For researchers and investors

The MiRacle project aims to bring a tumour-selective lethal miRNA into clinical Phase I testing by encapsulation of the miRNA in a tumour targeted delivery vehicle. This requires technological solutions on upscaling of the formulated miRNA into a stable formulation, exploration of the efficacy in validated tumour models and testing in toxicity models.

This outcome will both benefit head and neck cancer patients as well as the pharmaceutical and chemical companies that produce and sell the product.

From a scientific perspective, this will bring the first miRNA-based drug that is delivered directly to tumour tissue. The tumour-selective lethal miRNAs in this project offer great promise as they selectively eradicate tumour cells and not normal cells. Moreover they influence multiple genes, which may reduce the chance that tumours develop resistance. Finally they will be formulated in an innovative tumour cell targeting delivery vehicle that might enhance the safety profile by prevention of side-effects in vital organs.

To achieve this, the Small and Medium sized Enteprises (SMEs) and Research and Technological Development Performers (RTDPs) in this project combine and develop scientific and technological expertise, such as suitable therapeutic animal models, technical tools for up-scaling, detection of a formulated miRNA and pharmacokinetics and toxicity of the drug product. From a scientific and technical point of view, these technologies seem straightforward, but performance of these experiments with formulated nucleotides is novel and runs at the forefront of science since formulated oligonucleotide based drugs have so far not yet reached the market. Therefore the three SMEs line up with three RTDPs, which bring in the required technologies and models to test these new anti-cancer agents in vitro and in vivo.

Why use miRNA?
MicroRNAs (miRNAs or miRs) are a recently discovered class of endogenous 15-22 nucleotide small non-coding RNAs that function as master regulators of the human genome. The frequent aberrant expression and functional implication of miRNAs in many human diseases have lifted these molecules to interesting preferred drug targets. The functionality of a therapeutic miRNA is based on the catalytic process of the miRNA to pair with mRNAs carrying complementary sequences and consequently repress gene expression. Imperfect base pairing between miRNAs and mRNAs is common and enables miRNAs to regulate a broad, but nevertheless specific set of oncogenes. In view of cancer as a heterogenic disease that cannot be successfully treated by targeting a single gene, it is this ability of miRNAs that may hold the key to therapeutic success (1).

Head and neck cancers
This project focuses on therapeutic miRNAs in head and neck cancer. Head and neck squamous cell carcinomas (HNSCC) develop in the mucosal linings of the upper aerodigestive tract and contribute to approximately 5% of all cancers in the Western world (2). The prognosis of HNSCC remains disappointing, and development of novel anti-cancer agents is urgently awaited (3). Since HNSCC patients that are positive for human papillomavirus (HPV) infection, benefit well from existing therapy, this project will focus on novel treatments for tumours with a poor prognosis, those that are not caused by HPV infection.

In recent years, the treatment paradigm for HNSCC has shifted from surgery with adjuvant radiotherapy to chemoradiation with salvage surgery when indicated, depending on stage and subsite of the tumour. Also in cases that are primarily treated with surgery and postoperative radiotherapy, the addition of chemotherapy is used with increasing frequency. In addition, new EGFR-based targeted therapies are being used concurrently with radiotherapy (bioradiation), especially in patients who are unfit to receive chemotherapy (4). The five year-survival rate for patients with early stage disease is over 90%. Unfortunately the majority of HNSCC patients present with advanced stages of disease. These patients frequently develop locoregional recurrences, distant metastasis and/or second primary tumours resulting in 5-years survival rates of less than 60% (4). The development of novel anti-cancer agents to improve outcome is therefore urgently awaited.

Selecting the best miRNA candidate

The project will first select the most suitable miRNA candidate. After deciding for the best miRNA candidate, the synthesis process has to be upscaled from laboratory to pilot plant scale without significant quality loss.

There are several features to be considered when developing successful miRNA candidates. The synthesis has to be cost-effective, meaning that the solid-phase synthesis process itself has to be optimized to deliver a full-length raw product with an already significant purity of about 70-75%. Some combinations of sequences, structures or modifications tend to produce preliminary terminated oligonucleotides, which require a tedious and cost-intensive purification procedure. In the worst case, the oligonucleotide cannot be purified to the required purity of normally 85%-90%.
Also structural information about secondary and higher structures of the candidate molecules are of valuable interest and can be helpful in choosing the best miRNA candidate. DSC (Differential scanning calorimetry) may give valuable information about the structural change of a certain oligonucleotide and thereby expanding the oligonucleotide analysis methods such as HPLC-analysis for purity of the single strands and the resulting duplex, and LC-MS for mass determination of the single strands. .

