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

Abstract

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.

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

“…Extending the CED delivery beyond the current dosing period of 14 d may offer additional survival benefit when siRNA dosing limits per unit vehicle are in place with the current nano packaging technologies. Although there could be concerns about the potential for nonmodified siRNA duplexes to induce innate immune response for antitumor effects (75,76), our use of nu/nu mouse models with human BTIC xenografts and the universal nontargeting negative siRNA controls mitigated this common misinterpretation of antitumor results. For clinical translation, this potential impact will be assessed in immune-competent mouse models, which could reveal the impact of these LPNPs on the immune response of GBM.…”

Correlating animal and human phase Ia/Ib clinical data with CALAA-01, a targeted, polymer-based nanoparticle containing siRNA

“…The siRNA component of CALAA-01 is not chemically modified. Much preclinical work has demonstrated that nonchemically modified siRNAs can be immunostimulatory under certain conditions (16). Specifically, systemic administration of siRNA with lipid carriers can cause significant immunostimulation.…”

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…RNAi can cause expression suppression in genes homologous to the desired gene, and produce effects which were not the result of the silencing of the target gene. In early tests of siRNA therapies in small animals, the effects produced by the therapies may not have been from the target knock-down at all, but from the off-target effects [82,113]. Additionally, RNA sensors in the innate immune system can prohibit effective RNAi by triggering the release of interferons.…”

“…However, entering the cell from the bloodstream is still a hurdle. Because of their negative charge and high molecular weight, siRNAs cannot cross the plasma membrane [82,113]. Cells can uptake siRNAs through endocytosis, but then the siRNA is trapped within an endosome, and cannot produce gene knock-downs.…”