Sequence-non-specific effects generated by various types of RNA interference triggers.

Area of Science:

• Molecular Biology
• Immunology
• Genetics

Background:

• RNA interference (RNAi) is a powerful tool for gene silencing using reagents like short interfering RNA (siRNA), short hairpin RNA (shRNA), and artificial microRNA (sh-miR).
• Beyond targeted gene silencing, RNAi reagents can elicit unintended side effects, notably triggering immune responses.
• Understanding these off-target effects is crucial for safe and effective RNAi therapeutic development.

Purpose of the Study:

• To identify molecular markers indicative of immune response activation by various RNAi reagents.
• To compare the cellular responses, including immune activation and microRNA (miRNA) deregulation, induced by different RNAi formats (siRNA, D-siRNA, shRNA, sh-miR).
• To differentiate sequence-specific from sequence-non-specific effects of RNAi reagents.

Main Methods:

• Analysis of existing experimental data from the RNAimmuno (v 2.0) and GEO Profiles databases.
• Design and application of a series of RNAi reagents (siRNA, D-siRNA, shRNA, sh-miR) targeting three distinct transcripts (Luc, ATXN3, HTT) to ensure sequence-non-specificity.
• Assessment of cellular responses, including immune markers and miRNA expression profiles.

Main Results:

• RNAi reagents induce a range of sequence-non-specific effects, including significant deregulation of cellular miRNA levels.
• Standard siRNAs show weak interferon response stimulation but can saturate the miRNA biogenesis pathway, downregulating highly expressed miRNAs.
• Plasmid-based RNAi reagents (shRNA, sh-miR) activate known immune response markers and alter miRNA levels and their isomiR composition.

Conclusions:

• RNAi reagents, particularly plasmid-based ones, can trigger immune responses and disrupt normal miRNA homeostasis.
• The choice of RNAi reagent impacts the type and magnitude of off-target effects, including immune activation and miRNA deregulation.
• Distinguishing sequence-specific gene silencing from sequence-non-specific immune and miRNA effects is essential for optimizing RNAi applications.