Novel RNA molecular bioengineering technology efficiently produces functional miRNA agents

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Bioengineering

Background:

• MicroRNAs (miRNAs) are crucial regulators of gene expression involved in cellular processes and disease.
• In vivo produced miRNA agents are emerging as more natural alternatives to in vitro synthesized mimics.
• Previous work established high-yield in vivo production of recombinant miRNAs using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers.

Purpose of the Study:

• To compare the production and function of bioengineered RNA (BioRNA) molecules utilizing glycyl (BioRNA ^Gly) versus leucyl (BioRNA ^Leu) htRNA fused hsa-pre-miR-34a carriers.
• To assess the efficacy of these BioRNA molecules in inhibiting non-small cell lung cancer cell viability.
• To evaluate the gene regulatory efficiency and structural characteristics of different BioRNA variants.

Main Methods:

• Design, cloning, overexpression, and purification of 48 BioRNA/miRNA constructs.
• Comparison of expression levels, yields, and purities between BioRNA ^Gly and BioRNA ^Leu.
• Functional assessment of BioRNA/miRNAs in inhibiting cancer cell viability and regulating target gene expression (EGFR, MRP1, VDAC1).
• Computational modeling to analyze the 3D structures of BioRNA/miRNAs.

Main Results:

• Expression levels, yields, and purities were comparable between BioRNA ^Gly and BioRNA ^Leu molecules.
• Both BioRNA variants demonstrated similar efficacy in inhibiting non-small cell lung cancer cell viability.
• BioRNA ^Gly/miR-7-5p showed enhanced efficiency in regulating target gene expression (EGFR) compared to BioRNA ^Leu/miR-7-5p.
• BioRNA ^Gly/miR-7-5p exhibited comparable or slightly greater activity in modulating MRP1 and VDAC1 expression than a commercial mimic.
• Computational modeling revealed comparable overall 3D structures with distinct differences in htRNA and miRNA components.

Conclusions:

• Hybrid htRNA/hsa-pre-miR-34a carriers are reliable for RNA molecular bioengineering.
• Resultant BioRNAs are functional biologic RNAs suitable for research and development.
• The choice of htRNA moiety (glycyl vs. leucyl) can influence the specific gene regulatory efficiency of BioRNA molecules.