Short-hairpin RNAs delivered by lentiviral vector transduction trigger RIG-I-mediated IFN activation

Area of Science:

• Molecular Biology
• Immunology
• RNA Therapeutics

Background:

• Type I interferon (IFN) pathway activation by small interfering RNA (siRNA) contributes to off-target effects in RNA interference.
• Intracellularly expressed short-hairpin RNAs (shRNAs) were thought to be less prone to IFN activation, particularly when delivered via lentiviral vectors.
• IFN induction by RNA interference (RNAi) complicates gene function studies but may offer therapeutic benefits.

Purpose of the Study:

• To identify and characterize a specific short hairpin RNA (shRNA) capable of activating the type I interferon (IFN) pathway.
• To investigate the mechanism and cellular sensors involved in shRNA-mediated IFN activation.
• To determine if lentiviral vector delivery mitigates IFN activation by shRNAs.

Main Methods:

• Sequence and 5'-triphosphate dependency analysis of shRNA-mediated IFN activation.
• Assessment of interferon regulatory factor-3 (IRF3) dimerization and IFN promoter activation.
• Measurement of biologically active IFN secretion.
• Evaluation of IFN response following lentiviral vector transduction of shRNAs.
• Identification of the cytoplasmic sensor (RIG-I, M MDA5, or TLR3) for intracellular shRNAs triggering IFN activation.

Main Results:

• A specific shRNA was identified that potently activates the IFN pathway in human cells in a sequence- and 5'-triphosphate-dependent manner.
• This shRNA induced IRF3 dimerization, IFN promoter activation, and secretion of biologically active IFNs.
• Lentiviral vector transduction did not prevent IFN activation by the identified shRNA or another unrelated shRNA.
• Retinoic-acid-inducible gene I (RIG-I) was identified as the primary cytoplasmic sensor for intracellular shRNAs that trigger IFN activation, not MDA5 or TLR3.

Conclusions:

• Certain intracellularly expressed shRNAs can potently activate the type I IFN pathway, contrary to previous consensus.
• Lentiviral vector delivery does not inherently prevent IFN activation by all shRNAs.
• RIG-I is the key cytoplasmic sensor mediating IFN activation by intracellular shRNAs, highlighting a critical pathway for RNAi-mediated immunostimulation.