 1) [102, 103, 104].

Personalized peptide vaccination (PPV), appropriate peptide antigens for vaccination are screened and selected from a list of vaccine candidates in each patient, based on preexisting host immunity [105]. A phase III trial of PPV for HLA‐A24+ recurrent GBM found that the trial met neither the primary nor secondary endpoints. Unfavorable factors for the mOS of 58 PPV patients compared to 30 placebo patients included SART2‐93 peptide selection, ≥ 70 years old, > 70 kg body weight, and performance status [106]. The mOS for PPV patients without SART2‐93 selection plus one of these three favorable factors was significantly longer than that for the corresponding placebo patients. Preexisting immunity against all 12 warehouse peptides was significantly depressed in patients with SART2‐93 selection compared to those without. Biomarkers correlating for favorable OS included a lower percentage of CD11b + CD14 + HLA‐DR low immunosuppressive monocytes and a higher percentage of CD4 + CD45RA‐activated T cells. Another phase III study utilizing personalized peptide vaccine (PPV) for HLA‐A24+ recurrent GBM did not achieve the main or secondary goals. Detrimental variables for the mOS of 58 patients receiving PPV compared to 30 patients receiving placebo were SART2‐93 peptide selection, age ≥ 70 years, and body weight > 70 kg. The mOS for PPV patients without SART2‐93 selection, along with one of three favorable characteristics, was considerably longer than that of the placebo. Preexisting immunity against all 12 peptides was considerably decreased in individuals with SART2‐93 selection compared to those without. Biomarkers associated with improved mOS included a reduced proportion of CD11b + CD14 + HLA‐DR reduced immunosuppressive monocytes and an elevated proportion of CD4 +CD45RA‐activated T cells. Taken together, the vaccine elicited tumor‐specific immune responses, with prolonged PFS in responders, highlighting its potential in tailored immunotherapy [106, 107, 108, 109].

The weak pathogenic (infectious) viruses with genetic modification are made oncolytic viruses, which increase anticancer effects despite ceasing to destroy normal cells. The oncolytic effect results from direct lysis of the cancer cells due to the virus self‐replication in host cancer cells [110, 111]. In recent years, modified viruses have become more widely used, opening the door for their application in oncotherapy [112]. Viruses can activate the immune system by inducing innate responses and enhancing specific responses to tumor antigens, thereby significantly increasing the efficacy of vaccinations [113].

In clinical trials enrolling patients with glioma, Parvoviridae, Picornaviridae, Retroviridae, Paramyxoviridae, Adenoviridae, Reoviridae, and Herpesviridae are the most frequently utilized viruses. However, the most important problem is that most viruses, apart from Parvoviridae, cannot pass the BBB due to their lack of tropism; thus, issues have to be solved in this type of therapy [114]. Additionally, the expansion of the extracellular matrix is linked to a desmoplastic condition, one of the changes tumor microenvironments (TME) endure [115]. Herpesviruses rely on the tumor's surrounding microenvironment for entry, particularly due to the elevated expression of integrins in glioma cells, which facilitates their cellular access. However, the treatment of GBM remains highly challenging because of the brain's immune‐privileged status and the strongly immunosuppressive tumor microenvironment [116]. To overcome these obstacles, research into novel delivery systems that can either penetrate the BBB or inoculate directly into the tumor site is just as important as looking for therapies that stimulate an immune response [117]. The subsequent properties of viruses and the research conducted on them position them as viable candidates for oncolytic viral treatment in GBM.

Adenoviruses (Adv) are non‐enveloped, double‐stranded DNA viruses that have an icosahedral capsid. At least three oncolytic viruses have been developed for the treatment of GBM, making it one of the most often used oncolytic viruses [118]. Herpes simplex virus (HSV)‐1 is a virus containing double DNA strands in its envelope that infects human neural tissues without warning. Therefore, it shows potential as a viable choice for oncolytic virotherapy in GBM. Multiple modified oncolytic HSVs (oHSVs) with varying attenuation levels have been developed for treating GBM, with six of them progressing to clinical trials. HSV‐1 is among the most extensively studied oncolytic viruses (OV) [119, 120, 121].

