Design

Glioblastoma

Diagnosis

GBM is grade-IV astrocytic glioma, which means it derives from astrocyte cells. According to the classification of the World Health Organization (2021), it has unique molecular characteristics when compared to other gliomas. More specifically, it is characterized by the lack of mutations in isocitrate dehydrogenase (IDH1/2) and in histone 3 (H3) gene. In the diagnostic criteria are also mentioned mutations on the TERT promoter, EGFR gene amplification, chromosomal changes of chromosomes 7 and 10 (+7/–10 cytogenetic signature), and O-6-methylguanine-DNA methyltransferase (MGMT) mutations (Chrysanthakopoulos & Chrysanthakopoulos 2020; Louis et al. 2021; Weller et al. 2021; Stoyanov et al. 2022). The gold standard for diagnosis is Magnetic Resonance Imaging (MRI) and biopsy. The tumour is characterized by microvascular proliferation and necrotic areas (Stoyanov et al. 2022).

Therapy

GBM follows a different metastatic pathway instead of colonizing other tissues via blood circulation. After being detached from the original tumour, GBM cancer cells infiltrate into the healthy tissue by degrading the extracellular matrix or squeezing through the brain interstitial spaces (Catacuzzeno & Franciolini 2018). Thus, it is one of the most aggressive malignancies, due to its high migratory and invasive potential, which prevents complete surgical removal of tumor cells. In general, the gold standard of GBM therapy is surgery followed by radiotherapy and temozolomide (TMZ). However, surgery has limited therapeutic effects, as complete resection is impossible. Moreover, due to its high proliferation and angiogenesis, GBM is almost completely inaccessible. This renders radiotherapy ineffective as well. At the same time, TMZ has difficulties crossing the blood-brain barrier. Its action is largely limited in patients with MGMT promoter-methylated glioblastoma.

In recurrent GBM, standard-of-care treatments are not well defined and the treatment depends on prior therapy, age, MGMT promoter methylation status and disease progression. Second surgery increases the risk of unnecessary intervention due to pseudoprogression and is a sustainable option for only 20–30% of patients (Weller et al. 2021). Follow-ups are recommended every 2-3 months.

Despite GBM being the most frequent and aggressive brain tumor, finding new treatment methods has shown minimum progress. The research for more effective GBM therapies has received less funding than for other types of cancer while the complexity of its nature is another reason for the limited therapeutic and diagnostic approaches (Jaroch et al. 2019; Grochans et al. 2022).

Pseudoprogression challenge

MRI is an established imaging technique and constitutes the gold standard for GBM radiographic characterization in terms of diagnosis and post-treatment care (Ryken et al. 2014). Basic MRI modalities, such as native T1-weighted (T1w) and contrast-enhanced (T1CE), T2-weighted (T2w), and T2-fluid-attenuated inversion recovery (T2-FLAIR) sequences, are accessible from any clinical scanner and offer key clinical information on numerous processes of the tumour development (Shukla et al. 2017).

GBM, diagnosed usually when the tumour is large, often has a heterogeneous-ring enhanced pattern with irregularly enhancing margins and a central necrotic core with or without haemorrhagic component in the first MRI. They are surrounded by substantial edema, which usually contains infiltration by neoplastic cells. Multifocal GBM, where multiple areas of enhancement occur with abnormal white matter signals connection, represents microscopic spread of tumour cells. Less clear are the MRI results for early-stage glioblastoma, where the diagnostic findings are more heterogeneous and characterised by inter- and intra- examiner variability (Hu et al. 2020).

According to each specific MRI modality, the radiographic features of GBM include (Worsley et al. 2022, October 7):

  • T1w:hypo-/iso-intense mass within white matter, centrally placed heterogeneous signal, indicating necrosis or intratumoral haemorrhage
  • T1CE: Enhancement presence, peripheral and irregular with nodular components
  • T2/FLAIR: Hyperintense mass surrounded by vasogenic oedema and occasional flow voids

