Robust Functionality and Regulation of Selectively Expressed RNA as AAV Vectors and In Vitro Transcribed Molecules.

Area of Science:

• Molecular Biology
• Gene Therapy
• RNA Therapeutics

Background:

• Selectively expressible RNA (seRNA) offers cell type-specific protein expression via antisense interaction and internal ribosomal entry site activation.
• Current seRNA systems, often plasmid-based, face limitations in expression intensity and broad medical use due to transfection methods.

Purpose of the Study:

• Characterize plasmid-based seRNA uptake and activation.
• Explore alternative vector systems to enhance seRNA technology's medical applicability and effector expression.

Main Methods:

• Generated seRNA constructs using expression plasmids, adeno-associated virus (AAV), DNA minicircles, and in vitro transcribed RNA (IVT-RNA).
• Delivered constructs into eukaryotic cell lines via transfection/transduction.
• Analyzed uptake, activation, RNA stability, and expression using fluorescence microscopy, flow cytometry, and qRT-PCR.

Main Results:

• Plasmid-based seRNA systems showed efficient transfection but reduced steady-state RNA levels, likely due to transcription efficiency.
• Viral vectors and DNA minicircles enhanced seRNA effector expression and allowed linear regulation.
• In vitro transcribed seRNA yielded optimal results, maintaining target cell specificity.

Conclusions:

• seRNA technology functions effectively across various transfer vectors.
• Expression strength is tunable while maintaining functionality, paving the way for broad medical applications.