At the outset of this project, we noted that small non-coding RNA has strikingly altered our understanding of cellular homeostasis. We elected to focus on (a) short-interfering RNA (siRNA) as a tool for whole-genome interrogation and discovery of therapeutic principles, and (b) on microRNAs, which regulate gene expression. The impact of microRNAs extends to virtually all aspects of biology. In the meantime, not only has strong evidence emerged that non-coding RNAs are altered in human diseases ranging from cancer and inflammation to metabolic and heart disease, but an entirely new RNA-based tool of genome editing has emerged: the CRISPR system based on short-guide RNA (sgRNA) in concert with the CAS9 recombinase. CRISPR has the potential to transform biology as deeply as PCR did 30 years ago, and our consortium intends to fully exploit the opportunities offered by CRISPR in the realm of disease research.
The main rationale for our consortium continues to lie in the synergies generated by bringing together leading basic and clinical scientists into a cohesive multidisciplinary team devoted to the investigation and exploitation of small RNA in human diseases. Our team will continue to conduct a joint effort to utilize siRNA (and now sgRNA) in cell signalling discovery, to identify microRNA relevant to human disease, and to provide preclinical models to test their therapeutic potential.
One important goal is also to promote the science of early-to-mid-career scientists. In this context we have been extremely successful, with two of the four original CRPP founders having been recruited to prestigious chairs at other Universities. On the other hand, this outcome has created the challenge of revitalizing the consortium with innovative colleagues.
Therefore, we have opted to strengthen the fundamental RNA biology of the consortium by recruiting Prof. Markus Stoffel, a highly visible mi-croRNA expert, and Prof. Magdalini Polymenidou, an SNSF Research Professor focusing on disease-causing RNA-binding proteins.
We envision this research program to translate into novel diagnostic and therapeutic strategies which have the potential to impact future health care.
Our CRPP continues to be organized in a flat hierarchy which provides minimal intermediate levels and, in our opinion, is best suited to an academic environment with experienced, scientifically inde-pendent members. Two of the four original CRPP founders, Prof. Landmesser and Prof. Kyburz, have been recruited to prestigious chairs at the Charite Campus Benjamin Franklin, Berlin, and the University of Basel, respectively.
By reinforcing our CRPP with two new members, Prof. Markus Stoffel and Prof. Magdalini Polymenidou, we strengthened the fundamental RNA biology of the consortium and also increased its thematic cohesiveness. Focussing on the areas of neurodegeneration, skeletal muscle metabolism and pancreatic beta cell growth will further enhance the extensive collaborations of the various subprojects and increase scientific ex-changes. Prof. Stoffel is a member of both UZH and ETH, and his involvement will also enhance the much needed synergies between the two Zurich schools.
Achievements and perspectives
The useful life of this KFSP is expected to extend beyond the initial years of funding. A pivotal benchmark is the establishment of robust high-throughput platforms to dissect the interactions of microRNAs, mRNAs, and cell signalling pathways through viral shRNA libraries and a platform to systematically manipulate microRNAs through inhibitor libraries (antagomirs).
Research highlights during the first funding period (2012-2015) include the following:
Aguzzi lab: The traditional prion infectivity assay requires inoculating samples intracerebrally into indicator animals and determining the time to appearance of clinical symptoms (incubation time meth-od). Such animal bioassays require vast numbers of animals and take several months. To circumvent these limitations, we developed a “digital prion infectivity assay” (dPIA) with high sensitivity, rapidity, and full automation. Prion infectivity titers are determined “digitally” by subjecting the TRFRET data to an advanced algorithm that extracts binary signals from noisy sources. The dPIA provides a powerful high-throughput platform for high-throughput screens.
Krützfeldt lab: We have discovered that miR-29a is under control of IGF1 in human skeletal muscle and correlates with insulin sensitivity in both mice and humans. We are now exploiting human skeletal muscle progenitor cells that can be expanded, passaged and differentiated in vitro. We have successfully established culturing and automated small RNA transfection of human myotubes in a 384-well format on a robot platform. We are using this system to screen for miRNA as well as lncRNA affecting human skeletal muscle metabolism in different insulin-responsive states using small RNA-based libraries. Candidate hits are being validated using standardized assays of loss- and gain-of-function in vitro.
Polymenidou lab: Despite extensive efforts from many groups over the last years, it has been ex-tremely challenging to develop reliable models to study TDP-43 and FUS proteinopathies that reca-pitulate their subcellular mislocalization and aggregation. This has hindered the study of the potential toxic gain-of-function resulting from accumulation of TDP-43 or FUS aggregates. Therefore, in the past year we have established new TDP-43 and FUS aggregation models on long-term mouse organotypic brain and spinal cord slice cultures. We chose this system because it offers a complex cellular environment morphologically similar to the intact nervous system, while allowing for manipulations that are typically not possible in vivo. We exposed organotypic brain slices to TDP-43 and FUS protein aggregates or protein “seeds” created by bacterially expressed and purified protein. Our data suggest that induced TDP-43 aggregation leads to subcellular mislocalization in a well-defined timeframe that offers a window for molecular manipulations.