Click Chemistry-Based Strategy for Modular Ligand Attachment to siRNAs: Toward Extrahepatic RNAi

Radler, J. A.; Filipiak, E.; Marquant, A.; Ojansivu, M.; Czapik, T.; Hill, A.; Ahlskog, N.; Roudi, S.; Barradas, C.; Huang, Y.; Saher, O.; Wood, M.; Zain, R.; Honcharenko, M.; EL Andaloussi, S.

doi:10.64898/2026.05.21.726808

bioRxiv · 10.64898/2026.05.21.726808

Click Chemistry-Based Strategy for Modular Ligand Attachment to siRNAs: Toward Extrahepatic RNAi

Radler, J. A.·Filipiak, E.·Marquant, A.·Ojansivu, M.·Czapik, T.·Hill, A.·Ahlskog, N.·Roudi, S.·Barradas, C.·Huang, Y.·Saher, O.·Wood, M.·Zain, R.·Honcharenko, M.·EL Andaloussi, S.

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Abstract

Efficient extrahepatic delivery of siRNAs remains a major limitation for broadening their therapeutic potential. Using a modular, orthogonal click chemistry platform, we generated 28 siRNA conjugates varying in ligand class, valency, and spatial arrangement. Following systemic administration, fatty acid conjugates - particularly palmitic acid (C16) - outperformed sterol- and phospholipid-based designs in promoting extrahepatic gene silencing, with preferential activity observed in heart and skeletal muscle. Increasing ligand valency through 3',5'-bis-conjugation generally enhanced activity compared to 5-mono conjugation. Nevertheless, bis-C22 conjugates showed increased hepatic activity, suggesting a shift in tissue distribution linked to hydrophobicity. Architectural parameters further modulated outcomes: Branched 5' C16 conjugates, bearing two lipids on one terminus, were markedly less active than their bis counterparts and required short PEG spacers to restore activity. Notably, bis-lipid conjugation strategies that enhanced extrahepatic activity for an siRNA did not translate to an ASO gapmer, underscoring modality-specific constraints. Together, these findings delineate structure-activity relationships and establish bis-fatty-acid conjugation as a robust design principle for achieving extrahepatic RNAi. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/726808v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@b7af11org.highwire.dtl.DTLVardef@146b9c6org.highwire.dtl.DTLVardef@4e8948org.highwire.dtl.DTLVardef@16fa07_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Radler, J. A., Filipiak, E., Marquant, A., Ojansivu, M., Czapik, T., Hill, A., Ahlskog, N., Roudi, S., Barradas, C., Huang, Y., Saher, O., Wood, M., Zain, R., Honcharenko, M., EL Andaloussi, S.. 2026-05-22. Click Chemistry-Based Strategy for Modular Ligand Attachment to siRNAs: Toward Extrahepatic RNAi. https://doi.org/10.64898/2026.05.21.726808

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