Development of a high-resolution method to monitor structural changes of regulatory RNAs and therapeutic oligo-nucleotides directly in-cell.

Project Information

RNAatHD

Grant agreement ID: 747446

DOI

10.3030/747446

Project closed

EC signature date 17 March 2017

Start date 1 May 2017

End date 30 April 2019

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 185 857,20

EU contribution

€ 185 857,20

Coordinated by

KAROLINSKA INSTITUTET
Sweden

Periodic Reporting for period 1 - RNAatHD (Development of a high-resolution method to monitor structural changes of regulatory RNAs and therapeutic oligo-nucleotides directly in-cell.)

Reporting period: 2017-05-01 to 2019-04-30

Summary of the context and overall objectives of the project

MicroRNAs (miRs) are targeting different mRNAs, and thereby affect many biological processes and are involved in various human diseases such as cardiac diseases, Alzheimer’s, diabetes or cancer. This fact is making them prime drug targets and candidates for molecular diagnostics, however, this opportunity can only be fully exploited by a better comprehension of how and when each mRNA is targeted by a miRNA, a fact currently still poorly understood. Besides regulatory RNAs, synthetic antisense oligonucleotides (AONs), which also bind segments of mRNAs, are currently tested as cancer drugs. Progress has however been slow due to unspecific off-target-effects, difficulty to quantify the cellular uptake and again, the lack of understanding the mechanism of AONs targeting mRNAs. NMR is the method of choice to obtain high-resolution information of biomolecules and therefore to shine a light on this targeting process. While in-vitro experiments can give detailed in-sight on structure and dynamics of an oligonucleotide in a reconstituted minimal complex, a clear limitation is that simplified aqueous solutions do not allow full comprehension of the cellular environment and how it influences the behavior of oligonucleotides in the living cell. In contrast, functional data is routinely obtained in tissues or cells using visualization techniques, e.g. confocal microscopy that requires tagging for visualization by chemical modification of the molecule of interest. Thus, while the context in which data is acquired is highly relevant, the tagged system may exhibit altered behaviour and function. As a consequence, classical structural biology research needs to interface more with cellular biology, as it is crucial for the structural data obtained in vitro to be validated within the cellular context. Within this project we developed exactly this missing tool allowing us to carry out structural biology of macromolecules and complexes directly in their natural, most relevant environment, in intact human cells, and to overcome this lack of understanding of the mRNA targeting process. Combining the knowledge of the host (Petzold Lab) in structural and molecular biology of miRNAs with the experience in methods development in Nuclear Magnetic Resonance (NMR) of the experienced researcher (ER), an in-cell NMR method was successfully developed, using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP). Using this new In-Cell Enhanced DNP (“ICE-DNP”) NMR approach, we were able to detect oligonulceotides directly in intact frozen cells. With these methods it is possible to address differences between in-cell and in vitro experiments and to determine interaction partners, which have to be used to reconstitute a more realistic in-vitro sample. The scope of the method was demonstrated on a miRNA, miR-34a, as well as a synthetic oligonucleotide drug candidate in collaboration with AstraZeneca, to show the value and insight this method can add to the drug discovery process.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In order to achieve the objectives and develop this RNAatHD approach, we used an AON drug provided by AstraZeneca and set out to work on the following steps:

Preparation of an in-cell sample, where the AON is successfully delivered into human cells. This also meant that the delivered AON was shown to be functional in the cells and that the cells are viable. We tested functionality by measuring the downregulation of the target mRNAs by qRT-PCR. A very interesting result was that tagged (biotinylated or Cy3-tagged) versions of the AON drug, as used for microscopy, showed significantly less downregulation, i.e. functionality compared to the actual, unmodified drug molecule. Therefore they might not reflect how untagged AON behaves in the cell stressing the need for a tag-free technique to detect (drug-) molecules in cells as developed in this project.

In-cell NMR method: In order to develop a non-invasive in-cell NMR approach we decided to freeze the transfected cells and acquire solid-state dynamic nuclear polarization (DNP) NMR experiments. Cryoprotected, frozen cells stay intact for extended amounts of measurement times and, can be recultivated later, indicating a biologically relevant environment. Applying DNP to frozen cells, we overcome limitations of solution-state in-cell NMR (e.g. size, stability and sensitivity) as well as of visualization techniques since no tagging of the AON is required. Using this “ICE-DNP”(frozen In-Cell Enhanced NMR by DNP) method, we were indeed able to obtain an in-cell NMR signal of the investigated AON. To our knowledge, this is the first time that DNP is used to detect an exogenous oligonucleotide delivered into cells, and also that a transfected functional antisense drug in macromolecular complexes could be detected in intact human cells. The possibility to detect an untagged, active drug, interacting in its natural environment, means that the main objective of the project was reached.

In-vitro versus in-cell: For a complete RNAatHD approach, in parallel to developing an in-cell NMR technique, we also set out to do in-vitro comparison experiments. The idea thereby was to identify and add more and more interaction partners to reconstitute a more realistic in-vitro sample.

Application: The RNAatHD approach with the steps described above was successfully set up with and applied to an AON drug provided by AstraZeneca (as described above). In addition, the approach was also applied to a 13C/15N isotopically labelled miRNA prepared in the host lab.

The results so far were disseminated in two peer reviewed journal publications and presented at various seminars and conferences by the post-doc and the PI of the lab.
In addition, to ensure exploitation of the method we worked closely with pharmaceutical industry (AZ).

The post-doc was also selected as one of 30 “highly promising researchers” to represent 100000 funded fellows. She presented the project at the “Science is wonderful” event at the European Researchers’ Night in Brussels, 2017.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Cancer, heart disease and Alzheimer’s account for some of the highest rates of deaths of EU inhabitants. Drug design is however slow with high failure rates in clinical trials. A limitation is that drug design is usually carried out based on structural information obtained from aqueous in-vitro samples not allowing to comprehend the cellular environment. As a consequence, classical structural biology needs to interface more with cellular biology. Currently there is a lack of methods to understand structures and function of target-engaged drugs directly in the cellular context. The new method developed here, provides exactly this missing tool and combines high-resolution information of in-vitro structural biology with biological relevance of an in-vivo condition. It will provide unprecedented detailed information on the drug targeting mechanism and metabolism in the living cell, thereby accelerate the drug development process, lower cost-factors for pharmaceutical development and enhance the EU’s longterm competitiveness and directly impact societal and industrial needs.

Overview of the application of the new in-cell method to an AON drug