Due to the complexity of the anatomy, evaluating distinct tumour subregions visually in MRI scans can be extremely difficult (Zikou et al. 2018). A more accurate and objective evaluation, could be possible with automation in brain tumour segmentation. After therapeutic interventions, MRI is essential for monitoring of the patients, for the evaluation of the therapy and the adjustment of the treatment regimen. However, after radio/chemotherapy the MRI interpretation can be disrupted by a phenomenon called “pseudoprogression” (Ohgaki et al. 2013; Thust et al. 2018). Pseudoprogression constitutes a significant clinical problem, affecting the clinical evaluation and thus the therapeutic regimen. The term refers to enlarged areas of contrast agent enhancement that occur without any therapeutic change. This phenomenon is mostly due to edema caused by the therapy. GBM tumours are characterised by extreme neovascularization and radiochemotherapy increases vessel permeability causing edema and mimicking true progression, while actual tumour growth is absent.

Unfortunately, no imaging techniques are currently available to reliably distinguish between recurrence and pseudoprogression. To achieve higher levels of diagnostic accuracy, confidence and reliability, new imaging techniques are required. Thus, we propose the use of artificial intelligence, to discriminate between pseudoprogression and real progressive tumor growth, although Theriac is designed to avoid pseudoprogression issues.

Key Molecules

microRNAs

For the distinction between cancer and healthy cells, respectively, and for the release of the therapeutic agents, we designed Theriac to detect two different microRNAs simultaneously. In the following sector, we analyze the reasons why we choose microRNAs instead of other molecules and the characteristics of the selected microRNAs.

The main advantages of microRNA detection can be summarized in the following main points:

High safety profile:

  • high accuracy and high detection value; the selected microRNAs are overexpressed only in GBM cancer cells and cancer stem cells. In fact, miR-10b is not expressed in healthy neural and glial cells at all
  • low possibility for developing therapy resistance; microRNAs are more stable than other biomarkers because their expression changes slowly during the progression of the tumour

Better thermodynamic characteristics:

  • microRNAs are small molecules, thus the thermodynamic profile of our hairpins is better compared with bigger molecules
  • microRNAs have well-characterized sequences and they usually don’t have mutations. In contrast, proteins usually have mutations in cancer cells making detection more difficult and reducing accuracy

More therapeutic options:

  • we wanted a two-step therapy (Y-hairpin attaches the complementary microRNAs, obstructing them from acting in favor of the cancer cells while the selected siRNAs cause gene silencing. If instead of microRNAs we had selected proteins, the only therapeutic part of our mechanism would be the siRNAs, reducing the possible effect of the therapy)
  • we wanted a therapeutic approach that could easily be used for all types of cancers. The selected microRNAs offer a high selectivity, detecting only GBM cancer cells and not healthy cells. At the same time, these microRNAs are overexpressed in the cancer cells of other rare cancer types, such as pancreatic cancer, offering Theriac the resilience needed for future implementation in different types of cancer.

On the other hand, the selection of microRNAs also faces some obstacles. These limitations are mainly focused on the limited lab techniques for analyzing and studying microRNAs, the differential expression and specificity of the microRNAs overexpressed in GBM cancer cells, and the off-target effects of targeting microRNAs (Koshiol et al. 2010; Catuogno et al. 2012). Our team took these limitations under serious consideration and after discussing with experts in the field we concluded that the benefits were of great importance and we should proceed with the implementation of microRNAs.

Specific microRNAs selection

microRNAs are small regulatory RNA molecules that regulate the multiple normal biological activities of the cells. They are single-stranded RNA molecules 20-24 bases long and make up about 3% of the human genome. These molecules can have a cancer-suppressing or cancer enhancement role, but their function depends on the respective tissue. microRNAs are binding to a complementary sequence of their target mRNA molecule and through interactions with the RISC complex, they lead to the inhibition of translation or the degradation of the target (Aloizou et al. 2020).

In GBM, there are many overexpressed microRNAs, summarized in Table 1, each one with a unique role and function. The selection of the most appropriate microRNAs is crucial for our work. Thus, we decided that a multidimensional research path should be followed for this procedure, analyzing both bibliographic elements and databases. Here, we analyze the procedure we followed for the analysis of the data we obtained from databases.

Table 1: microRNAs overexpressed in GBM cancer cells and cancer stem cells

Use of Databases

Finding databases to correlate microRNA concentrations with glioblastoma tissue samples was extremely difficult due to the lack of data. After extensive research, we identified dbDEMC, OMIM, ONCOMIR and TCGA databases. Below we describe the way we used some of these data.

