Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry

Abstract

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Limb Girdle Muscular Dystrophy Type 2B (LGMD2B): Diagnosis and Therapeutic Possibilities

“…Modifying the sugar moiety of nucleotides by incorporating 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE) or 2′-fluoro (2′-F) modifications, can further improve the stability of AOs while maintaining their RNA binding affinity [ 111 , 112 ]. Substituting specific nucleotide bases with modified analogs, such as locked nucleic acid (LNA) or peptide nucleic acid (PNA) can also enhance the binding affinity and specificity of AOs to their target RNA sequences [ 94 , 113 , 114 ]. Phosphorodiamidate morpholino oligomers (PMO) replace the deoxyribose/ribose moiety with a morpholine ring and substitute the charged phosphodiester inter-subunit linkage with an uncharged phosphorodiamidate linkage.…”