Poliovirus is an icosahedral virus that contains single‐stranded RNA (ssRNA) and is not enveloped. PVSRIPO, a genetically altered version of the Sabin type 1 poliovirus, is the second oncolytic virus to receive Food and Drug Administration (FDA) breakthrough therapy status for recurrent GBM [122]. The natural target of PVS is the poliovirus receptor CD155, which is found in APCs and is increased in GBM. The ability of the virus to infect the nervous system is connected to its internal ribosome entry site, which is exchanged in the engineered version with that of human rhinovirus type 2 [123, 124, 125, 126].

Encased RNA viruses are known as retroviruses. The gamma‐retroviral vector Toca 511 can replicate itself and carries a yeast cytosine deaminase (CD) gene. This gene catalyzes the transformation of 5‐flucytosine (5‐FC), an antifungal medication, into 5‐fluorouracil (5‐FU) that triggers a local antitumor response [127]. In preclinical glioma models, Toca 511 has shown significant oncolytic efficacy [127, 128, 129]. Additionally, it has been demonstrated that Toca 511 at high local concentrations of 5‐FU depletes immunosuppressive myeloid cells in the TME, triggering the development of a T cell‐mediated antitumor immune response [130, 131]. Another clinical trial was conducted using H‐1PV, a DNA virus that infects rats, as a treatment for GBM. The reason why cancer cells are susceptible to H1PV infection is their high levels of essential components for viral replication within the cell, although H‐1PV does not pose any danger to humans [132].

The measles virus (MV), which belongs to the Paramyxovirus family and is a negative single‐stranded RNA virus, has been shown to exhibit oncolytic properties in numerous types of cancer. Naturally occurring oncolytic, weakened vaccine strains of MV have been altered to enhance their specificity for tumors and allow for tracking within living organisms [133, 134]. The MV Edmonton strain (MVEdm) utilizes the carcinoembryonic antigen (CEA) as a reporter gene to monitor viral activity within the body [135]. Glioma animal models treated intratumorally with MV‐CEA showed noticeable tumor shrinkage [136, 137].

Another Oncolytic vaccine virus belongs to the Poxviridae family of enveloped double‐stranded DNA viruses. By incorporating the suicide gene (FCU1) to enhance tumor specificity, TG6002 is a modified Oncolytic vaccine virus with mutations in the ribonucleotide reductase and thymidine kinase genes [138]. In preclinical studies, TG6002 has shown effectiveness in fighting cancer and is currently being tested with 5‐FC in a phase I/II trial involving IV administration of the virus in 78 GBM patients (NCT03294486) [138, 139].

Human cytomegalovirus (CMV) nucleic acids and proteins are initially detected in GBM tissues in over 90% of patients, but not in the normal brain around the tumor [140, 141].

This discovery led researchers to create a peptide immunization against this virus. The vaccination aims to focus on pp6537, the main structural protein of CMV. Research has been done on two methods of treatment: utilizing pp6537‐stimulated DCs and cultivating CMV‐specific CD8⁺ T cells (Table 1) [142, 143, 144]. The study conducted by Reap et al. provides compelling evidence that dendritic cell vaccination (DCV) can enhance the functionality of adoptively transferred CMV‐specific T cells. These T cells exhibit increased polyfunctionality, characterized by concurrent expression of multiple effector molecules such as IFN‐γ, TNF‐α, and IL‐2, suggesting improved antitumor efficacy. The finding underscores the therapeutic potential of CMV‐targeted immunization strategies in GBM, as they have been shown to potentiate T cell polyfunctionality and cytotoxic activity. These approaches may mitigate the profound immunosuppression of the GBM tumor microenvironment, thereby enhancing the efficacy of adoptive and vaccine‐based immunotherapies. By augmenting polyfunctional T cell responses, these approaches offer a promising avenue to strengthen antitumor immunity in this highly immunoevasive malignancy [145]. In addition, complementary evidence from the IMMU‐04 clinical trial reinforces the therapeutic promise of CMV‐targeted vaccination in glioblastoma. In this investigation, nGBM patients were administered a novel CMV peptide‐based vaccine in conjunction with the standard‐of‐care chemotherapeutic agent TMZ, demonstrating the feasibility and potential synergistic efficacy of this combinatorial approach. The results demonstrated the feasibility and safety of this combinatorial approach, and early data indicated the induction of robust CMV‐specific immune responses. Notably, the use of CMV antigens, which are selectively expressed in GBM tissues but not in surrounding normal brain, offers a tumor‐selective target that minimizes off‐tumor toxicity. These outcomes highlight the promise of CMV‐directed immunotherapies and warrant further investigation in larger, controlled trials to validate clinical efficacy and inform optimization of vaccine design [146].

