RIBOLUTION – Platform for the identification of ncRNA-based diagnostics

Effective and specific early detection using molecular diagnostics

Due to demographic developments our healthcare system faces major challenges. An increasingly aging population also means increasing numbers of oncological, degenerative and chronic inflammatory diseases. This results in rising costs that place an increasing burden on our healthcare system. Effective molecular diagnostics offer a potential solution for this problem. It uses the presence or concentration of specific molecules, so-called biomarkers, as indicators of disease or the response to a particular form of therapy. Improved early detection therefore enables difficult and costly disease progressions to be avoided. In addition, an improved differential diagnosis based on biomarkers enables therapies to be individually adjusted for each patient. Despite a high demand for clinical diagnostics, there is currently a lack of such markers, which show sufficient sensitivity and specificity, for many conditions.

One example is chronic obstructive pulmonary disease (COPD) that, with over 600 million cases and over 2.75 million deaths worldwide per year, represents the fourth most common cause of death. Reliable diagnosis is only possible in advanced stages of the disease, through progressively decreasing lung function, which is usually too late for interfering therapy. The situation is similar for prostate cancer, one of the most common cancers in men. In this case there is also a lack of reliable biomarkers, which enable the unequivocal diagnosis of the tumor and its subsequent treatment in its early stages.

Non-coding RNAs as biomarkers

The aim of the RIBOLUTION project, where Fraunhofer IGB cooperates with the Fraunhofer institutes IZI (coordination), IPA, FIT, ITEM, as well as numerous clinical partners (Universities of Dresden, Leipzig, Charité Berlin) and the pharmaceutical company GlaxoSmithKline, is the identification of novel diagnostic indicators for diseases such as COPD and prostate cancer. Since January 2011 our project has focused on a new class of molecules, so-called non-(protein)-coding ribonucleic acids (ncRNAs), which are still largely uncharacterized. Previous studies have shown that ncRNAs represent the central level of cellular control in complex organisms and regulate diverse cellular processes, such as transcription, translation, RNA-editing, chromatin structure or epigenetic processes [1, 2]. It is suspected that they play a crucial role in disease development and therefore have great potential as diagnostic biomarkers.

Procedure

The first genome-wide identification phase of novel, diagnostically applicable RNA-based biomarkers will be carried out at the Fraunhofer IGB with the aid of high-throughput sequencing technology (Next-Generation Sequencing), which enables up to 109 DNA sequence fragments to be sequenced in parallel. This high density of data enables the denovo identification of significantly expressed ncRNAs in selected COPD or prostate cancer patient samples. In the second and third phases of the screening process these ribonucleic acids are further screened using specific probes on DNA microarrays (customized arrays) and subsequently validated by quantitative real-time PCR of up to 2000 patient samples.

GeneScapes Viewer.
Visualization of the sequence data using the GeneScapes viewer, developed at the Fraunhofer IGB.

Results

We are currently establishing suitable procedures for the preparation of ribonucleic acid for sequencing non-coding RNAs from patient samples (e.g. from whole blood in COPD), by Illumina high-throughput sequencing (HiSeq2000). Additionally, we are developing and validating various methods of sample preparation for both strand-specific and non-strand-specific sequencing. All procedures are to be carried out in a GLP-like environment, in order to assure the traceability and secure documentation for later certification or licensing of the diagnostic markers.

Outlook

The high standard of process and quality control will ensure that the identified biomarkers will be valid. It will also be possible to transfer the information and experience gathered throughout the RIBOLUTION project to other relevant diseases. In this context, the project will make major contribution to the field of “personalized medicine”.

Literature

[1] Mattick, J. S. (2001) Non-coding RNAs: the architects of eukaryotic complexity. EMBO Rep. 2(11): 986-991

[2] Mattick, J. S.; Makunin, I. V. (2006) Non-coding RNA. Hum Mol Genet. Apr15;15 Spec No 1: R17-29

Project and cooperation partners

  • Fraunhofer IZI, Leipzig (coordinator)
  • Fraunhofer IGB, Stuttgart (lead: biomarker discovery)
  • Fraunhofer IPA, Stuttgart
  • Fraunhofer FIT, Sankt Augustin
  • Fraunhofer ITEM, Hannover
  • Universitätsklinikum Carl Gustav Carus, Dresden
  • Universität Leipzig
  • Charité Universitätsmedizin Berlin
  • GlaxoSmithKline, London

Funding

We would like to thank the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) for their funding of the project “RIBOLUTION – Integrated platform for the identification and validation of innovative RNA-based biomarkers for personalized medicine”.