Purifying circRNA by ultrafiltration with membranes having well-defined pores

Area of Science:

• Biotechnology
• Molecular Biology
• Materials Science

Background:

• Circular RNA (circRNA) offers therapeutic potential due to its inherent stability.
• Current purification methods like size-exclusion chromatography are inefficient for large-scale circRNA production, yielding low purity and output.
• Developing advanced purification strategies is crucial for advancing circRNA therapeutics.

Purpose of the Study:

• To develop and validate an ultrafiltration strategy for efficient circRNA purification.
• To establish a scalable, non-chromatographic method for producing high-purity circRNA.
• To assess the therapeutic potential of ultrafiltration-purified circRNA.

Main Methods:

• Utilized polycarbonate track-etched membranes with precisely controlled pore sizes, adjusted via gold plating, for circRNA purification.
• Measured sieving coefficients to define optimal operating conditions for continuous diafiltration.
• Evaluated purity, yield, and gene expression efficiency of purified circRNA in HEK293T cells.

Main Results:

• Achieved 94% purity and 59% yield of circRNA using the ultrafiltration method after six diavolumes.
• Demonstrated comparable gene expression levels for ultrafiltration-purified circRNA versus enzymatically purified circRNA in cell-based assays.
• Identified a modest reduction in transfection efficiency for the purified circRNA.

Conclusions:

• Membrane-based ultrafiltration with tailored pore sizes provides a scalable and effective platform for circRNA purification.
• This non-chromatographic approach addresses limitations of existing methods, facilitating therapeutic circRNA manufacturing.
• Further development of this technique holds promise for advancing circRNA-based therapies.