Base Modification Strategies to Modulate Immune Stimulation by an siRNA

Abstract

Immune stimulation triggered by siRNAs is one of the major challenges in the development of safe RNAi-based therapeutics. Within an immunostimulatory siRNA sequence, this hurdle is commonly addressed by using ribose modifications (e.g. 2′-OMe or 2′-F) which results in decreased cytokine production. However, since immune stimulation by siRNAs is a sequence-dependent phenomenon, recognition of the nucleobases by the trigger receptor(s) is also likely. Here, we use the recently published crystal structures of Toll-like receptor 8 (TLR8) bound to small molecule agonists to generate computational models for ribonucleotide binding by this immune receptor. Our modeling suggested that modification of either the Watson-Crick or Hoogsteen face of adenosine would disrupt nucleotide/TLR8 interactions. We employed chemical synthesis to alter either the Watson-Crick or Hoogsteen face of adenosine and evaluated the effect of these modifications in an siRNA guide strand by measuring the immunostimulatory and RNA interference properties. For the siRNA guide strand tested, we found that modifying the Watson-Crick face is generally more effective at blocking TNFα production in human peripheral blood mononuclear cells (PBMCs) than modification at the Hoogsteen edge. We also observed that modifications near the 5′ end were more effective at blocking cytokine production than those placed at the 3′ end. This work advances our understanding of how chemical modifications can be used to optimize siRNA performance.

Safety of Striatal Infusion of siRNA in a Transgenic Huntington’s Disease Mouse Model

“…Persistent microglia activation can lead to the production of both neurotoxic (M1-like) and neuro-protective (M2-like) factors, but has an overall detrimental impact on neuron survival[11,17,43]. siRNA is known to activate microglia via TLR7 and TLR8[6,19]. TLR stimulation leads to an M1-like response[11]; microglia activated by siRNA would have neurotoxic effects.…”

“…This characteristic makes microglia rapid responders to CNS perturbations through detection of a variety of molecules, including ATP[15], pathological and misfolded proteins[16], neuronal adhesion molecules[17], and cytokines[18]. siRNA can activate microglia via toll-like receptors 7 and 8 (TLR7/8) leading to cytokine production[6,19]. Activated microglia both produce and respond to cytokines.…”

Systemic Delivery of Small Interfering RNA Therapeutics: Obstacles and Advances

“…Immunostimulation is another barrier in the delivery of siRNA as it leads to degradation of siRNA after recognition as an alien moiety by the macrophages and kupffer cells (Whitehead et al, 2011;Khairuddin et al, 2012). In order to tackle this issue, 2'-position of the ribose sugar had been modified with 2'-O-methyl and 2'-fluoro groups to suppress the immune response and improve serum provide stability (Valenzuela et al, 2014;Haraszti et al, 2018).…”

Progress and Prospects of Anti-HBV Gene Therapy Development

“…Typically, synthetic activators are designed to resemble miR duplexes (short interfering RNAs/siRNAs), whereas expressed activators resemble pre-miR (short hairpin RNAs/shRNAs and pre-miR mimics) and pri-miR (pri-miR mimics). Advantages of using siRNAs include ease of production, dose control and chemical modification to increase stability, specificity and reduced immunostimulatory effects (reviewed in [ 74 , 75 , 76 ]). As they act in the cytoplasm and do not require nuclear delivery, efficient cellular delivery is easier to achieve than with DNA expression cassettes.…”