Thereafter, the efficacy, immunogenicity, and the stability of the candidate molecule in the biological system will be assessed.

Developing analytical formulation and processes and scaling up the formulation of freeze dried drug products

MiRNAs are double stranded RNA molecules consisting of 15-22 nucleotide basepairs. So far, the partners have used commercially available synthetic miRNA molecules to test the anti-proliferative activity of the tumour-selective lethal miRNA in head and neck squamous cell carcinoma (HNSCC) cell lines using a standard cell transfecting agent. The transfecting agent is needed to bring the highly negatively charged RNA molecules into the cell. Meanwhile, research from the project parnters showed that non-chemically modified miRNAs may cause a strong innate immune response in a human whole blood assay. It is well known that this stimulation of the immune system can be prevented by correct chemical modification of RNA molecules (5). In a prior study, project partners synthesised several chemically modified miRNA molecules. These studies showed that each miRNA sequence requires an optimal chemical modification pattern to ensure a low immune response while retaining or improving its anti-proliferative potency.

In addition, the project will be using a delivery system that is suitable to bring small RNA molecules into tumour cells in humans. This new drug delivery system specifically targets CD44 positive tumours by coverage of lipid captured RNA molecules with hyaloronic acid (HA). This formulation is named gagomers (GAGs) and was invented by Prof. R. Margalit and Dr. D. Peer (6) and licensed to Quiet Therapeutics (Quiet), a partner in this project.

Even if different therapeutic entities can be loaded in this delivery system, Quiet focuses its efforts on nucleic acid-based drugs to establish the proof-of-concept in vivo, upscale GAGs production and conduct safety studies. HA is natural to the body, distributed widely throughout connective, epithelial, and neural tissues. HA binds mainly to CD44, a cell surface glycoprotein involved in cell -interactions, -adhesion and -migration.

Although CD44 is present on normal and cancer cells, CD44 conformation varies between malignancies (7). Quiet has shown that CD44 was highly expressed on both normal and cancer cells. However, GAGs displayed higher affinity to the cancer cells. This is explained by the splice variants of CD44 present on the surface of cancer cells as opposed to normal cells (8).

Studies conducted on head and neck cancers have demonstrated the presence of v3, v6 and v10 CD44 variants (9). In fresh head & neck cancers, the selective binding of GAGs to cancer cells is observed with a very low binding to adjacent normal cells (10). A key step in the development of GAGs is development of a formulation that can be easily stored and retains activity. Exploratory research using lyophilisation is on-going and will continue in the consortium with a special emphasis on HNSCC cancer applicability.

The above illustrates that the first steps towards a novel drug candidate for treatment of head and neck cancers requires the following research and development steps:

1. Identification of chemical modification with low immunogenicity and high activity
2. Formulation development and production of lyophylised formulation
3. Synthesis of GLP batch of miRNA
4. Synthesis of GLP batch of delivery vehicle (GAGs)
5. Stability of GAG-formulated miRNA

Tissue distribution and efficacy studies with formulated miRNA

Once the correct chemical modification of the tumour-selective lethal miRNA is established and a protocol for freeze dried pilot batches of GAG formulated miRNAs is established, these pilot batches will be tested in vivo to study the tissue distribution and tumour targeting of miRNA delivery. As a first step the detection method will be developed using e.g. stem-loop PCR and Elisa-probe detection. In collaboration with the RTD performers the best method will be selected. This method will be used to determine the amount of miRNA that accumulates in the tumour after single dosing and multiple dosing.

In parallel the efficacy of the miRNA in the tumour cells will be determined by measuring the differential expression of a miRNA target gene or biomarker gene after a single dose of formulated miRNA. The single dose that results in highest and longest gene knock-down will be used to explore different dosing regimen in in vivo tumour inhibition studies.

In vivo assessment of the anti-tumour efficacy in animal models

The project will deliver a single dose resulting in strong regulation of a miRNA target gene or biomarker gene. This dose will be used in different regimens to study the inhibition of head and neck tumour growth in two different head and neck cancer mouse models (VU-SCC-OE and UM-SCC-22A). Since current therapy are combinations of surgery and irradiation or cisplatin with irradiation, first the maximum tolerated dose and tumour growth inhibition curves when using irradiation/cisplatin (IR/Pt) treatment will be determined.

