Next-generation oligonucleotide-conjugates for enhanced potency and versatile functionality

Thesis

Nucleic acids are powerful biopolymers. As therapeutics, they enable interventions at various stages of gene expression through Watson-Crick base pairing, while as polymers they allow programmability, stimuli responsiveness, and incorporation of diverse functionalities. Covalent modifications with small molecules or ligands can further expand therapeutic potential. This DPhil research aimed to develop inno- vative strategies for targeted mRNA and protein degradation using such oligonu- cleotide conjugates — exploring new designs to unlock biological functionality and advance molecular medicine.

The thesis is divided into two parts. Part I focused on the development of anti- sense oligonucleotide (ASO) conjugates for targeted sub-cellular nuclear localisation (Chapter 2) and for establishing new mechanisms of RNA degradation (Chapters 3, 4).

In Chapter 2, ASOs were linked to (+)-JQ1, a BET bromodomain ligand that promotes nuclear import. These conjugates enhanced splice-switching and mRNA knockdown across diverse targets and backbone chemistries. Notably, (+)-JQ1- Oblimersen, a late-stage clinical ASO, showed improved e cacy in an acute myeloid leukemia model.

Chapters 3 and 4 focused on attempts to optimize ASOs for enhanced cytoplasmic activity. Chapter 3 explored coupling ASOs to an RNase L–recruiting ligand for cytoplasmic mRNA degradation. This RNase L-recruitment approach for mRNA degradation also involved the optimisation of an ASO sequence for targeting MLL- AF4 fusion leukemias. Chapter 4 aimed at the development of LyTONs (lysosomal tra cking antisense oligonucleotides) by conjugating ASOs to an autophagosome- tethering compound. LyTONs were shown to achieve potent lysosome-dependent knockdown of Menin (MEN-1) mRNA, an important leukemia target, and boost the activity of conventional gapmers.

Together, these strategies demonstrated how chemical conjugation expands ASO activity and mechanism, off ering opportunities to target otherwise inaccessible transcripts.

Part II aimed at the development of Oligonucleotide-linked proteolysis targeted chimeras (OligoPROTACs) for oligonucleotide-guided protein degradation. Classical PROTACs, while powerful, su ffer from off -target eff ects and limited control. Chapter 5 presents OligoPROTACs: hybridisation-assembled DNA sca ffolds that configure PROTAC warheads in space for distance-dependent degradation. Crucially, this system has been engineered to contain a reversible o -switch for activity — achieved via toehold-mediated strand displacement. This first demonstration of programmable, controllable PROTACs highlights the potential of nucleic acid conjugates to enhance the precision and safety of targeted protein degradation therapies.

Authors: Kashyap, D

• DOI: 10.5287/ora-avzqyvz9q
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: gene therapy; PROTACs; Oligonucleotide therapeutics; covalent conjugation
• Subjects: Chemical biology