Unconventional Lysine-Type Lipid Assemblies Enable Efficient Antisense Oligonucleotide Delivery with Distinct...

Area of Science:

• Biotechnology
• Drug Delivery Systems
• Molecular Biology

Background:

• Antisense oligonucleotides (ASOs) offer therapeutic potential for genetic and neurological disorders by modulating gene expression.
• Effective delivery of ASOs remains a significant challenge, with current research primarily focusing on lipid nanoparticles (LNPs).
• Alternative formulations, preparation methods, and lipid compositions for optimizing ASO delivery require further exploration.

Purpose of the Study:

• To investigate the potential of lysine-type cationic lipids in formulating lipoplexes (LPXes) and LNPs for enhanced ASO delivery.
• To compare the formulation stability, cellular entry mechanisms, ASO delivery efficiency, and immune responses of novel lipid formulations against a benchmark.

Main Methods:

• Screening of a lysine-type lipid mini-library to identify stable and safe cationic lipids (K3C14, K3C16).
• Characterization of LPX and LNP formulations' physicochemical properties, cytotoxicity, ASO delivery efficiency, and immunogenicity.
• Structural analysis of novel lipid assemblies using cryogenic electron microscopy (Cryo-EM).

Main Results:

• Lysine-type lipids with K3 spacer or C14 fatty acid tails demonstrated excellent stability and safety.
• The K3C16 lipoplex formulation exhibited ASO delivery efficiency and immune responses comparable to the SpikeVax LNP benchmark.
• Cryo-EM revealed unique structures: K3C14 lipoplexes formed rouleaux-like structures, and K3C16 lipoplexes formed lipid nanosheet-like structures.

Conclusions:

• Novel lipid assemblies, specifically lysine-type lipoplexes, show significant potential for efficient ASO delivery.
• Unconventional lipid structures may offer alternative strategies for overcoming ASO delivery challenges.
• These findings open new avenues for developing advanced ASO-based therapeutics.