Data analysis from TCGA

The Cancer Genome Atlas Program (TCGA) was the database with the more information related to our search.

From this database, we extracted the microRNAs concentration values of 10 samples of healthy brain tissue, aiming to confirm bibliography reports. From analysing the data and displaying it in bar graphs, we concluded to the following:

  • Some microRNAs have high concentrations in healthy tissue, too
  • miR-21 expression in healthy tissues was low, while in GBM very increased
  • miR-10b wasn’t expressed in healthy tissues while it was slightly, but statistically significant increased in GBM tissue samples

DBDEMC

We used this database to study the expression of specific microRNAs in glioblastoma tumour tissue. In this database, we found the concentration of microRNAs both for healthy and cancer tissue, too. The analysis confirmed that both miR-21 and miR-10b are overexpressed in GBM cells.

Online Mendelian Inheritance in Man
With the help of this tool, we search the microRNAs overexpressed in GBM and we verified the results from the previous databases.

Stability of the microRNAs

Another important criterion for the selection of the microRNAs was also their stability in the cytoplasm. The selected microRNA should be relatively stable to achieve detection with high accuracy. Thus, we also calculate the Gibbs free energy, using Nupack. We calculated the stability of miR-10b and miR-21 molecules at 37oC (body temperature) and 25oC (room temperature). The analysis showed that both molecules have negative free energy, close to 0, and thus high stability.

After thorough research, we selected miR-21 and miR-10b as biomarkers, based on their high safety profiles and the suggestions of experts. Some of the rest microRNAs overexpressed in GBM didn’t have such a high safety profile because of their expression in a variety of pathological conditions. Two other promising candidates were miR-155 and miR-20a. We rejected miR-155 because it is also overexpressed in hematopoiesis and neuroinflammation, both common phenomena in people over 50 years old (Faraoni et al. 2009). We rejected miR-20a because it has a very similar way of action to miR-21, which is better-studied. Between miR-20a and miR-10b, we preferred miR-10b because of its high specificity for GBM.

Links of databases used: dbDEMC OMIM ONCOMIR TCGA

miR-21 and miR-10b in GBM

To validate the expression levels, the safety profile and the molecular pathways in which the selected microRNAs are involved, we reviewed data from the aforementioned databases and articles, mostly from PubMed. The involvement of miR-21 in glioblastoma has been studied in more than 224 papers, while miR-10b in more than 42. miR-10b has a very similar sequence with miR-10a, also overexpressed in GBM. Thus we also reviewed the data and papers for miR-10a.

miR-21 has been characterised as an oncomiR, due to its overexpression in the majority of cancer types, such as in the brain, head and neck, oesophagus, breast, lung, stomach, pancreas, colon and prostate cancer, playing a key role in tumorigenesis (Fang et al. 2017). In GBM, miR-21-5p has been reported to be increased not only in cancer cells (Sippl et al. 2019) but in cancer stem cells (CSCs) (Zhang et al. 2012; Shang et al. 2015), in comparison with cultured non-neoplastic glial cells, and healthy and nonneoplastic foetal brain tissues (Chan et al. 2005; Yang et al. 2014). In cancer stem cells, while most researchers support its overexpression, interestingly, Tomei et al. (2021) reported that miR-21 was downregulated in CSCs. In contrast with all previous results, Basso et al. (2022) found that miR-21-3p, and not -5p, is increased in GBM tumours.

Regarding miR-21 expression levels and tumour stages, the literature is divided, with other researchers suggesting that miR-21 levels are overexpressed equally in all tumour grades (Conti et al. 2009; Koshkin et al. 2014) while others suggesting that the higher the increase in miR-21 levels, the higher the tumour stage (Gaur et al. 2011; Park et al. 2012; Hermansen et al. 2013; Shi et al. 2015; Zottel et al. 2020). Matos et al. (2018) reported a significant change in the expression of miR-21 in the recurrent tumour when compared to the primary, although in both cases miR-21 was increased. While Ma et al. (2012) reported that miR-21 is upregulated only in primary GBM tumours and is significantly downregulated in secondary GBM tumours.