In another major study, Zadeh et al. found that oncolytic virotherapy combined with ICPIs represents a novel strategy for GBM. This study evaluated DNX‐2401, a replicative oncolytic adenovirus, with pembrolizumab in recurrent GBM. Patients received DNX‐2401 followed by pembrolizumab. Tumor responses and immune profiles were monitored. The findings revealed that the combination was well‐tolerated, with partial responses observed in a subset of patients [147]. A phase I/II trial evaluated the reliability of G47∆, a triple‐mutated oncolytic HSV‐1, in patients with progressing GBM after radiation and TMZ treatments. Thirteen patients completed treatment, with 12 experiencing adverse events. The secondary endpoint was efficacy, with a mOS of 7.3 months and a 1‐year survival rate of 38.5%. Median progression‐free survival was 8 days, mostly attributable to the rapid expansion of the contrast‐enhanced region of the target lesion on MRI. Three patients endured for over 46 months, and one entire response was observed at 2 years. Biopsies indicated postadministration MRI characteristics probably represented tumor cell death by viral multiplication and lymphocyte infiltration towards tumor cells. The mechanism of immunological progression is indicative of this treatment. This research shows that G47Δ is suitable for treating recurrent GBM [148]. This collection of studies highlights advancements in immunotherapy for GBM, exploring vaccines targeting specific tumor antigens, such as mutant IDH1, EGFRvIII, and survivin.

Approaches include peptide vaccines such as rindopepimut, SurVaxM, autologous dendritic cell therapies, and oncolytic virotherapy (DNX‐2401) combined with ICPIs. While trials such as ACT IV and Audencel showed limited impact on survival, others demonstrated immune activation and potential clinical benefit, particularly in personalized or combination therapies. These findings underscore the promise of immunotherapy in GBM treatment while emphasizing the need for improved strategies to overcome immune resistance and enhance therapeutic efficacy. Also, an investigation revealed that another cause of GBM may be a mutation in IDH1, so it is a promising target for immunotherapy. A clinical trial was executed to assess the immunogenicity and preliminary efficacy of the IDH1 peptide vaccine. Patients with IDH1‐mutant gliomas were registered and obtained the vaccine with standard therapies. Immune responses were evaluated using T‐cell assays, and clinical outcomes were monitored. The results of this study indicate that the vaccine was well‐tolerated and elicited strong, mutation‐specific T‐cell responses. Early efficacy signals included stabilization of disease in several patients, supporting the potential of this approach for IDH1‐mutant gliomas [70].

Viruses are used as carriers of therapeutic genes to improve cell function or target abnormal cells, with retroviral replicating vectors (RRVs) and Adv being the most studied for GBM [112, 149]. Recombinant viruses, as opposed to oncolytic viruses which kill tumor cells, are designed to safely transport antigens. Without the use of extra adjuvants, these vectors can infect target cells and create antigen peptides, inducing a potent immune response. Viral vectors can strengthen the immune system by utilizing pathogen‐associated molecular patterns (PAMPs); however, prolonged usage may develop antiviral immunity [150, 151]. For example, for the treatment of rodent GBM models, researchers have created a bicistronic Adv vector containing HSV‐1 and a Tet‐inducible expression cassette for Flt3 ligand. The vector has therapeutic efficacy, cytotoxic and immunostimulatory effects, and no excess viral particle burden after injection [152]. There is an ongoing phase I clinical trial that combines immune‐mediated killing triggered by the Flt3L gene with direct destruction of tumor cells. Adv‐tk plus an antiherpetic prodrug are the two drugs used in gene‐mediated cytotoxic immunotherapy (GMCI), and it has demonstrated favorable therapeutic effects on tumor cells [153]. Clinical trials on newly diagnosed, recurring malignant and juvenile malignant gliomas have shown survival advantages and low toxicity [154, 155, 156].

