For quite some time, the main focus in molecular biology was on DNA, being the carrier of the genetic code, with RNA being viewed merely as an intermediary player. However, lately it has become clear that RNA plays a much more prominent role and is a key player in the cell's regulatory mechanism.

Various new types of RNA have been discovered, which has provided a further boost to RNA research. As a result, research into small regulatory RNA molecules and in particular microRNA (miRNA) has experienced an exponential gain in attention. The realization that RNA molecules play an essential role in gene regulation has fuelled research in this area, leading to a steep increase in the amount of papers published on regulatory RNA molecules.

The award of the 2006 Nobel Prize in Physiology or Medicine to Andrew Fire and Craig Mello for their discovery of RNA interference - gene silencing by double-stranded RNA - is a clear sign of the importance of this field. MicroRNA (miRNA) are naturally occurring, non-coding strands of RNA that trigger the RNA interference pathway. They result from an extensive processing route in which a long RNA transcript, folded in a hairpin structure, is cleaved into short strands of approximately 22 nucleotides - the miRNA molecules.

MiRNAs regulate gene expression by controlling the efficiency of messenger RNA (mRNA) translation - the process of translating genetic information into proteins. The miRNA binds to its target sequence in mRNA transcripts, which leads to translational repression or mRNA degradation. As a result, the production of protein encoded by that particular mRNA sequence is inhibited.

So far, approximately 550 human miRNA sequences have been documented. Of many it is not clear what genes they target. However, some miRNAs have been reported to regulate the expression of genes involved in differentiation and cell growth, tumor suppressors and oncogenes. Each miRNA may regulate the expression of multiple genes. miRNAs are now recognized as one of the key regulators of gene expression, involved in almost every aspect of a cell life from cell differentiation to apoptosis, although specific functions have been elucidated only for a handful of miRNAs.

Check the references below for some relevant research papers and reviews on miRNAs

Technology and IP portfolio

The core of InteRNA's technology position is the use of proprietary, highly advanced bioinformatics methods to analyze, predict and validate miRNA sequences. This has led to the identification of a large population of novel miRNAs and subsequent patent applications. InteRNA applies the following technologies:

• Identification of novel miRNAs by massive parallel sequencing
• Profiling on miRNAs from human tissue
• Computational analyses of large sets of small RNA sequencing data
• Biological validation of candidate miRNAs by (modified) RAKE assay and other molecular biology techniques
• Bioinformatical integration of multiple molecular datasets
• Tools to perform functional assays
• Functional assays for validation of miRNAs

IP Portfolio

At present, InteRNA has patented approximately 1,000 mature human miRNA sequences and over 4,000 human hairpin sequences. In addition, we have patented novel microRNAs identified in various tissues of mouse and primates. Approximately 750 miRNA sequences discovered by us can be found in the miRNA database miRBase 10.
Our IP portfolio is continuously being expanded by validation of the above described human miRNAs in disease tissue.

Discovery and validation process

Novel miRNA candidates were identified by a number of different methods. Computational prediction resulted in the identification of a great number of novel miRNAs. These miRNA candidates were confirmed using microarray based RAKE (RNA-primed, array-based Klenow enzyme assay) and other molecular biological techniques, such as detection by Northern Blot and Q-PCR. Most of our proprietary miRNA sequences do not originate from computational predictions, but were experimentally cloned from small RNA of various sources. Further computational approaches - including stable hairpin formation and phylogenetic conservation criteria - resulted in the classification of candidate miRNAs. Additional biological validation of a subset of candidate miRNA sequences in human tumor samples was achieved by microarray profiling.

Tools

We are currently pursuing various experimental approaches to identify biological roles of the candidate miRNAs. Functional assays employing both miRNA overexpression and knockdown techniques are being implemented.

Data analyses platform

Recent advances in DNA sequencing technology make it possible to read billions of DNA bases in a single run. These ultra-high-throughput (U-HTP) sequencing technologies are changing the approaches to genome-related research and open up an era of personalized genomics, where each individual can have its own genome sequenced. Today, these new technologies enable novel approaches for small RNA discovery and detection, gene expression profiling, ChIP-SEQ, etc. These developments have lead to the generation of novel software tools by our bioinformatics experts. These new tools for analyses of large sets of raw sequencing data are exploited by InteRNA Genomics, a subsidiary of InteRNA Technologies. InteRNA Genomics offers services and software for processing, analyzing and presenting U-HTP sequencing data generated by various platforms. Our informatics solutions convert large sets of raw sequencing data into interpretable formats and integrate this information with existing proprietary and public data resources.

Opportunities

We are open to discussions on collaborations with academic as well as commercial partners. Opportunities for licensing also exist outside InteRNA's focal area, such as the development of diagnostic and prognostic assays.