In GBM, miR-21 has been reported to contribute to:

Tumour growth and cell proliferation, mainly through downregulation of Pdcd4 (Gaur et al. 2011)
Cancer cells survival; miR-21 acts as an anti-apoptotic factor
Migration - invasion
Angiogenesis
Poor prognosis (Yang et al. 2014; Cheng et al. 2016)
Tumour microenvironment
Tumour resistance to chemotherapy/actinotherapy


A unique characteristic of miR-10b is that its expression is silenced in normal neuroglial cells, neural progenitor cells and neural cells in the human brain. Its expression is up-regulated in many pathological conditions, such as GBM playing an essential tumour-promoting role. Both miR-10a and miR-10b have been found upregulated in glioblastoma and anaplastic astrocytomas, in some cases reaching more than a 100-fold overexpression (Gabriely et al. 2011; Teplyuk et al. 2016; Deforzh et al. 2022).

In the majority of literature, miRNA-10b-5p levels have been found significantly elevated in glioma tumours of all stages; from low-grade glioma (LGG) to Glioblastoma (grade IV glioma) (Liu et al. 2021). The higher the grade, the higher miR-10b levels observed (Sasayama et al. 2009; Visani et al. 2014).

miR-10b has been reported to regulate:

Tumour growth – cell proliferation; miR-10b regulates genes that promote cell cycle progression
Survival – regulating apoptosis
Cancer cells migration and invasion
Angiogenesis
Chemo-and Radio- therapy resistance (Zhen et al. 2016)

miR-10b seems to have a pleiotropic effect on GBM cancer cells, although the exact mechanisms and way of action are not yet fully understood. In literature, the findings are contradictory, with others suggesting that miR-10b affects only tumour growth but not invasion and others suggesting that it regulates all the above. This conflicting evidence might be due to the deficiencies in tumour models used and the differences noted between different subtypes of gliomas (Lin et al. 2012).

Except for miR-10b, miR-10a is overexpressed in GBM, too. The two miRNAs likely co-function since they are predicted to have identical targets. Gabriely et al. (2011) have found that together these microRNAs expression may correlate with patient survival. Patients with higher miR-10b and miR-10a levels have been reported to have lower possibilities for survival, indicating that both microRNAs may contribute to tumorigenesis. On the contrary, Guessous et al. (2013) showed that inhibition of miR-10b alone can significantly decrease in vivo tumour growth, reducing the proliferation of GSCs, while it didn’t have any effect on healthy cells (Guessous et al. 2013).

Safety issues with miR-21 and miR-10b

miR-21 regulates a plethora of biological functions and pathways of high importance in all developmental stages, both in health and pathological conditions. After fertilisation, the maternally inherited mRNAs are degraded and the embryonic genome is activated. miR-21 and STAT-3 activation are very important for this procedure and for the activation of embryonic genome expression. In the next stages of development, miR-21 and pre-miR-21 seem to affect pluripotency by interfering with self-renewal regulators in a complex and contradictory manner. miR-21 has been also found to affect branching morphogenesis and organogenesis in exocrine glands, lungs and kidneys (Kumarswamy et al. 2011).

Although it is the most studied miR, the mechanism of its upregulation is still not well known. However, the available data imply that it is regulated by transcriptional and post-transcriptional mechanisms. Accumulating data indicate a regulatory loop between these factors and miR-21 (Kumarswamy et al. 2011). The expression of miR-21 has been found deregulated in a variety of pathological conditions, such as in:

1. Inflammation: mainly through its interaction with STAT3, it seems to affect antigen clearance; it has also been found increased in asthma (Kumarswamy et al. 2011).
2. Epithelial-to-mesenchymal transition and organ fibrosis through its interplay with TGFβ pathways. It is weakly expressed in normal myocardium while its expression is elevated in failing cardiac fibroblasts, suggesting a protective role to cardiac cells (Kumarswamy et al. 2011).
3. CNS diseases: miR-21 mainly regulates the apoptosis and inflammatory processes in the nervous system and is also involved in astrocyte activation, glutamate toxicity, synaptic dysfunction, microglial burst activity, and remyelination. miR-21 has been found upregulated in acute CNS injuries and ischemic stroke, with a neuroprotective role. However, miR-21 has been found downregulated in microglia subjected to hypoxia, suggesting that its expression changes in a time-dependent manner during the different stages of stroke (Lopez et al. 2017). In Alzheimer’s Disease (AD) and Parkinson’s Disease (PD), miR-21 has been found deregulated, too. High levels of miR-21 have an anti-inflammatory and anti-apoptotic effect (Bai and Bian 2022).