Moreover, powerful vehicles have been engineered using AAV, a viral vector intimately connected to the immune system. To improve T cell function, Ye et al. created a hybrid CRISPR screening system that can target and modify membrane proteins on primary murine T cells in vivo [157]. A vector with VEGF‐C was developed to enhance CD8⁺ T cell activation in mice models of GBM. This vector could enhance antigen removal and change the tumor microenvironment, potentially enhancing the efficacy of immune checkpoint inhibitors (ICPIs) in GBM therapy [158]. In addition to VEGF‐C, GBM frequently displays changes to the epidermal growth factor receptor (EGFR), suggesting that this receptor is essential for the development and proliferation of glial tumors. The RNA polymerase III‐dependent H1 promoter was incorporated into HSV‐1‐based amplicons to allow for the expression of double‐stranded hairpin RNA against EGFR at two distinct sites (pHSVsiEGFR I and pHSVsiEGFR II). Human GBM (gli36‐luc) cell growth was inhibited both in vitro and in vivo by this dose‐dependent posttranscriptional gene silencing using vector‐mediated RNA interference [159]. These findings indicate that effective posttranscriptional gene silencing can be achieved with HSV‐1 amplicons.

Furthermore, RRVs that encode the CD gene can selectively transfect neoplastic cells and convert the prodrug 5‐FC into the cytotoxic agent 5‐FU [160]. Mitchell et al. conducted a study examining the immunogenic properties of RRVs in murine glioma models. Researchers revealed that Toca 511 in conjunction with 5‐FC can elicit a moderated and escalating immune activation. Post‐treatment observations indicated an increase in the expression levels of 41BB, CD40L, and PD‐1, while there was a reduction in the population of immunosuppressive cells. Mice that achieved complete tumor eradication experienced extended survival durations [131].

Recombinant parvoviruses have been utilized to influence the immune response within GBM tumors. In a syngeneic murine model, the transduction of CXCL10 and TNF‐alpha cytokines resulted in tumor regression. This synergistic interaction contributed to a postponement of tumor proliferation in naïve, preestablished tumors; however, no regression was noted in naïve tumors [161]. Notwithstanding the demonstration of significant therapeutic efficacy in clinical trials, viral‐vector gene therapy has yet to attain approval from the FDA. Further progress is required due to limited effectiveness, viral vector delivery, tumor penetration, and safety issues. Even with these challenges, the bright future of viral‐vector gene therapies is supported by the many creative solutions being explored in academia, biotechnology, pharmaceuticals, and manufacturing industries [118].

In addition to these approaches, several viral vector‐based strategies have been developed to deliver immunomodulatory genes directly into the tumor microenvironment. These include inducible IL‐12 expression systems using the RheoSwitch Therapeutic System (RTS), allowing controlled immune stimulation and minimizing systemic toxicity [162]. In this regard, Rivera‐Molina et al. engineered a GITRL‐armed Delta‐24‐RGD oncolytic adenovirus, which extended survival and induced antiglioma immune memory in preclinical models [163]. Passaro et al. constructed an oncolytic HSV‐1 vector encoding a PD‐1‐blocking scFv antibody, providing local checkpoint blockade and improved immune infiltration [164]. In another study, King et al. demonstrated that codelivery of Flt3L and thymidine kinase (TK) via gene therapy led to eradication of multifocal glioma in syngeneic models through combined immunostimulation and direct cytotoxicity [165]. These strategies demonstrate the expanding versatility of viral vectors in not only delivering tumor‐targeting genes but also enhancing antitumor immune responses in glioma therapy.

