Syntheses of Morpholine-Based Nucleotide Analogs for Hepatic siRNA Targeting and Stabilization

Area of Science:

• Medicinal Chemistry
• Nucleic Acid Therapeutics
• Hepatology

Background:

• Small interfering RNA (siRNA) holds promise for gene silencing therapies, but requires effective targeting and stabilization strategies.
• Hepatic delivery of siRNA is crucial for treating liver diseases, necessitating specific targeting ligands.
• Morpholine-based nucleotide analogs offer potential for enhancing siRNA properties.

Purpose of the Study:

• To develop and evaluate morpholine-based nucleotide analogs as building blocks for hepatic siRNA targeting and stabilization.
• To investigate the structure-activity relationships of GalNAc-conjugated morpholino scaffolds for siRNA delivery.
• To assess the impact of these modifications on siRNA knockdown potency and in vivo duration of action.

Main Methods:

• Synthesis of morpholine-based nucleotide analogs functionalized with a GalNAc ligand via various linkers.
• Conjugation of these analogs to the sense strand of a transthyretin-targeting siRNA.
• In vitro and in vivo evaluation of siRNA knockdown potency and stability.
• Structure-activity relationship studies focusing on linker characteristics and attachment sites.

Main Results:

• A clear structure-activity relationship was established for linker type, length, and attachment site of morpholino GalNAc moieties.
• Alkylation of the morpholine nitrogen yielded a nucleotide analog that enhanced siRNA stability as a 3'-overhang.
• Optimized siRNA constructs, combining targeting and stabilizing elements, showed improved in vivo duration of action without phosphorothioate stabilization.

Conclusions:

• Morpholine-based nucleotide analogs can be effectively utilized for targeted hepatic siRNA delivery and enhanced stability.
• The developed analogs and conjugation strategies significantly improve siRNA therapeutic efficacy and duration of action.
• This approach offers a promising platform for developing next-generation siRNA therapeutics for liver diseases.