miR-10 family is a set of well-conserved genes located in the Hox genes’ area. More specifically, miR-10b is located near the homeobox D (HOXD) cluster on chromosome 2. miR-10 translation is strictly regulated sometimes together with the Hox genes, suggesting a key role in developmental procedures. Hox genes are organised into genomic clusters which affect their expression. In mammals, miR-10a resides upstream from Hoxb4 and miR-10b upstream from Hoxd4 and both their position and sequences are well conserved among species, indicating a requirement for the same cis-regulatory elements that regulate the Hox genes (Lund 2010; Wimmer et al. 2020).

miR-10 family and specifically miR-10b, have been linked to a variety of functions and pathways and their regulation has been associated with angiogenesis, cancer and promotion of cell invasion. In cancer, miR-10b overexpression was mainly associated with the stemness of the cancer cells and metastasis and thus is characterised as a metastasis-linked miRNA (Wimmer et al. 2020).

Interestingly, there are a few types of cancer that are not associated with miR-10b expression and miR-10b plays even a tumour-suppressive role. For example, increased miR-10b levels have been associated with increased survival in renal cell cancer, while in bladder cancer results are inconsistent (Sheedy and Medarova 2018).

miR-10a seems to play a crucial role in normal developmental procedures such as in hematopoietic pathways. For example, miR-10a and miR-10b levels in CD34+ cells are decreased during their differentiation into megakaryocytes. miR-10a levels are higher in hematopoietic stem cells than in peripheral blood lymphocytes, suggesting a role in progenitor cells (Lund 2010). Like miR-10b, miR-10a is associated with inflammatory pathways, too. For example, elevating miR-10a-5p levels inhibited proinflammatory gene expression in cultured macrophages (Cho et al. 2019).

Conclusions

In general, the literature highlights the importance of miR-21 and miR-10b inhibition as a therapeutic approach for GBM. Both microRNAs are good biomarkers and although are not very specific for GBM, they are very specific for detection of cancer cells against healthy ones. Theriac’s first hairpin doesn’t only detect these two microRNAs, but at the same time it deactivates them, inhibiting their action in favour of cancer cells. Due to the importance of their elevated levels in some neuropathological conditions, Theriac should be carefully administered to patients with these comorbidities. To learn more, read our future implementation section.

Small interfering RNA

RNA interference is a natural mechanism in which double stranded ribonucleic acid (dsRNA) controls a certain gene's activity, endogenously in eukaryotic cells. Both siRNA and miRNA could be associated with RNAi based gene silencing. SiRNAs, which are used in our project, are double stranded nucleotide molecules consisting of 21–23 nucleotides. These molecules are bound to the RNA induced silencing complex (RISC) and are unwounded into single-stranded RNAs by Argonaut 2 (AGO2) that bind to the complementary mRNA sequence (unnecessary strands are degraded). The pairing causes cleavage at the targeted area, which prevents the targeted mRNA from being translated and causes its degradation. When the paired mRNAs are cleaved, RISC and single-stranded siRNAs are free to bind to other mRNAs to lower protein expression level (Kim et al. 2019).

While completing the Human Genome Project, gene therapy has received a great deal of attention since sequencing of certain genomes could provide useful information for RNA interference (RNAi)-based therapeutics. siRNA allows the inhibition of multiple targeted proteins which are difficult to reach and control with traditional small-molecular-weight or protein medicines. Compared to conventional therapies, the identification of gene-specific siRNAs is faster, requires less time for development, less money, and has considerably greater selectivity and efficacy. Moreover, siRNA therapies are less painful and invasive than normal practices and don’t cause intense side effects such as those caused by extracorporeal therapy.