The efficacious dose for irradiation in mouse models is known, but the combination with cisplatin is unknown. Therefore first the optimal dosing regimen for current standard of care (IR/Pt) will be determined. The maximum dose for GAG-formulated miRNA is presently 2 mg/kg, but a higher or lower efficacious dose will emerge from the project. The final efficacy experiment of GAG formulated miRNA will be added to and compared to standard of care. When the GAG formulated miRNA shows efficacy in vivo, we foresee that the GAG formulated miRNA treatment will be combined with IR/Pt treatment or might substitute the Pt. The results will be used to build a project suitable for partnering with the Pharmaceutical industry to move the miRNA further in pre-clinical development.

Safety and toxicity studies of formulated miRNA

The goals of this non-clinical safety evaluation include a characterisation of toxic effects with respect to target organs, dose dependence, relationship to exposure, and, when appropriate, potential reversibility. This information will be used to estimate an initial safe starting dose and dose range for the human trials and to identify parameters for clinical monitoring for potential adverse effects. The non-clinical safety studies, although usually limited at the beginning of clinical development, should be adequate to characterise potential adverse effects that might occur under the conditions of the clinical trial to be supported.

The next step would then be, although outside the scope of MiRacle, human clinical trials to investigate the efficacy and safety of a pharmaceutical, starting with a relatively low systemic exposure in a small number of subjects. This would be followed by clinical trials in which exposure to the pharmaceutical usually increases by duration and/or size of the exposed patient population. Generally, in toxicity studies, effects that are potentially clinically relevant can be adequately characterised using doses up to the maximum tolerated dose (MTD).

Assessment of biomarkers and mechanism of action

MiRNAs are multi-pathway inhibiting agents, i.e. they antagonize many different genes by pairing with mRNAs carrying complementary sequences and consequently repress gene expression. The base-pairing occurs through a conserved seed-sequence of 7-8 nucleotides plus imperfect base pairing outside the seed sequence region. Since many mRNAs carry the complementary seed sequence (preferentially in the 3’UTR), many mRNAs bind and thus a broad but nevertheless specific set of genes is regulated. To assess the mechanism of action through which the tumour-specific lethal miRNA induces cell death, an RNA-seq experiment will be performed to determine the set of differentially expressed genes in the head and neck cancer cell lines after treatment of the cell line with the miRNA (using the same cell lines as in the in vivo efficacy models).

From previous experience the project partners have noted that the miRNA target gene prediction tools (based on pairing of the seed sequence to the human genome) are not very accurate, and the project will also filter the differentially expressed set of predicted and non-predicted genes by overlapping down-regulated genes with genes that are expressed at low level in normal cells and at high level in malignant cells, assuming that such genes apparently essential for the tumour are likely upregulated. MiRacle has such a dataset available of paired microdissected tumour and mucosa samples from 23 head and neck cancer patients.

The expression profiles of selected candidate target genes will be confirmed by qRT-PCR and the biological role will be validated by transfecting cells with shRNA for these candidate genes to reproduce the phenotype. Subsequently the binding of the miRNA to the mRNA of the candidate gene will be investigated by a 3’UTR binding assay and mutation of the binding site will show that the binding is indeed specific.

The RNA-seq approach will deliver down-regulated genes that are biomarkers for efficacy, particularly when validated by functional experiments. These markers will be validated by qRT-PCR and can directly be used as efficacy marker in the tissue distribution and tumour delivery studies. The mechanism of action of the microRNAs and the biomarkers will serve as efficacy markers and add to the pre-clinical drug development package and generate new business opportunities.


  1. Bader et al. Gene Therapy 2011
  2. Kamangar JCO 2006
  3. Leemans et al Nat Rev Cancer 2011
  4. Leemans et al. Nat Rev Cancer 2011
  5. Robbins et al. Oligonucleotides. 2009
  6. Tel Aviv University, Mizrahy et al Journal of Controlled Release 2011; Rivkin et al Biomaterials 2010
  7. Ponta et al Nat Rev Mol Cell Biol 2003
  8. Wallach-Dayan et al J Cell Sci 2001, Salles et al. Blood 1993, Kuniyasu et al Clin Cancer Res 2001, Kuhn et al Mol Cancer Res 2007
  9. Wang et al Laryngoscope 2009
  10. Bachar and Peer BioMaterials 2011