Transforming delivery and activity of antisense oligonucleotides (ASOs) outside of the liver with high throughput in vivo ligand screens

Academic Supervisors: Prof. Matthew Wood; Dr. Alyssa Hill; Dr. Dhanu Gupta

Project summary

Nucleic acid-based therapeutics, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), are revolutionary medicines capable of addressing both rare diseases (e.g., spinal muscular atrophy) and more common conditions (e.g., hypercholesterolemia). Widespread adoption of standard-format chemistries such as 2′-O-methoxyethyl phosphorothioate (MOE PS) and conjugation to N-acetylgalactosamine (GalNAc), a ligand that achieves delivery to the liver, have brought oligonucleotide therapeutics to the fore of clinical productivity. ASOs and siRNAs can now be rapidly designed and tested in preclinical models without years of medicinal chemistry optimization, which fundamentally accelerates drug development. However, the delivery of ASOs and siRNAs to extrahepatic tissues remains an urgent challenge, particularly given the potential impact of ASOs and siRNAs in the context of diseases affecting difficult-to-reach tissues such as the brain, skeletal muscle, and heart. Directed evolution approaches has been able to overcome similar challenges for viral vectors; here we apply similar concepts to oligonucleotide ligands. Using molecular barcodes, we will establish a high-throughput, in vivo screening platform to identify lipids, peptides, and sugars that enhance oligonucleotide biodistribution, uptake, and efficacy, with an initial focus on the brain and skeletal muscle to enable new treatments for neurological and neuromuscular disorders. Initial work in rodent models will pave the way to more complex human models being developed by the MRC CoRE.

Lay project summary

Antisense oligonucleotides (ASOs) are a new drug class with the power to revolutionize medicine. However, in the body, ASOs mainly traffic to the liver, which means they are suitable for liver diseases but less suitable for diseases affecting other tissues, such as the brain, muscle, and heart. We aim to identify ligands such as lipids, peptides, and proteins that, when attached to an ASO, direct its delivery to difficult-to-reach tissues in the same way postage directs a letter to an address. We will achieve this by conjugating ligands to ASOs tagged with unique molecular “barcodes,” pooling multiple ligand-ASO conjugates together, and administering them to mice in a single experiment. The barcodes will allow us to track each conjugate within different tissues, which will enable the identification of ligands that direct ASOs to specific extrahepatic sites. Ligands that help ASOs accumulate in tissues of interest will be designated as hits and advanced into follow-up studies to confirm that the delivered ASOs are pharmacologically active in those tissues. By identifying ligands capable of delivering ASOs beyond the liver, this approach has the potential to make previously inaccessible tissues accessible to oligonucleotide therapeutics, thus expanding treatment options for a wide range of diseases.

Project Objectives

• ID ligands: Identify ligands for ASO delivery from a vast library (e.g., lipids, peptides, and de novo designer proteins).
• Barcode: Develop a barcoding strategy for detecting a single ligand-ASO conjugate species among a defined population of ligand-ASO conjugates.
• Detect and refine: Test and optimize the detection system in vitro.

• ID hits in vivo: Systemically administer ligand-ASO conjugates to mice and identify hits. Carry out follow-on studies on hit ligand-ASO conjugates to evaluate their activities.

Research Methodologies

To maximize our chances of success, we will employ a large, chemically diverse library of ligands; established conjugation techniques (e.g., click chemistry); and clinically validated oligonucleotide detection systems (e.g., splint ligation- or chemical ligation-qPCR). For follow-on activity studies, we will target a well-known proxy gene and monitor activity on the gene using state-of-the-art methods (i.e., RT-qPCR or branched DNA-based assays). We will work with an internal team to innovate around chemistry where desired or necessary. We will prioritize innovating around the type and number of the conjugated ligands, the barcoding strategy, and the throughput of the in vivo screening strategy.

Potential Project Impact

This project aims to take the long, laborious process of identifying ligands that increase ASO activity in difficult-to-reach tissues – which is typically done in a low-throughput manner (i.e., one by one, starting with in vitro screening, which is poorly predictive of in vivo efficacy, before progressing to in vivo studies) – and replace it with a higher-throughput, in vivo workflow that is efficient, economical, and affords more chances at success in identifying hits. This project has the potential to impact on the oligonucleotide therapeutics field by unlocking new tissues as sites of action, and thereby new indications, for ASOs and siRNAs.

Proposed Project Timelines

• Year 1: Ligand ID and design of synthetic route(s) to access ASO conjugates
• Year 2: Development of barcoding strategy, in vitro testing and optimization, and in vivo proof-of-concept/truth-telling experiments
• Year 3: In vivo screening and hit ID
• Year 4: Continuation of in vivo screening and hit ID and thesis/publication writing

Project Risks & Mitigations

• Risk: Failure to ID ligands with accessible synthetic routes. Mitigation: Consult external chemistry team(s); outsource synthesis; employ a library of well-known ligands with established synthetic routes; innovate around number, as well as type, of conjugated ligands.
• Risk: Failure to achieve sufficient resolution of conjugate species. Mitigation: Optimize barcode features (e.g., length, character, chemistry); optimize detection system; where necessary, sacrifice the number of species in the pool to demonstrate a proof of principle.
• Risk: Failure to ID hits. Mitigation: Build diversity into the ligand library (e.g., type, number); demonstrate a proof of principle on established ligands, e.g., GalNAc.

Training Opportunities

This project will expose the successful candidate to cutting-edge techniques in oligonucleotide therapeutics research and offer opportunities for independent, self-driven work as well as collaboration with a world-class, interdisciplinary team.

Opportunities for student participation in Public and Patient Involvement and Engagement (PPIE)

The successful candidate will have the opportunity to engage in Public and Patient Involvement and Engagement (PPIE) activities, such as preparing materials to inform patients and the public, helping to organize outreach events, and participating in discussions with patients and patient advocacy groups. These exchanges will develop the candidate’s skills in science communication and help them contextualize their work as well as foster scientific literacy and strengthen the public’s perception of scientific research.

Studentship code: MRCCoRETG2025004

Funding Notes

Long-term funding obtained

Application Deadline

12 noon, Tuesday 2nd December 2025

Enquiries

mrccoretg@paediatrics.ox.ac.uk

Please read these guidance notes for detail on how to apply

Click here to apply: IPP login screen (ox.ac.uk)

For more information on DPhil in Paediatrics: DPhil in Paediatrics | University of Oxford

For more information about our MRC-Oxford Doctoral Training Programme see: https://www.medsci.ox.ac.uk/study/graduateschool/mrcdtp