Despite its significant therapeutic potential, siRNA therapies are still facing various challenges based mainly on their stability and natural properties. Considering that siRNAs are small molecules, with a length between 21–23 nucleotides, they are quickly detected by endogenous enzymes in the blood, causing a brief half-life and a rapid release from the body through the kidneys, or through binding with serum proteins. Also, because of their anionic nature, siRNAs must overcome obstacles in order to penetrate plasma membranes (which are also negatively charged). Last but not least, the blood-brain barrier and immune recognition system make it difficult to deliver siRNAs to their targets under normal administration (Zhou et al. 2014; Aghamiri et al. 2021).

Selection of siRNAs and their targets

After we concluded on using siRNAs as therapeutic molecules, we had to decide the ideal target. Thus, we search all the possible targets for GBM already silenced with RNA interference technologies. In Table 2, we summarise all the possible targets found through our searches:

Table 2: List of mRNAs targeted with siRNAs in GBM as therapeutic approaches

After a literature review and talking with experts, we concluded that 3 targets were more appropriate for Theriac. We rejected as targets the trans-membrane proteins and proteins with many isoforms due to the easy adaptation of cancer cells to these interventions. The 3 possible targets we concluded on were STAT3, HIF-1a, and PLK1. We preferred these mRNAs because they were multiple effective siRNAs used in GBM cancer cells. We also took into consideration possible interactions with the microRNAs selected to achieve better inhibition and apoptotic results.

Analysis of targeted proteins

During the process of choosing the best protein target and the most appropriate siRNAs, we examined a plethora of different targets, with most researchers suggesting STAT3. STAT3 is a protein, consisting of 770 amino acids and characterised by the presence of 6 functionally conserved domains (Zou et al. 2020) (U.S. National Library of Medicine Oct. 8).

It is important in many cellular processes, like cell growth and apoptosis, by interfering with a lot of genes in response to cell stimuli. Its signalling pathway is activated by cytokines, upstream growth factor kinases and by many carcinogens, too (National Center for Biotechnology Information. 2022, Oct. 8). Non-receptor tyrosine kinases can also lead to constitutive activation of STAT3. Phosphorylated STAT3 dimerizes and translocates to nucleus, which causes the transcription of target genes including immunosuppression, angiogenesis, metastasis, proliferation and survival mainly through regulation of survivin and Bcl apoptotic pathway. (Zou et al. 2020).

STAT3 is involved in the regulation of cellular growth, differentiation and survival. It is considered important for embryonic development, inflammation, immunity, wound healing, haematopoiesis and cell migration. It has been proved that the deficiency of STAT3 causes the death of mouse and human embryos. As far as its role in the immune system, the silencing of this protein results in deficient TH17 cells, central memory T cells and memory B cells. Research also indicates that STAT3 is an important factor in emotional reactivity (Li & Bhaduri-McIntosh 2016); absence of STAT3 from the serotonergic system reduces negative emotional reactivity in mice behaviour (Reisinger et al. 2021).

Activated STAT3, according to Gepia (http://gepia.cancer-pku.cn/detail.php?gene=STAT3) is found upregulated in many human tumours such as, multiple myelomas, breast, ovarian, prostate, and head and neck tumours. The median expression of STAT3 in GBM tumour and normal tissue is, 66.1 and 17.86, respectively, according to Gepia (http://gepia.cancer-pku.cn/detail.php?gene=STAT3), indicating that it is overexpressed in glioblastoma cells. This upregulation in GBM affects tumour survival and chemotherapy resistance (Cui et al. 2020). Suppression of STAT3 causes apoptosis induction, via upregulation of autophagy (Yuan et al. 2015).

STAT3 is involved in the regulation of cellular growth, differentiation and survival. It is considered important for embryonic development, inflammation, immunity, wound healing, haematopoiesis and cell migration. It has been proved that the deficiency of STAT3 causes the death of mouse and human embryos. Also as far as its role in the immune system, the deficiency of this protein results in deficient TH17 cells, central memory T cells and memory B cells. Research also indicates that STAT3 is an important factor in emotional reactivity (Li et al. 2016). Moreover it was found that with the absence of STAT3 from the serotonergic system of the midbrain in mice, the mice showed reduced negative emotional reactivity in their behaviour. This protein is an important mediator in the serotonergic system for emotional reactivity control, proving a link between the immune system, serotonergic transmission and affective disorders (Reisinger et al. 2021).

