Base modification strategies to modulate immune stimulation by an siRNA

Area of Science:

• Biochemistry
• Immunology
• Medicinal Chemistry

Background:

• Immune stimulation by small interfering RNA (siRNA) hinders the development of safe RNA interference (RNAi) therapeutics.
• Ribose modifications like 2'-OMe or 2'-F decrease cytokine production but may not fully address sequence-dependent immune recognition.
• Toll-like receptor 8 (TLR8) is implicated in recognizing immunostimulatory nucleic acids.

Purpose of the Study:

• To investigate the impact of adenosine modifications on siRNA binding to Toll-like receptor 8 (TLR8).
• To evaluate the efficacy of chemically modified siRNAs in reducing immune stimulation while maintaining RNA interference activity.
• To determine the optimal positioning and type of adenosine modification for minimizing cytokine production.

Main Methods:

• Computational modeling of ribonucleotide binding to TLR8 using crystal structure data.
• Chemical synthesis of modified adenosine nucleosides and their incorporation into siRNA guide strands.
• Assay of immunostimulatory properties by measuring TNFα production in human peripheral blood mononuclear cells (PBMCs).
• Evaluation of RNA interference activity.

Main Results:

• Computational models predicted that modifications to either the Watson-Crick or Hoogsteen face of adenosine disrupt TLR8 interactions.
• Chemical modification of the Watson-Crick face of adenosine in siRNA was more effective at blocking TNFα production than Hoogsteen face modification.
• Modifications located near the 5'-end of the siRNA guide strand showed greater efficacy in reducing cytokine production compared to 3'-end modifications.
• RNA interference activity was maintained with the tested modifications.

Conclusions:

• Adenosine modification represents a viable strategy to mitigate siRNA-induced immune stimulation.
• Targeting the Watson-Crick face of adenosine, especially near the 5'-end, is a promising approach for designing less immunostimulatory siRNAs.
• This research enhances the understanding of structure-activity relationships for optimizing siRNA therapeutics.