Ligand Valency and Linker Design Dictate the Efficacy of CI-M6PR-Mediated Targeted Delivery of M6P–siRNA Conjugates

Targeted delivery of small interfering RNAs (siRNAs) to nonhepatic tissues remains a major challenge in RNAi therapeutics owing to inefficient cellular uptake and off-target effects. In this study, mannose-6-phosphate (M6P)-conjugated siRNAs are developed to target the cation-independent M6P receptor (CI-M6PR/IGF2R), which is overexpressed in various cancers, including chronic myeloid leukemia (CML). Several mono-, di-, and tetravalent M6P-siRNA conjugates are synthesized via solid-phase methods using either flexible hexanediol or rigid proline-based linkers. Thermal melting analysis indicated that M6P conjugation modestly reduced duplex stability, with high-valence constructs and those containing more flexible linkers being particularly affected. By contrast, rigid proline linkers mitigated destabilization. Circular dichroism spectroscopy confirmed that the native A-form RNA structure was maintained after M6P conjugation, while in vitro gene silencing studies in CI-M6PR-positive K562 cells targeting <i>KNTC2</i> mRNA demonstrated that ligand valency and linker rigidity significantly influenced the activity under transfection-free conditions. Tetravalent siRNA <b>7</b> (partially proline-linked) achieved the most efficient knockdown (∼36% reduction in <i>KNTC2</i> mRNA), whereas the reduction in the activity of tetravalent siRNA <b>5</b> (fully hexanediol-linked) was similar to that of the divalent constructs, likely owing to impaired receptor engagement. Flow cytometry analysis further established that this superior activity was attributable to the markedly higher cellular uptake of siRNA <b>7</b> relative to the other conjugates. Electroporation experiments confirmed that all siRNA variants retained RNAi activity, indicating that delivery efficiency, rather than functional impairment, is responsible for the observed activity differences. These findings illustrate the pivotal role of ligand valency and linker architecture in optimizing CI-M6PR-targeted siRNA delivery and provide a framework for designing ligand-conjugated siRNA therapeutics against CI-M6PR-expressing tumors.