Another protein target that we examined and found to be appropriate for our project, with good results, is HIF-1A. Execution of aerobic metabolism and energy generation in mammalian cells is achieved via the maintenance of specific oxygen hemostasis in cells. In cancer, metastases, and other diseases, cells have low oxygen levels, and become hypoxic, due to cellular oxygen imbalance. This is common in a lot of solid tumours, causing the obstruction and compression of the blood vessels around it. Hypoxic tumour cells tend to adapt to the lower oxygen conditions, with the activation of several pathways, including the HIF-1 transcription factor (Masoud et al. 2015).

The HIF-1 transcription factor has 2 subunits, HIF-1A and HIF-1β, both of which don’t seem to be modified by hypoxia at the mRNA level. In normal cells, HIF-1β protein occurs, while HIF-1A is degraded and it also has a small half-life. But during hypoxia, this degradation is inhibited leading to increased HIF-1 protein. This protein induces the transcription of genes that help cells survive hypoxia. Moreover, under hypoxia conditions, the stability and transcriptional activity of HIF-1a is being controlled by post-translational modifications (Masoud et al. 2015).

HIF-1A has been found overexpressed in gliomas. Specifically for GBM, the protein is related to tumour growth and angiogenesis. It also helps in cancer cell’s survival under hypoxic conditions, with the elevation of glycolysis and angiogenesis (Jensen et al. 2006; Ahmed et al. 2018). The median expression of HIF-1A in GBM tumour and normal tissue is 104.26 and 15.94, respectively, according to Gepia (http://gepia.cancer-pku.cn/detail.php?gene=HIF1A). Our bibliographic research indicated that the downregulation of HIF-1A can lead to the regulation of cell cycle and apoptosis-related pathways (Gillespie et al. 2007; Zhou et al. 2013). This downregulation makes the glioblastoma cells more sensitive to temozolomide and radiotherapy, increasing effectiveness (Kessler et al. 2010). Under hypoxic conditions, it activates the transcription of over 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, HILPDA, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia (Masoud et al. 2015). It plays an essential role in embryonic vascularization, tumour angiogenesis and pathophysiology of ischemic disease.

In general, hif-1a is an important transcriptional regulator of the adaptive response to hypoxia. The transcription of 40 genes is activated, under hypoxic conditions. Hif-1a is increased in a lot of solid tumours and it is associated with poor prognosis of various cancer types. Genetic variability of HIF1A was also found to be associated with cardiovascular system diseases like: ischemic heart disease, coronary artery disease (CAD) with stable exertional angina, premature coronary artery disease, pre-eclampsia, acute myocardial infarction and frequent intradialytic hypotension. It also determines the outcome of inflammatory and infectious diseases (Kessler et al. 2010).

The third protein target that we examined as suited to our project is PLK1. This serine/threonine protein kinase belongs in the PLK (Polo-like Kinases) family. PLK1 has an important role in mitosis, since it regulates cell division and maintains genome stability, spindle assembly and DNA damage response.
PLK1 is overexpressed in a lot of human cancers and it is connected with poor prognosis in patients (Iliaki et al. 2021). Its role as an oncogene has been identified by various studies, and it is also upregulated in GBM (Lee et al. 2012). The median expression of PLK1 in GBM tumour and normal tissue is 17.36 and 2.44, respectively, according to Gepia (http://gepia.cancer-pku.cn/detail.php?gene=PLK1). As a therapeutic target, its down-regulation would decrease cancer cell survival, induce apoptosis and decrease resistance to chemotherapy drugs, while having little effect on healthy cells (Wang et al. 2020; Jiang et al. 2021).

Moreover, there is a mutual regulatory relationship between PLK1 and STAT3, since their dual inhibition can inhibit the invasiveness of glioma cells and induce apoptosis, via the regulation of MYC. In general, PLK1 phosphorylates various substrates to regulate a lot of essential steps throughout mitosis and cytokinesis. It is enhanced in tissues like bone marrow, lymphoid and the testis. This protein is expressed in embryonic tissues, but reduced in the thymus and ovaries in adults (Wang et al. 2020).

