New microRNA Cocktail Shows Promise Against Glioblastoma

• Glioblastoma (GBM) is the most aggressive brain tumor, with around 200,000 deaths worldwide annually. Standard treatments, surgery, radiotherapy, and chemotherapy, rarely prevent recurrence, and the blood-brain barrier limits drug delivery. A new study led by Davide De Pietri Tonelli demonstrates that an 11-microRNA cocktail, delivered via ApoE-coated lipid nanoparticles, can slow GBM growth and reduce resistant tumor stem cells in laboratory models. Although not yet ready for clinical use, this research highlights the promise of multi-targeted therapies for GBM.

• “Simplifying the formulation would certainly facilitate regulatory acceptance and streamline future development. From a regulatory perspective, our nanoformulated cocktail occupies a grey zone. On the one hand, it could be classified as a gene therapy product, given that its active principle is based on miRNAs. On the other hand, it may also fall under the category of complex medicinal products (EMA) or complex drug products (FDA). Although the regulatory landscape has become more receptive to RNA-based formulations following the success of RNA vaccines, important challenges remain.” Professor Davide De Pietri Tonelli

Glioblastoma (GBM) is the most deadly brain tumor. It is estimated that about 200,000 people die each year, 15,000 in Europe and 9,000 in the United States. The biggest problems associated with glioblastoma arise from its exceptional aggressiveness. The tumor grows rapidly, penetrating deep into the surrounding brain tissue. Standard treatments, surgery, radiotherapy, and chemotherapy, can temporarily slow the disease, but rarely prevent its recurrence. An additional challenge is the blood-brain barrier, which prevents the entry of many potentially effective drugs. The scientific community is therefore intensively investigating new therapeutic strategies, including immunotherapy, gene therapy, and nanoparticulate drug delivery systems, in the hope that a significant breakthrough will be achieved in the future.

A new study is a significant step forward

“Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults, with a median survival of approximately 14.5 months, a figure that unfortunately has not improved over the past two decades.” That is how the new study “Synergic microRNAs suppress human glioblastoma progression by modulating clinically relevant targets” begins.

This study showed that a combination of 11 microRNA cocktails can successfully inhibit the growth of glioblastoma (GBM) and reduce the number of resistant tumor stem cells under laboratory conditions. The key was the use of ApoE-coated lipid nanoparticles (LNPs), which were specifically designed to enable stable and efficient delivery of the cocktail directly into the brain tumor.

The research was conducted by mostly Italian scientists, and the list of authors is extensive: Silvia Rancati, Rui C. Pereira, Michele Schlich, Stefania Sgroi, Silvia Beatini, Letizia La Rosa, Lidia Giantomasi, Roberta Pelizzoli, Clarissa Braccia, Andrea Di Fonzo, Carlotta Spattini, Kiril Tuntevski, Amanda Lo Van, Meritxell Pons-Espinal, Annalisa Palange, Adriana Bajetto, Antonio Daga, Andrea Armirotti, Tullio Florio, Paolo Decuzzi, and Davide De Pietri Tonelli.

The team is based at the Italian Institute of Technology (IIT) in Genoa, and the work also involved institutions such as the University of Genoa and IRCCS Policlinico San Martino. The study is led by Davide De Pietri Tonelli at the Istituto Italiano di Tecnologia.

We spoke with study authors Davide De Pietri Tonelli and Silvia Rancati.

First Study to Prove a Synthetic microRNA Cocktail Can Combat Glioblastoma In Vivo

As explained, microRNAs (miRNAs) are natural regulators of gene expression, crucial for processes such as cell growth, differentiation, and migration. In the healthy brain, they control the development of neural networks, but in glioblastoma, or the tumor, they enable uncontrolled growth and escape from therapy.

The goal was to find out more about miRNAs that can restore normal cell differentiation and prevent abnormal proliferation. After analysis, 11 miRNAs were selected that together affect multiple tumor processes at once. This cocktail, as stated in the study, not only slows down the growth of glioblastoma in laboratory conditions but also makes tumor cells more sensitive to the standard chemotherapeutic drug, temozolomide.

When the tumor cells were treated with the 11-miRNA cocktail, the researchers observed changes in cell dynamics, such as slower growth. It is important to note that the number of cells believed to be responsible for therapeutic resistance decreased. To more closely mimic conditions in the human brain, the researchers created three-dimensional spheroids, tiny “mini-tumors” that behave much more like real glioblastomas than traditional two-dimensional cultures. In these spheroids, the miRNAs had a pronounced effect: the invasive cells at the outer edge became far less active.

