MULTIPLEX ANALYSIS OF PRIMARY miRNA REGULATORY NETWORKS IN VIVO

Every cell within our body carries the same genetic information (DNA), yet following iterative developmental transitions hundreds of morphologically and functionally distinct cell types are being generated. At the foundation of this fascinating cellular diversification lies a milieu of finely orchestrated and sophisticated regulatory programmes, which act to turn on or off thousands of genes (~20,000 in humans) with minute spatial and temporal precision. Errors in these programmes can give rise to developmental defects and many human diseases including cancer. One such essential regulatory layer is provided by microRNAs (miRNAs). miRNAs are fascinating RNA molecules comprised of only 21 to 23 letters of the genetic alphabet, and are never translated into a polypeptide chain (protein). Strikingly, these tiny pieces of RNA appear to act as molecular rheostats of gene expression programs (protein production) in almost every living organism, by binding to numerous messenger RNAs (mRNAs) via defined "miRNA response elements" (MREs). Therefore, central to understanding cellular pathways regulated by miRNAs, is identification of their direct functional targets (MREs) in vivo. Paradoxically, this essential facet of miRNA biology has met with relatively limited success to date.

Due to the complexity of the cellular environment, identifying all functional targets of a miRNA in the context of a living cell requires a systems biology approach, which hitherto had been technically unfeasible. Here, we propose to develop an innovative high-throughput discovery pipeline, which will enable for the first time a systems-level functional characterization of any primary miRNA target network. At the core of this revolutionary approach lies the development and assembly of two cutting edge technology platforms: i) a multiplex genome engineering-based strategy for assessing MRE activity; ii) a platform for parallel interrogation of miRNA target site accessibility. Integration of these tools in a combinatorial mode will engender unprecedented insight into the principles and rules governing miRNA target selection in vivo, thus addressing this fundamental, yet unmet, dimension in miRNA biology. Therefore, we anticipate that this endeavour will have a major impact on the research community, empowering scientists with the ability to understand, predict, and assess the impact of miRNAs in the context of a living cell. Considering the interdisciplinary nature underlying this research, the proposal brings together four leading international research centres: Weatherall Institute of Molecular Medicine Oxford, Genome Engineering Centre Oxford, Oxford Genomics Centre (WTCHG), and the EMBL European Bioinformatics Institute (EMBL-EBI) Cambridge.

microRNAs (miRNAs) provide an essential layer of gene regulation underlying cellular function, development and disease. The specificity of miRNA target recognition is largely dictated by promiscuous sequence complementarity with cellular RNAs (6-8nt). Consequently, computational algorithms and high-throughput biochemical approaches have predicted thousands of putative miRNA targets. Surprisingly, despite tremendous progress in understanding the complexity and importance of miRNAs, the physiological relevance of only a small proportion of possible miRNA-target interactions has been functionally established. Compounding the challenge, the rules governing miRNA target selection in a complex cellular environment remain poorly understood. Deciphering this fundamental aspect of miRNA biology requires novel high-throughput strategies for functional interrogation of miRNA regulatory pathways in the context of a living cell. Here we propose to develop a set of technology platforms, and assemble them into a cutting edge discovery pipeline for multiplex functional analysis of primary miRNA target networks. Each of these tools is designed to provide definitive insight into a specific layer of miRNA-target recognition, and act in a combinatorial feedback circuit within the pipeline. Specifically, we will develop: i) a platform for multiplex investigation of MRE activity via programmable RNA-guided nucleases (CRISPR/Cas9); ii) a strategy for parallel interrogation of miRNA target site accessibility. Using this pipeline, we propose to study for the first time the impact of one miRNA (miR-184) on its complete repertoire of cellular targets, and decipher the molecular mechanisms underlying selectivity within a miRNA target network. We anticipate this endeavour will unlock unprecedented potential for complex miRNA studies in virtually any cellular context.

The proposed project is focused on developing an innovative multiplex technology platform and deciphering a fundamental aspect of miRNA-mediated gene regulation. However, we anticipate that the outcome of this research could have a broader spectrum of applications and impact biomedical sciences at multiple levels. Examples include: basic academic research, biotechnology and therapeutic discovery.

1. Academic impact. The impact of this study on academic research is carefully detailed in the "Academic beneficiaries" section of this application. The technologies we propose to develop will mark a paradigm-shift in the strategies currently used for the discovery and functional characterization of primary miRNA target networks. As such, they will impact miRNA studies at multiple levels (see Academic beneficiaries). Furthermore, although the proposal focuses on miRNA research, the basic concepts and resources generated throughout the course of this study can be adapted to benefit research on genome regulation outside of the miRNA field.

2. Industry and commercial impact. In principle, the individual technologies developed here, or the entire combinatorial pipeline could be streamlined and packaged into ready-made kits for commercial use. Since the final readout of each technology relies on a standard next generation sequencing platform, this step could be carried out in a single reaction by pooling rationally designed targeted NGS libraries, defined by assay-specific barcode systems. Under this scenario, much like the current NGS kits or Nanostring nCounter profiling assays, companies could offer generic or custom-designed probe sets for the analysis of any candidate miRNA regulatory pathways in various species.

3. Impact on therapeutic discovery. Because miRNAs are much smaller, less antigenic and display a stereotypical mechanism of action compared to protein-coding genes, the field of miRNA-based therapeutics is rapidly gaining momentum. Both miRNA replacement and systemic miRNA inhibition therapies have now entered clinical trials for a variety of human diseases. Examples include the Hepatitis C antiviral drug miRavirsen designed to recognize and sequester liver-expressed miR-122, or the liver cancer tumour suppressor drug MRX34. However, although miRNAs appear to have superior therapeutic potential, their broad transition into clinic remains a challenging task, primarily due to their pleiotropic effects on a large number of cellular RNAs. By providing the means to accurately define all the nodes in a primary miRNA target network, the technologies developed here could be integrated into drug discovery pipelines, allowing more targeted lead compound identification. Consequently, therapeutic agents could be designed directly against unique or multiple downstream targets of disease-associated miRNAs, thus alleviating the potentially harmful consequences of interfering systemically with the activity of a cellular miRNA.

4. Research training and societal impact. The multidisciplinary nature of the proposed research will provide an excellent training ground for the assigned PDRA and RA, as well as for at least one Oxford University undergraduate student who will have an opportunity to work on this project during a final year lab placement. Furthermore, the PI and assigned researchers will engage with the general public in an effort to promote a better understanding of the scientific approach and the benefits of cutting edge technology advances for biomedical discovery and human health (see Pathways to Impact document).