In Vivo Fermentation Production of RNA Interference Agents

Area of Science:

• Molecular Biology
• Biotechnology
• RNA Therapeutics

Background:

• Conventional RNA agent manufacturing methods like in vitro transcription and chemical synthesis have limitations.
• There is a need for novel in vivo approaches for RNA agent production.
• Bioengineered RNA molecules offer potential for therapeutic applications.

Purpose of the Study:

• To describe a streamlined method for producing BioRNA/siRNA molecules.
• To provide a detailed protocol for the design, cloning, overexpression, and purification of BioRNA/siRNA.
• To enable efficient production of high-quality RNA agents for research and experimental therapy.

Main Methods:

• Utilized unique tRNA-fused pre-miRNA carriers for RNA bioengineering.
• Employed rational design, molecular cloning, and heterologous overexpression in bacteria.
• Purified BioRNA/siRNA using chromatography to achieve high purity and low endotoxin levels.

Main Results:

• Successfully produced multi-milligram quantities of BioRNA/siRNA from small bacterial culture volumes (0.25 L).
• Achieved high quality of BioRNA/siRNA with >98% homogeneity.
• Ensured low endotoxin levels (<5 EU/μg RNA) for the purified RNA agents.

Conclusions:

• The developed method provides a robust and efficient platform for producing BioRNA/siRNA.
• This technology facilitates the consistent generation of high-quality RNA agents.
• The produced BioRNA/siRNA molecules are suitable for both basic research and preclinical therapeutic studies.