Hybridization Chain Reaction

To deliver with safety and efficiency the siRNAs with Theriac we exploited a technique called Hybridization Chain Reaction (HCR). HCR is an isothermal amplification method without the need of enzymes. This technique is based on the recognition and hybridization events between different types of DNA hairpins in the presence of a specific molecule that initiates the reaction (initiator). We thought we could alter these hairpins to carry therapeutic molecules so that this process could be used as a tool in a new therapeutic approach for GBM (Evanko et al. 2004). In detail, Theriac consists of 4 DNA hairpins (HCR-hairpins) complementary to each other; when all hairpins are hybridised in between, two siRNA molecules are released.

Nanocarrier

As mentioned before, siRNAs are easily degraded when free in the blood (Ali et al. 2012). Did it not trouble us for months, how we would administer our molecules in a way that could improve the therapeutic index! There is one obvious way to pass biological barriers and achieve maximum concentration in the target cells -with the least possible side effects; someone must carry the package intact to the target. Henceforth comes our nanocarrier!

An extended literature search into the most relevant papers available pointed out that nanocarriers would be the best option: they meet requirements such as safe transport to every corner of the brain, and enhancement of the MRI signal without pseudoprogression issues.

In the past, many nanocarriers have been tested for drug delivery in glioblastoma. Polymeric nanoparticles (micelles, dendrimers), liposomes, lipid nanocapsules , inorganic materials (iron, silica, gold) are prime examples. For some context, therapeutic agents are loaded in these particles by either encapsulation, by covalent linking or by surface adsorption.

What makes a systemic nanocarrier ideal for brain drug delivery is having as many as possible of the following properties:

  • it is nontoxic, biodegradable, biocompatible
  • it should not be causing immune response (e.g. inflammation)
  • it protects the drug from degradation
  • it has targeting strategies to be selectively delivered to the brain
  • it should be improving therapeutic efficacy and safety
  • it avoids opsonization and consequent clearance by the reticuloendothelial system (RES) and has a long plasma circulation time.
Lastly, specifically for our project, we wanted the nanocarrier to enhance the MRI signal to achieve diagnostic properties.

For safety reasons, we decided to go with nanocarriers that have already been approved by the Food Drug Administration (FDA) or European Medicines Agency (EMA). An advantageous characteristic we were looking for is whether the nanocarrier has already been used for transporting molecules to the brain before. Some nanocarriers had indications for such use while others had not- for some of the ones that didn’t, recently published papers had found they might be also applicable as transporters to the brain. Two other reasons for elimination appeared from the need for two certain characteristics: MRI enhancement and liposomic structure. This meant that e.g. colloid substances with inorganic materials other than iron, did not suit our purpose and were abandoned (Mehrabian et al. 2022).

After following the aforementioned inclusion-exclusion criteria, we concluded that we should use as a prototype specific FDA-approved nanoparticles:

  • Ferumoxytol – Feraheme: magnetic nanoparticles – USPION (Huang et al. 2022)
  • Ferrixan: magnetic nanoparticle – SPION (Stueber et al. 2021)
  • ONPATTRO – Patisiran: Lipid nanoparticle RNAi for the knockdown of disease‐causing TTR protein (Anselmo et al. 2019)

Delivery

A non-invasive alternative approach for directly addressing the CNS has been proposed very recently: intranasal medication delivery. Bypassing the BBB, this method of delivery minimises systemic adverse events that have long been a drawback for intravenous administration. A number of formulations have been proposed, mostly using drug delivery technologies that are nano-sized, to further improve nose-to-brain transfer modalities. In intranasal administration the molecules are crossing into the brain through axonal transfer (olfactory nerve cells) or though the paracellular pathway crossing through the “gaps” of the cells, reaching the subarachnoid space (Trevino et al. 2020; Lee& Minko 2021). Overall, pre-clinical animal studies showed that after intranasal delivery, anticancer medications will have better biodistribution and a more potent therapeutic effect. Additionally, clinical trials carried out in selected GBM patients using this route of administration demonstrated patient compliance and favourable results (Upadhaya et al. 2020).

In several exploratory studies over the course of the past five years, intranasal administration has been used as a highly accessible, non-invasive delivery method, paving the way for a better future management of GBM. However, there are still drawbacks since medications may be deteriorated or fail to reach the site of action.

References

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