E. Coli fermentation is the conventional way of producing pDNA and is widely adopted in the biotech industry. However the long lead time and high cost associated with outsourcing pDNA production have posed great challenges to many biotech companies.
Doggybone DNA (or dbDNA) is an innovative and proprietary technology from Touchlight that uses enzymatic reactions to achieve plasmid DNA (pDNA) manufacturing. And this technology has the potential to address the challenges of conventional bacterial fermentation production. The company website has detailed information about the technology. Overall, it uses Φ29 DNA polymerase for rolling circle amplification (RCA) to create double stranded concatemers including both GOI and backbone, and then cleave out the backbone and produce covalently closed dbDNA. Couple of benefits of this technology include:
- Enzymatic reaction to produce pDNA is much faster, eliminating steps like clone selection/screening, cell banking, seed train through fermentation, etc. The total time from production to release has the potential to be shortened from a couple of months to weeks.
- Compared to using cells, enzymatic reactions have less process related impurities, such as host cell DNA and proteins, making the downstream purification much easier, as well as increasing purity of final plasmid.
- Much smaller footprint is expected due to elimination of large size bioreactors and tanks to hold intermediate materials.
However, there are questions and potential challenges as well:
- It’s not clear how the efficiency looks like when the backbone is cleaved out and dbRNA is formed through covalent interaction with the other strand of DNA at the same end. It’s also a question whether concatemers can be fully cleaved.
- However about the transcription efficiency when only 28bp TEL is present on both ends of the dbDNA? Will it be consistent across different lengths and sequences of DNA?
- How will the transfection be impacted for some applications like gene therapy?
- This technology is still very new and hasn’t been fully tested yet. Although there are two programs using this technology entering into clinical stages, concerns around safety and regulatory requirements still exist across the whole industry.
Overall, it takes time for any new technology to be accepted and adopted across a biotech industry. This technology, if approved to be safe and viable, will have the power to revolutionize how plasmids are produced in the future. The upcoming readouts from currently ongoing clinical trials will either give researchers confidence in this technology or promote more research work around it.