siRNA and Innate Immunity (original) (raw)

Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA

Nature Biotechnology, 2005

Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications 1. Many nucleic acids, including double-(dsRNA) 2 and single-stranded RNA (ssRNA) 3-5 , can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system 6,7. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.

Overcoming the Innate Immune Response to Small Interfering RNA

Human Gene Therapy, 2008

Many types of nucleic acid, including canonical small interfering RNA (siRNA) duplexes, are potent activators of the mammalian innate immune system. Synthetic siRNA duplexes can induce high levels of inflammatory cytokines and type I interferons, in particular interferon-␣, after systemic administration in mammals and in primary human blood cell cultures. These responses are greatly potentiated by the use of delivery vehicles that facilitate cellular uptake of the siRNA. Although the immunomodulatory effects of nucleic acids may be harnessed therapeutically, for example, in oncology and allergy applications, in many cases immune activation represents a significant undesirable side effect due to the toxicities associated with excessive cytokine release and associated inflammatory syndromes. The potential for siRNA-based drugs to be rendered immunogenic is also a cause for concern because the establishment of an antibody response may severely compromise both safety and efficacy. Clearly, there are significant implications both for the development of siRNAbased drugs and in the interpretation of gene-silencing effects elicited by siRNA. This review provides the background information required to anticipate, manage, and abrogate the immunological effects of siRNA and will assist the reader in the successful in vivo application of siRNA-based drugs.

Therapeutic siRNA: principles, challenges, and strategies

The Yale journal of biology and medicine, 2012

RNA interference (RNAi) is a remarkable endogenous regulatory pathway that can bring about sequence-specific gene silencing. If harnessed effectively, RNAi could result in a potent targeted therapeutic modality with applications ranging from viral diseases to cancer. The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo. An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response. The design of such a system requires rigorous understanding of all mechanisms involved. This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facin...

Inflammatory cytokine induction by siRNAs is cell type- and transfection reagent-specific

Biochemical and Biophysical Research Communications, 2006

Specific knock-down of cellular gene expression using short, interfering RNAs (siRNAs) has become a powerful tool for functional genomics studies and a promising future therapeutic approach. However, recent studies have revealed that siRNAs can trigger an innate immune response upon intravenous administration in mice and transfection into purified immune cells by upregulating inflammatory cytokine levels. In this study, we demonstrate that transfection of siRNAs into several established human cancer cell lines can also induce inflammatory cytokine production regardless of the sequence of the siRNA used. The amount of inflammatory cytokine induction is cell type-specific, whereas the induction pattern is siRNA sequence-specific. We also show that, in a given cell type, different transfection reagents have different effects on inflammatory cytokine induction. Our results highlight the promiscuity of siRNA-triggered innate immune responses in human cancer cell lines and call for caution in the design and analysis of siRNA-based experiments.

Misinterpreting the Therapeutic Effects of Small Interfering RNA Caused by Immune Stimulation

Human Gene Therapy, 2008

Activation of innate immunity has direct effects in modulating viral replication, tumor growth, angiogenesis, and inflammatory and other immunological processes. It is now established that unmodified siRNA can activate this innate immune response and therefore there is real potential for siRNA to elicit nonspecific therapeutic effects in a wide range of disease models. Here we demonstrate that in a murine model of influenza infection, the antiviral activity of siRNA is due primarily to immune stimulation elicited by the active siRNA duplexes and is not the result of therapeutic RNA interference (RNAi) as previously reported. We show that the misinterpretation stems from the use of a particular control green fluorescent protein (GFP) siRNA that we identify as having unusually low immunostimulatory activity compared with the active anti-influenza siRNA. Curiously, this GFP siRNA has served as a negative control for a surprising number of groups reporting therapeutic effects of siRNA. The inert immunologic profile of the GFP sequence was unique among a broad panel of published siRNAs, all of which could elicit significant interferon induction from primary immune cells. This panel included eight active siRNAs against viral, angiogenic, and oncologic targets, the reported therapeutic efficacy of which was based on comparison with the nonimmunostimulatory GFP siRNA. These results emphasize the need for researchers to anticipate, monitor, and adequately control for siRNA-mediated immune stimulation and calls into question the interpretation of numerous published reports of therapeutic RNAi in vivo. The use of chemically modified siRNA with minimal immunostimulatory capacity will help to delineate more accurately the mechanism of action underlying such studies.

Current Development of siRNA Bioconjugates: From Research to the Clinic

Frontiers in Pharmacology, 2019

Small interfering RNAs (siRNAs) acting via RNA interference mechanisms are able to recognize a homologous mRNA sequence in the cell and induce its degradation. The main problems in the development of siRNA-based drugs for therapeutic use are the low efficiency of siRNA delivery to target cells and the degradation of siRNAs by nucleases in biological fluids. Various approaches have been proposed to solve the problem of siRNA delivery in vivo (e.g., viruses, cationic lipids, polymers, nanoparticles), but all have limitations for therapeutic use. One of the most promising approaches to solve the problem of siRNA delivery to target cells is bioconjugation; i.e., the covalent connection of siRNAs with biogenic molecules (lipophilic molecules, antibodies, aptamers, ligands, peptides, or polymers). Bioconjugates are "ideal nanoparticles" since they do not need a positive charge to form complexes, are less toxic, and are less effectively recognized by components of the immune system because of their small size. This review is focused on strategies and principles for constructing siRNA bioconjugates for in vivo use.