Propensities of Fatty Acid-Modified ASOs: Self-Assembly vs Albumin Binding

Area of Science:

• Biophysics
• Oligonucleotide Chemistry
• Drug Delivery

Background:

• Antisense oligonucleotides (ASOs) are a promising therapeutic modality.
• Modifying ASOs with fatty acids (FAs) can enhance their properties.
• Understanding self-assembly and albumin binding is crucial for ASO development.

Purpose of the Study:

• To investigate the self-assembly and albumin-binding characteristics of fatty acid-modified LNA ASOs targeting MALAT1.
• To correlate fatty acid structure with biophysical properties and albumin interactions.
• To explore the potential of these modifications for influencing ASO pharmacokinetics and biodistribution.

Main Methods:

• Synthesis of label-free LNA ASOs covalently modified with saturated fatty acids of varying lengths and attachment sites.
• Analytical ultracentrifugation (AUC) to study self-assembly and stoichiometry.
• Isothermal titration calorimetry (ITC) to determine binding affinities and thermodynamics.

Main Results:

• ASOs with fatty acids longer than C16 showed increased self-assembly into vesicular structures.
• Fatty acid-modified ASOs (C16-C24) formed stable complexes with human and mouse serum albumin, with binding strength correlating to hydrophobicity.
• Self-assembled structures formed by longer fatty acid chains were stable and could be disrupted by albumin, forming 2:1 complexes, except for a C32 di-palmitic acid conjugate which formed a stable hexameric complex.

Conclusions:

• The hydrophobic effect governs the formation of mono- vs multimeric structures of modified ASOs, with fatty acid chain length dictating supramolecular assembly.
• Fatty acid modification of ASOs provides two key avenues for influencing pharmacokinetics: albumin binding as a carrier and self-assembly into albumin-inert structures.
• These strategies offer opportunities to modulate biodistribution, receptor interactions, and uptake mechanisms for improved *in vivo* efficacy, potentially enabling extrahepatic delivery.