UniqueO-Methoxyethyl Ribose-DNA Chimeric Oligonucleotide Induces an Atypical Melanoma Differentiation-Associated Gene 5-Dependent Induction of Type I Interferon Response

Abstract

Antisense oligonucleotides (ASO) containing 2Ј-O-methoxyethyl ribose (2Ј-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2Ј-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2Ј-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-␤ (IFN-␤) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h.We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-␤ in Toll/interleukin-1 receptordomain-containing adapter-inducing IFN-␤ or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-␤ promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-␤ and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.

Immunological and hematological toxicities challenging clinical translation of nucleic acid-based therapeutics

“…Similar to ISIS 116847, no change in inflammatory signaling was observed in knockout animals deficient in MyD88 and TRIF. Interestingly, this study demonstrated that two cytosolic receptors, melanoma-differentiation associated-5 (MDA-5) and IFN-b promoter stimulator-1 (IPS-1) were involved in the inflammatory reaction to ISIS 147420, but retinoic acid inducible gene-1 (RIG-1) was not [64]. Both IPS-1 and RIG-1 are pattern recognition receptors that are capable of recognizing foreign nucleic acids.…”

“…For NATs, the majority of available studies have focused on TLRs (TLR3, TLR7/8 and TLR9) to explain observed immunostimulation [63,64,[165][166][167][168]. However in many cases, inhibiting or eliminating TLRs from test systems reduced immunostimulation but did not eliminate it, leaving a gap in the understanding of the mechanism(s) [63,64].…”

“…Based on these findings, the authors suggested that 2¢-MOE non-CpG ASO induced cytokines through either TLR3 which functions through the My-D88-independent adaptor TRIF (Toll/IL-1R-containing adaptor protein inducing IFN-b), or another unidentified receptor. Another elegant study from this group showed a different 2¢-MOEmodified non-CpG ASO (ISIS 147420) induced high levels of type I interferons (IFN-b) independently from TLR3 [64]. Similar to ISIS 116847, no change in inflammatory signaling was observed in knockout animals deficient in MyD88 and TRIF.…”

Antisense Oligonucleotide Therapies

“…The shorter basepair length of most LNA gapmers (based on their higher binding efficiency) seems to reduce their proinflammatory potential as compared to other backbones. The basis for the sequence specificity in rodents are sequence motifs that are recognized by pathogen-associated molecular pattern (PAMP) receptors, such as the nucleic acidsensing TLR receptors (Agrawal and Kandimalla 2004;Senn, Burel, and Henry 2005;Richardt-Pargmann and Vollmer 2009;Burel et al 2012;Burel et al 2013). These attributes have been exploited for immunomodulatory drug indications by industry (Krieg 1998(Krieg , 1999Henry et al 1999).…”

DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

“…The microarray gene expression analysis showed several downregulated genes predicted to be because of off-target hybridization of HDO (Table 2). In addition, sequence-dependent and hybridization-independent immunestimulatory adverse effects mediated by activation of toll-like innate receptors 52,53 can also be a concern. Finally, a sequenceand hybridization-independent chemical property of nucleotide analogues and chemical modifications can cause liver dysfunction 51,[54][55][56][57] .…”