Novel Antisense Oligonucleotides to Improve Invasion and Cleavage of Structured RNAs

Antisense oligonucleotides (ASOs) are short sequences of DNA or chemically modified analogues, designed to bind specific RNA targets inside cells by Watson-Crick base pairing. Depending on the chemistry of the ASO, they can either cleave their target upon binding, or simply block its interactions with other cellular factors. The ASO approach has obvious potential for silencing experimental genes (as a research tool) or disease-causing genes (as a therapeutic). Indeed, over 100 oligonucleotides are in clinical trials.

One of the challenges involved in the antisense approach is that cellular RNA targets are often highly structured - folded up on themselves in ways that makes it harder for ASOs to bind to them. This contributes to the fact that researchers often have to screen many ASOs to find a good compound, and that some targets are not very responsive to inhibition by ASOs.

This project seeks to address the challenge of targeting structured RNAs with higher efficiency. How will we do this? We will incorporate a stretch of Peptide Nucleic Acid (PNA), a DNA analogue with a neutral backbone that allows it to bind structured RNAs with favourable kinetics and high binding affinity. The challenge is that bringing PNA and DNA together into a single oligonucleotide has typically led to sequences with reduced binding affinity, limiting their usefulness as ASOs.

We hypothesised that previous generations of PNA-DNA chimeras suffered a loss of binding affinity because the helical structures of PNA and DNA are different, and both are relatively flexible. This would almost certainly induce an unfavourable conformation at the junction between PNA and DNA. We further hypothesised that by introducing a constrained nucleotide at the junction, we could control the conformation of the helix in this region and avoid this destabilizing distortion. We predicted that this in turn would increase the binding affinity and specificity of our PNA-DNA chimeras.

We have now obtained data in support of these hypotheses. Incorporation of a conformationally constrained nucleotide at the junction between PNA and DNA caused a dramatic increase in binding affinity compared with the normal PNA-DNA junction (for binding complementary RNA, deltaG298 = -84 kJ/mol (constrained) vs -67 kJ/mol (normal)). Thus, we have now developed a way to bring together PNA and DNA into a single ASO, without compromising high binding affinity. This is an important finding and the current proposal will build upon it.

In a first aim, the proposal will explore the conformational requirements at the PNA-DNA junction. We will test various constrained nucleotides and PNA monomers at the junction site, to optimize binding affinity and specificity. Several conformationally contstrained nucleotides are known and we used computational analysis of these and other novel structures to find a shortlist of targets for synthesis.

In a second aim, the proposal will characterise chimeric PNA-DNA oligonucleotides based upon our optimised junction. This will include extensive biophysical testing, to understand how the PNA portion and the junction structure affect kinetics and thermodynamics of binding. We will use our optimised junction to make "gapmer" oligonucleotides containing DNA in the middle with PNA on one end, and LNA on the other end. LNA shows outstanding binding affinity and is one of the most exciting modifications in clinical trials. Chimeric strands containing both LNA and PNA should show improved the properties over LNA modified oligonucleotides, particularly in terms of binding to structured RNA targets. The optimised oligonucleotides will be transfected into mammalian cells (either naked or as cationic liposomes) to study their cell uptake, trafficking and gene silencing activity.

There are already a number of good chemistries available for use in antisense oligonucleotides. One of the "next frontiers" in the field is the ability to invade structured targets efficiently. We have designed this proposal to address the challenge.

We have already shown (preliminary data) that conformational control of the nucleotide at the PNA-DNA junction can have a huge effect on the binding affinity of the resulting chimeric strand. A locked nucleotide appears to maintain both flanking DNA and PNA in a conformation favorable for binding, as shown by our binding affinity data. This is a tremendous result, but is just the beginning. As we test alternative nucleotides and conformations, we hope to develop "structure-activity relationships" in terms of binding affinity. This will inform the design of future analogues with further improvements in their properties.

Furthermore, even more impact in this proposal will come from the applications of our PDL chimeras to silencing of challenging targets. One key area of impact for this work is in the silencing of noncoding RNAs. This is an area of tremendous interest and growth. The past decade has seen an explosion in our knowledge of how much RNA is transcribed and what functions it can assume. Less than 1.5 % of the genome codes for protein, but 60-90% of the genome is transcribed into RNA. The difference is made up of noncoding RNA, much of which plays important but poorly understood regulatory roles in the cell. Australian Biologist John Mattick has gone so far as to describe the human genome as an RNA machine. Yet noncoding RNAs (ncRNAs) are hard to study. To explore this new world of biology, new chemical tools are required.

There are three reasons why noncoding RNAs are hard to target using existing gene silencing technology: 1) RNA interference has a complex biology in the nucleus, and so duplex RNAs do not always give predictable silencing. 2) Normal ASOs are not optimized for structure. Noncoding RNAs are often highly structured. 3) ncRNAs may also be less dynamic in structure than messenger RNAs (mRNAs), which are repeatedly being unfolded by the ribosome in order to be translated. So while traditional ASOs might have a window of opportunity to access a mRNA while it is unfolded, they are less likely to be able to target a structured ncRNA. The structure-targeting ability of PNA is therefore likely to provide a substantial advantage for silencing ncRNAs.

This proposal thus holds significant potential for impact in basic knowledge of cellular processes and gene regulation, as well as therapeutic silencing of structured noncoding RNAs.

Given that ASO technology is increasingly recognised as having high potential for research and therapeutics, our technology may be in high demand. If this demand surpasses the applicant's capacity for synthesis, the technology can be commercialized. This could be carried out in collaboration with ADTBio, an oligonucleotide company based in the same building as the applicant's laboratory, or one of several other synthesis companies with whom the applicant is in contact (Sigma Custom Products, IDT, etc). We are also in touch with a number of oligonucleotide therapeutics companies (see Pathways to Impact document) who can pursue therapeutic applications of the work if appropriate. This process would be of benefit to the applicant's laboratory, to the UK and European economy, and to researchers who would have greater access to an excellent gene silencing technology.

It would be a major step forward for the field if we are successful at developing ASOs that can efficiently invade, bind and cleave structured RNA targets. We do not know of any other groups taking a similar approach. We are encouraged by our preliminary results and are anxious to test, optimize and apply these constructs as soon as possible.