One particular subset of APCs that regulates immunity and immunological tolerance is the DC. They are considered an interesting goal for eliciting immune responses towards malignancy because they are present in most tissues as immature (resting) cells. They exhibit mature peptides on their human leukocyte antigen (HLA) class I and II receptors after antigen collection and processing, resulting in MHC–peptide complexes. Mature activated DCs move from peripheral tissues to secondary lymphoid organs and lymph nodes in order to engage in physical interactions and trigger T‐cell responses [166].

There is much controversy regarding DCV as a therapeutic adjuvant in GBM. Hundreds of GBM patients have received vaccinations to induce an anticancer immune response through the use of DCV as active immunotherapy in numerous clinical trials. Effectiveness of DCV in GBM generally varies, from no clinical response to notable responses [167]. DCVs increase tumor‐specific IFN‐γ, activate CTL, slow the growth of tumors, and extend life expectancy. The vaccination seems to be safe, well‐tolerated, and free of major side effects (≥ grade 3) [168, 169]. Bota et al. A phase 2 trial evaluated the survival, adverse events, and efficacy of the Aivita GBM vaccine (AV‐GBM)‐1, produced by incubating autologous DCs with irradiated autologous tumor‐initiating cells, using autologous DCs. They determined the treatment was well tolerated; however, there were several treatment‐emergent CNS adverse events (AEs). AV‐GBM‐1 was reliably produced. Treatment was well tolerated. The median Progression‐Free Survival (mPFS) was longer than historical benchmarks, but no mOS improvement was observed [170]. In another study, researchers investigated the efficacy of the autologous tumor lysate‐loaded Dc vaccine (DCVax‐L) in the survival of GBM patients. In this investigation, compared to matched, contemporaneous external controls, adding DCVax‐L to SOC has been connected to a clinically relevant and statistically significant improvement in mOS for patients with both nGBM and recurrent GBM (Table 1) [171].

A phase III trial examined patients who received autologous DC vaccines tailored to tumor antigens alongside standard care. Researchers assessed the survival and immune responses. While overall results were mixed, patients with robust immune responses showed improved survival, underscoring the potential of individualized vaccines [172]. Combining dendritic cell vaccines with radiochemotherapy may enhance immune responses in GBM. Inogés et al. in this phase II trial were evaluated immune activation and survival outcomes. For this reason, patients underwent fluorescence‐guided surgery followed by radio chemotherapy and autologous DC vaccination. The most striking result to emerge from the data is that combination therapy was safe and led to enhanced immune responses. Modest survival benefits were observed, highlighting the potential of integrating DC vaccines into standard GBM treatment [173]. In another study, Audencel, a DC‐based vaccine, was evaluated for its impact on GBM outcomes in a phase II trial. The nGBM patients received Audencel alongside standard therapy. OS and PFS were the primary endpoints. The vaccine showed no significant impact on survival outcomes; therefore, the addition of Audencel to the standard of care did not improve the clinical outcomes of patients with primary GBM [174, 175, 176].

TSAs and TAAs, which are categorized into five kinds based on their expression in tumors, are used by the immune system to identify cancers [177]. The main goals of the research are to determine which TAA epitopes are the most immunogenic in humans, to characterize the immunogenicity of TAAs, and to investigate their potential as tumor‐defense antigens. A promising strategy to induce a potent antitumor response and long‐term memory is to create vaccines from entire tumor cells, including numerous TAAs recognized by CD8⁺ cytotoxic T lymphocytes (CTLs) and CD4⁺ T helper cells. For efficient tumor regression, this tactic seeks to stimulate the innate and adaptive immune systems [178].

By targeting various tumor antigens and removing the individual epitopes, whole tumor vaccination therapy presents a promising approach to cancer treatment that can be used for all patients, independent of their HLA type [179]. Allogeneic vaccinations offer a more standardized and scalable approach employing tumor cell lines from diverse tumors, whereas autologous vaccines using the tumor cells of patients give customized treatment choices with distinct neo‐tumor antigens. Allogeneic vaccines provide advantages in terms of scalability and quality control, while autologous vaccines have drawbacks regarding reproducibility and tailored therapy [179, 180]. All things considered, whole tumor vaccination therapy offers promise for efficient and customized cancer immunotherapy [181, 182, 183].

