Characterization of Storage-Induced mRNA Modifications in (4S)-KEL12 LNP: Adduct Formation Kinetics, mRNA Decay, and...

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmaceutical Sciences

Background:

• Lipid nanoparticles (LNPs) are crucial for mRNA therapeutics.
• Lipid-mRNA adducts form during LNP storage, reducing therapeutic efficacy.
• The drivers and consequences of adducts, especially with novel lipids, need further study.

Purpose of the Study:

• Investigate adduct formation between mRNA and impurities of the ionizable lipid (4S)-KEL12.
• Characterize the structural drivers and functional impacts of these adducts.
• Identify strategies to mitigate adduct formation and improve mRNA therapeutics.

Main Methods:

• Studied adduct formation under varying storage temperatures.
• Utilized mass spectrometry to identify adduct structures and targets on mRNA.
• Assessed protein expression and innate immune response of adducted mRNA in cell lines (293T and THP1).

Main Results:

• Elevated temperatures accelerated adduct formation and mRNA degradation independently.
• (4S)-KEL12 degradation impurities, Z4 and Z1, were key drivers of adducts, with Z4 being more potent.
• Z4 preferentially formed adducts with cytidines on mRNA, without compromising mRNA integrity.
• Adducted mRNA showed reduced protein expression but did not trigger significant innate immune responses.

Conclusions:

• Impurity control and thermal management are critical for advancing mRNA therapeutics.
• Understanding adduct formation mechanisms is key to ensuring LNP stability and efficacy.
• Adducted mRNA's limited impact on innate immunity offers insights into safety profiles.