Boronic Acid Mediated Cellular Delivery of Therapies

A University of Pittsburgh scientist has identified a novel approach to improve cell penetration of oligonucleotide-based therapies. Using boronic acids to bind to cell surface glycans, penetration of oligonucleotides into a cell can improve their use in research and enhance efficacy of oligonucleotide-based drugs.  

Description

Delivering nucleic acid therapeutics, including antisense oligonucleotides (ASOs) across the cell membrane has been a limiting factor in drug development. This approach harnesses the reversible binding between boronic acids and the cell surface to mediate cell penetration, potentially revolutionizing the development of potent oligonucleotide-based drugs. Structure of one ASO synthesized during the development of this novel approach. Includes a strand of RNA with a single 5boU unit to allow for reversible binding to surface glycans and a HaloTag substrate for the study of cell penetration.

Applications

• Drug delivery
• Drug development
• Research including pharmacokinetic studies

Advantages

Delivering nucleic acids across the cell membrane, either for therapeutic purposes (e.g., regulation of RNA expression) or research purposes (e.g., observation of cellular processes using fluorescent probes), has traditionally been a hinderance to the development of compounds containing ASOs, short interfering RNAs (siRNAs), microRNAs (miRNAs), and messenger RNA (mRNA). Nucleic acid therapies are a promising and growing area of pharmaceutical research. Overcoming the challenge of cell penetration could advance this field allowing new nucleic acid therapies to be developed.
This novel approach harnesses the reversible binding between boronic acid and cell surface glycans to facilitate drug delivery across the cell membrane. An artificial nucleotide, 5-dihydroxylboryluridine (5boU), can be easily added to any nucleic acid sequence to enhance cytosolic penetration. This increased cellular penetration could lead to the discovery of novel ASO-based therapeutics and improve understanding of intracellular mechanisms.

Invention Readiness

For the first time a concise, modular method of incorporating 5boU into short ASOs using solid-phase synthesis has been developed. Cell penetration was investigated using chloroalkane penetration assay (CAPA) in cells expressing HaloTag enzyme following treatment with a chloroalkane-tagged ASO (ct-ASO) carrying zero, one, two, or three 5boU units. Compared to non-boronated ct-ASO, addition of one 5boU unit improved cytosolic penetration by 2.4 times, while addition of two or three 5boU units to ct-ASO improved cell penetration roughly five-fold. Using a cell-based luciferase reporter assay these cell penetration results were confirmed, including that addition of a third 5boU unit exhibited no significant benefit. Further optimization of the boronate moiety and placement within the ASO may also enhance activity.

IP Status

https://patents.google.com/patent/WO2024254253A2