References

• Berezikov, E., Guryev, V., van de Belt, J., Wienholds, E., Plasterk, R. H. and Cuppen, E. (2005) Phylogenetic shadowing and computational identification of human microRNA genes. Cell 120, 21-24
• Berezikov, E., van Tetering, G., Verheul, M., van de Belt, J., van Laake, L., Vos, J., Verloop, R., van de Wetering, M., Guryev, V., Takada, S., van Zonneveld, A. J., Mano, H., Plasterk, R. and Cuppen, E. (2006) Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis. Genome Res 16, 1289-1298
• Berezikov, E., Thuemmler, F., van Laake, L., Kondova, I., Bontrop, R., Cuppen, E. and Plasterk, R. H. (2006) Diversity of microRNAs in human and chimpanzee brain. Nat Genet 38, 1375-1377
• Kloosterman, W. P., Steiner, F. A., Berezikov, E., de Bruijn, E., van de Belt, J., Verheul, M., Cuppen, E. and Plasterk, R. H. A. (2006) Cloning and expression of new microRNAs from zebrafish. Nucleic Acids Res 34, 2558-2569
• Takada, S., Berezikov, E., Yamashita, Y., Lagos-Quintana, M., Kloosterman, W. P., Enomoto, M., Hatanaka, H., Fujiwara, S., Watanabe, H., Soda, M., Choi, Y. L., Plasterk, R. H. A., Cuppen, E. and Mano, H. (2006) Mouse microRNA profiles determined with a new and sensitive cloning method. Nucleic Acids Res 34, e115
• Berezikov, E., Cuppen E. and Plasterk, R.H. (2006) Approaches to microRNA discovery. Nat Genet 38 suppl: S2-7
  
  

Clinical relevance of miRNA

Currently only a limited number of biological targets has been identified, but it is widely agreed that miRNA plays an important role in a variety of biological processes, including cell death, cell growth and fat storage. The individual functions of the majority of the discovered miRNAs remain unclear and substantial research efforts are required to uncover these functions. Numerous reports emphasize however that deregulation of miRNA is involved in a number of pathological conditions, such as cancer, viral infections, metabolic diseases and neurological disorders.

As research progresses, it is to be expected that the specific role on miRNA in these conditions becomes more clear and that additional functions of miRNA and its role in other pathological conditions come to light. The findings described below provide ample motivation for further research into miRNA as well as interesting leads for the development of miRNAs for diagnostic, prognostic and therapeutic applications.

A concise overview of what is known so far.

miRNA in Cancer

The initiation and progression of cancer are two processes that may involve miRNA. In several tumor types, miRNAs have been detected, including breast, lung, colon and thyroid cancer, lymphomas and leukemia. Interestingly, miRNA sequences have been identified in parts of deleted or translocated genes in leukemia and lymphoma. In those cases, miRNA may have had a tumor suppressor function. On the other hand, other studies have shown miRNA to be expressed at a higher level in certain tumor cells. These miRNA molecules may therefore function as oncogenes. Cancer specific fingerprints of miRNA have been identified in a large number of cancer types - B-cell chronic lymphoid leukemia (B-CLL), glioblastoma and breast, lung, colon, pancreatic, thyroid, hepatocellular and gastric carcinoma. Expression profiles of miRNAs have also been shown to change during cancer progression. It is therefore likely that other hallmarks of cancer are also affected by miRNA, such tumor angiogenesis and the ability to metastasize.

Current efforts concerning the use of miRNA in oncology are mostly focused on miRNA profiling for diagnostic and prognostic purposes. InteRNA Technologies believes there are vast possiblities to apply miRNA profiling for therapeutic applications and will concentrate its efforts in this direction.

Viral infections

There is evidence that miRNA is implicated in viral infections, particularly in persistent ones. Two forms of miRNA have been described in relation to viruses. First, there are miRNAs that are encoded by the virus itself. Examples of viruses that encode miRNA are several herpes viruses, SV40, cytomegalovirus (CMV), HIV and adenovirus. The function of these miRNAs is not fully clear yet, but it seems that virus miRNA promotes latency and therefore persistency. Second, there are the host miRNAs. These also seem to play a role in viral infections; one example is miRNA-122 in the liver which has been shown to prevent viral replication of the Hepatitis C virus (HCV).

Metabolic diseases

Recent reports suggest a role for miRNA in human fat metabolism and insulin secretion. It is therefore speculated that deregulation of miRNA might be involved in diabetes and obesity, but more research is needed to gain a clear picture.

Neurological disorders

Recently, miRNA has been demonstrated to play an important role in brain development. Based on the currently available data, it is anticipated that deregulation of miRNA might also be relevant to certain neurological disorders, especially those of neurodevelopmental origin, such as schizophrenia and autism.