Development and characterization of a miRNA-responsive circular RNA expression system with cell type specificity

Area of Science:

• Molecular Biology
• Gene Therapy
• RNA Therapeutics

Background:

• MicroRNAs (miRNAs) typically degrade messenger RNAs (mRNAs).
• However, miRNAs can enhance viral RNA expression via internal ribosome entry sites (IRES).
• This interaction suggests potential for miRNA-mediated control of RNA expression.

Purpose of the Study:

• To develop a novel strategy for inducible and tissue-specific gene expression using circular RNAs (circRNAs).
• To investigate the potential of using miRNA binding sites within circRNA IRES elements for translational control.
• To explore the therapeutic applications of miRNA-regulated circRNAs.

Main Methods:

• Constructed circRNAs incorporating the hepatitis C virus (HCV) 5' UTR with miR-122 binding sites as an IRES.
• Transfected cells with engineered circRNAs to assess miRNA-inducible gene expression.
• Utilized alternative miRNA binding sites to demonstrate broad applicability.
• Administered lipid nanoparticle-formulated circRNAs (circRNA-LNPs) intravenously in mouse models.

Main Results:

• Engineered circRNAs demonstrated specific, inducible gene expression in response to miR-122.
• Substituting miR-122 sites with other miRNA binding sites enabled corresponding miRNA-mediated translational activation.
• In vivo studies showed enhanced tissue-specific expression of circRNA-LNPs with functional miRNA binding sites compared to mutated controls.

Conclusions:

• Introduced a novel method for regulating circRNA expression using miRNA targeting.
• Demonstrated the potential for tissue-specific gene expression control via engineered circRNAs.
• This strategy holds promise for advancing gene therapy and developing precise therapeutic interventions.