Nucleic acid immunity as relevant to oligonucleotide therapeutics
Under the constant selection pressure of infection by diverse and ever-changing pathogens, eukaryotic cells and organisms have evolved complex immune systems to defend themselves. Of particular interest for the oligonucleotide therapeutics field is the response of the innate immune system to the introduction of exogenous nucleic acids and how to bypass or manipulate this reaction for therapeutic purposes.
The innate immune system can detect invading nucleic acids via membrane-bound or cytoplasmic pattern-recognition receptors.1, 2 The membrane-bound toll-like receptors TLR3, 7, 8 and 9 and the cytoplasmic sensors protein kinase R (PKR; also known as eIF2AK2), retinoic acid-inducible gene I (RIG-I; also known as DDX58) and melanoma differentiation-associated protein 5 (MDA5; also known as IFIH1) have been identified as the main cellular sensors for immune responses to oligonucleotide therapeutics.2 Unfortunately, even though there are numerous excellent reviews on the receptors for exogenous pathogen nucleic acids, not much literature is available on how those receptors are accidentally triggered by therapeutic RNA and DNA oligonucleotides.
In humans, TLR7 and 8 recognise single-stranded RNA in a sequence-specific manner but are also activated by siRNA,3, 4 whereas TLR9 identifies unmethylated CpG motifs in DNA.5, 6 Unmethylated CpG motifs may be found in antisense and splice switching oligonucleotides as well as DNA aptamers and can activate TLR9 at less than 1% of the dose required for antisense or exon skipping effects (e.g., ~1 mg vs 200 mg in human). TLR9 has also been implicated in CpG-independent activation of the nucleic acid immune response by oligonucleotides, particularly those containing phosphorothioate backbones.7 This appears to be a result of binding between charged sugar-phosphate backbones, such as phosphorothioates, and the receptor for advance glycation end product (RAGE) located on the cell surface. Enhanced endosomal uptake of the RAGE-nucleic acid complex increases exposure to and thus activation of TLR9,8, 9 but this is still an area under investigation.
TLR7, 8 and 9 are predominantly located in the endosomal compartment of certain immune cells. For TLR7 and 9, these are plasmacytoid dendritic cells (pDCs), a DC subtype producing large amounts of type I interferons as well as activated and memory B cells, where these TLRs are implicated in B cell maturation and memory.10 TLR8 is expressed in monocytes, macrophages and conventional dendritic cells (cDCs – previously called myeloid dendritic cells, these are antigen-presenting cells). TLR3 on the other hand, can be found in both the cell membrane of fibroblasts and endothelial cells as well as the endosomal membrane of macrophages and cDCs. It is generally believed to recognize double-stranded RNA longer than 30 bp in humans, while murine TLR3 is activated by shorter dsRNA such as siRNA.11 However, recent evidence suggests that human TLR3 localized to the cell membrane is able to recognize 21 bp siRNAs,12 perhaps explaining previous reports of human TLR3 activation by siRNA. This difference is a prime example of the fact that mice do not accurately predict immune effects of nucleic acids in humans. In fact, most of the TLRs seem to be expressed in different cell types between mice and humans and proteins in the downstream signalling pathways may also be different.
Upon activation, all TLRs induce nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation via an adaptor protein called myeloid differentiation factor 88 (MyD88), except for TLR3, which signals through TIR domain-containing adaptor-inducing interferon-β (TRIF). This results in the production and secretion of inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-12 and tumour necrosis factor α (TNF-α). TLRs also signal via interferon-regulatory factors (IRF3, 5 and 7) thus upregulating the expression and secretion type I interferons (IFNα and IFN‑β) which in turn activate several hundred IFN-stimulated genes (ISGs) in the same as well as bystander cells.2 Release of these cytokines results in the recruitment and activation of B cells, T cells, and natural killer (NK) cells and in this way, links the innate and adaptive immune systems.
The cytoplasmic immune sensors PKR, RIG-I and MDA5 are also able to induce NF-κB and type I interferons. In addition, PKR can directly inhibit protein synthesis via phosphorylation of eIF2α upon activation. PKR recognises dsRNA longer than 19 bp such as typical 21 bp siRNAs with two–base pair 3′ overhangs as well as siRNAs with blunt ends. RIG-I reacts particularly well to 5′-triphosphates on dsRNA longer than 10 bp, but also forms filaments on AU-rich dsRNA which amplify the activation signal in a length-dependent manner such that loss of the 5′-triphosphate motif is completely compensated with dsRNAs longer than 200 bp. Earlier reports that single-stranded RNA is a RIG-I ligand have now been attributed to contamination with dsRNA intermediates during in vitro transcription. In vitro transcription also adds a 5′-triphosphate and thus siRNA, aptamer and mRNA therapeutics produced in this way are likely to activate RIG-I. MDA5 is thought to only recognize double-stranded RNA longer than 1 kb and like RIG-I forms filaments which are required for signal amplification. Curiously though, a report by Burel and colleagues13 showed activation of MDA5 but not RIG-I by a 2′-O-methoxyethyl gapmer design.
This example aptly demonstrates that careful sequence design and addition of select chemical modifications meant to reduce activation of the innate immune response by oligonucleotide therapeutics is not sufficient to completely avoid such effects. Apart from siRNA, published studies of possible innate immune responses to oligonucleotide therapeutics are few and far between. It does seem to be a rather understudied area, even though all experimenters using oligonucleotide therapeutics should be alert for such unintended immune effects. In particular, activation of innate immune responses is known to trigger tumour regression in mouse models and investigators may thus mis-identify the mechanism of action of their therapeutic oligonucleotide.
However, how to investigate and properly control for such immune stimulation by oligonucleotide therapeutics is a story for another day.
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- Unique O-methoxyethyl ribose-DNA chimeric oligonucleotide induces an atypical melanoma differentiation-associated gene 5-dependent induction of type I interferon response.
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- Silencing or stimulation? siRNA delivery and the immune system.