The biggest challenge

However, the biggest challenge is delivering the cocktail to the brain. The complexity of this phase, particularly related to brain delivery, was explained in detail by PhD Silvia Rancati, one of the study authors: “The main challenges associated with our therapeutic approach relate to in vivo delivery. The nanoformulation used in our study is composed of three primary synthetic components: a miRNA cocktail (11 miRNAs resembling siRNAs), the lipid constituents that form the LNP, and a protein coating (ApoE). Each of these elements is already individually available and used in clinical settings.

However, to advance this combined product toward preclinical validation, the formulation will require careful characterization and rigorous quality assessment. Therefore, reducing the complexity of the formulation is an essential objective, as doing so could significantly facilitate its transition toward clinical application. Fortunately, the field has progressed substantially since we began this work, and new RNA modifications are now available that increase stability and do not require lipid carriers, as well as slow-release delivery systems that may support more prolonged therapeutic effects.”

When the nanoparticles were injected into the brains of mice implanted with human glioblastoma cells, fluorescent signals confirmed that the miRNAs had successfully entered a large number of tumor cells, while healthy brain areas remained unaffected. The effect lasted for about two weeks, suggesting that future therapies may require periodic dosing, but also showing that the formulation is stable enough to achieve a significant therapeutic effect. Bioluminescent monitoring of tumor growth showed a slowdown during the period in which the nanoparticle formulation was active. When combined with temozolomide, the effect was even more pronounced in certain glioblastoma lines.

Temozolomid: It took more than a decade for its clinical approval

Temozolomid was developed by Professor Malcolm Stevens in a multidisciplinary drug discovery laboratory in the pharmacy department at Aston University (UK). It took more than a decade for its clinical approval. It was approved in the United States (FDA) and in Europe (EMA) in 1999. Even though temozolomide has become part of the therapy for the most aggressive brain tumor, glioblastoma (GBM), the median survival of patients is, as we mentioned, about 14.5 months.

As scientists point out in the study published in 2021, TMZ resistance remains a major limitation in the treatment of GBM and contributes to the dismal prognosis. Therefore, the need to develop innovative therapeutic approaches, such as combinatorial microRNA therapy, is more urgent than ever.

The EU could provide stronger and more accessible support for translational innovation

Are current investments in Europe sufficient to ensure an efficient and rapid transition from the laboratory to clinical trials?

Davide De Pietri Tonelli: While funding mechanisms exist, the current landscape suggests that the EU could provide stronger and more accessible support for translational innovation. Funding opportunities in the EU to support this transition fall into three main categories: public funding (EU-level and national), financial instruments (banks, venture capital, and related mechanisms), and private investment from industry (e.g., pharma or biotech companies). Public funding has not seen a major budget increase in recent years, while the number of applications—particularly to highly competitive EU schemes such as the EIC Pathfinder—has roughly doubled since COVID-19.

As a result, success rates have fallen from around 4% to approximately 2%, making these programmes feel increasingly like a gamble rather than a merit-based selection process. Financial investors generally seek projects that are already at an advanced preclinical stage, with clear IP, manufacturability, and regulatory pathways. In contrast, pharmaceutical companies tend to engage much later, usually once a product has reached clinical Phase II or beyond, when clinical relevance and commercial potential are more evident.

Looking ahead to 2026, what will be crucial for advancing further research in this area?

Davide De Pietri Tonelli: Simplifying the formulation would certainly facilitate regulatory acceptance and streamline future development. From a regulatory perspective, our nanoformulated cocktail occupies a grey zone. On the one hand, it could be classified as a gene therapy product, given that its active principle is based on miRNAs. On the other hand, it may also fall under the category of complex medicinal products (EMA) or complex drug products (FDA). Although the regulatory landscape has become more receptive to RNA-based formulations following the success of RNA vaccines, important challenges remain.

The urgent need for innovative strategies against one of the deadliest brain tumors

In the study, a series of in vitro and in vivo experiments were conducted, including cell culture cultivation, microRNA transfection, migration and invasion tests, cell growth and adhesion tests, 3D cultures in collagen hydrogel matrices, and clonogenic tests. Furthermore, the methods involved the use of lipid nanoparticles (LNPs), in vitro transfection, and intratumor administration in animals. For example, to assess safety, scientists injected the nanoformulated, fluorescently labeled molecule (siRNA) into the brains of healthy mice. The results were positive: no tissue damage or cell death was observed in the area around the injection. This study also required extensive monitoring. In the case of assessing the efficacy and delivery of miRNA via ApoE-LNPs, the creation and growth of the tumor were closely monitored over 49 days using the IVIS Spectrum in vivo imaging system.

Despite these promising laboratory results, the 11-microRNA cocktail is not yet ready for clinical use. The study demonstrates, however, the potential of multi-targeted therapies to tackle glioblastoma’s aggressive growth and treatment resistance. By combining innovative molecular strategies with advanced delivery systems, researchers are opening new paths toward more effective treatments.

Image: Gliobastoma/Immunitybio.com