A novel approach being studied in GBM patients is the production of DNA vaccines. Viral origins were highlighted in earlier research as a biological vector for introducing predetermined antigens into host cells. The emphasis has now switched to plasmids made of synthetic DNA [187]. DNA vaccines are perfect for developing cancer vaccines because of a number of their features. Plasmid vaccines pose fewer risks regarding immunogenicity and replication competency than other delivery systems (recombinant proteins and viral vectors). Additional benefits include improved compatibility with people, stability, ease of large‐scale production, and lack of infectious agents [188]. Patients with recently confirmed IDH1 or IDH2 mutations in GBM are participating in a phase I trial of a DNA vaccination. To treat patients with this mutation, this trial makes use of two DNA plasmids: one encoding the tumor‐specific antigen GNOS‐PV01 and the other containing a synthetic DNA plasmid generating the pro‐inflammatory cytokine interleukin‐12, or INO‐9012. Through the promotion of the development of specialized T cells against antigens particular to a patient, IL‐12 functions as a molecular adjuvant to activate the immune system [189]. mRNA vaccine has been described in the following sections.

The delivery of mRNA vaccines can occur via two general approaches, including in vivo and ex vivo. Both approaches have unique advantages and are chosen based on the specific type of cancer and the desired immune response [209, 210]. In vivo involves direct administration of the mRNA vaccine into the patient, where it is taken up by immune cells such as DCs directly within the tissues. Typically, mRNA is encapsulated in LNPs to protect it from degradation and facilitate efficient cellular uptake [220]. So, LNPs are typically designed to have a size and surface charge that promote uptake by APCs, including DCs, through endocytosis or micropinocytosis [221]. Also, after intramuscular or subcutaneous injection, LNPs drain into lymph nodes, where DCs are highly concentrated. Subsequently, the LNPs enable the mRNA to enter cells and subsequently translate into antigens, triggering an immune response. In vivo delivery is advantageous because it is relatively straightforward and can lead to a rapid, systemic immune response [210].

In contrast, the ex vivo approach involves isolating specific immune cells, often DCs, from the patient and modifying them outside the body. In this process, patient‐derived DCs are extracted and transfected with mRNA in a controlled laboratory setting. Once the DCs have successfully processed the mRNA and presented the desired antigens, they are reintroduced into the patient [222, 223]. This approach offers greater control over the activation of immune cells and is particularly useful for personalized therapies where immune responses must be tightly regulated [224]. Each approach has its benefits. In vivo delivery is less invasive, and it is suitable for broader applications. Despite this, ex vivo delivery allows for a highly customized immune response tailored to individual patients, which is especially advantageous in cancer immunotherapy [225].

A key challenge in mRNA vaccine therapy is effective delivery systems. RNA vaccination encounters hurdles such as overcoming barriers that inhibit the entry of foreign nucleic acids and breakdown by RNases. Additionally, the large size of RNA hinders its diffusion within cells, increasing its detection and destruction by the host [226, 227]. Therefore, proper delivery systems are essential [227]. Traditional methods such as in vitro‐loaded DCs, polymer delivery, and mechanical techniques [gene gun, electro injection] are complex, expensive, or unsuitable for humans. Viral vectors are efficient for nucleic acid delivery but face issues such as immunogenicity and manufacturing challenges [228, 229]. Nonviral vectors, while less efficient, are less safe and can carry larger genetic loads, making them easier to synthesize [230, 231]. LNPs are the most common method of non‐viral mRNA delivery [232]. These sub‐micrometer solid structures form an emulsion with solid lipids. LNPs usually have a hydrophilic core and a lipid bilayer shell with different lipids that play different roles. Most formulations use cationic lipids to effectively complex with negatively charged RNA, while anionic and neutral types have also been used. In fact, this electrical charge difference helps to retain the mRNA in the nanoparticles [233, 234]. Novel lipidoids with amine groups maintain a low or neutral surface charge at physiological pH, reduce nonspecific interactions, and facilitate oligonucleotide release into the cytosol. In acidic endosomes, amine groups are ionized, forming a hexagonal phase that disrupts the endosomal membrane and facilitates the escape of mRNA into the cytoplasm (Table 2) [235, 236].