Rational Design of Chimeric Antisense Oligonucleotides on a Mixed PO-PS Backbone for Splice-Switching Applications.

Area of Science:

• Oligonucleotide chemistry
• Molecular biology
• Drug discovery

Background:

• Synthetic antisense oligonucleotides (ASOs) are effective for treating diseases by modulating gene expression.
• Phosphorothioate (PS) backbones are common in approved ASO drugs, but their synthesis can create stereoisomers impacting properties.
• Splice-switching oligonucleotides (SSOs) target pre-mRNA splicing, with only one approved SSO currently using a PS backbone.

Purpose of the Study:

• To design and evaluate novel stereo-random SSOs with a mixed phosphodiester (PO) and phosphorothioate (PS) backbone.
• To assess the impact of mixed PO-PS backbones on SSO binding affinity, biological potency, and nuclease stability.
• To explore a strategy for limiting undesirable stereoisomers in SSO synthesis without compromising efficacy.

Main Methods:

• Design and synthesis of stereo-random SSOs incorporating both PO and PS linkages.
• Evaluation of synthesized SSOs for target mRNA binding affinity.
• Assessment of biological potency and stability against nucleases in cellular assays.

Main Results:

• Several mixed PO-PS SSOs were successfully designed, synthesized, and evaluated.
• The mixed backbone approach demonstrated potential for reducing stereoisomer complexity.
• Efficacy and stability of the SSOs were maintained, suggesting a favorable balance.

Conclusions:

• A mixed PO-PS backbone is a promising strategy for developing next-generation SSOs.
• This approach may overcome limitations associated with purely PS-based SSOs.
• Further development could lead to improved SSO therapeutics with enhanced pharmaco-properties.