Scientists at North Carolina State University have developed a revolutionary alternative technique dubbed “controlled evolution.” This collaborative approach greatly increases the quality and yield of plasmid DNA, integral to the field of biomedical manufacturing. This groundbreaking development has potential to help make the production of gene therapies more efficient. It would lower costs for other uses that are dependent on pDNA.
The study, titled “Inducible genome-wide mutagenesis for improvement of pDNA production by E. coli,” was recently published in the journal Microbial Cell Factories. Zidan Li, a postdoctoral researcher at NC State, led the research team. They explored how bacterial mutations can increase pDNA production.
Methodology and Findings
To optimize the pDNA-producing E. coli strains further, the researchers started with genetically modified strains of E. coli that were already known to be effective pDNA producers. They then engineered multiple mutations into these bacteria and later screened them to find ones that resulted in greater pDNA production.
“Our modified E. coli produced 8.7 times as much pAAV pDNA as the E. coli we started with,” said Li, highlighting the substantial improvements achieved through their method.
The team experiented on five different types of pDNA. In doing so, they exceeded expectations working with three types that are traditionally easier to produce and two types that come with much higher difficulty. They were very pleased to observe a 44% increase in p15A pDNA yield. This was especially notable, given that manufacturing it at scale had previously proven to be quite difficult.
“At the high end, we found our modified E. coli produced 8.7 times as much pAAV pDNA as the E. coli we started with.” – Zidan Li
Implications for Biomedical Manufacturing
The implications of this research go well beyond ivory tower curiosity. These results represent a significant increase in pDNA production. This breakthrough has the potential to greatly lower the cost of manufacturing, opening up new avenues for biomedical applications. Nathan Crook, one of the project’s partners, highlighted the difference these technological improvements would make.
Crook for optimism that this advancement will significantly reduce the cost of manufacturing biomedical applications with pDNA. He mentioned it could accelerate research that is based on pDNA resources.
Now, pDNA production relies on genetically modified bacteria and is extremely expensive. This cost burden can be especially challenging for broader research and development of gene therapies and other medical advancements.
“pDNA is largely produced by genetically modified bacteria and can cost as much as $100,000 per gram.” – Nathan Crook
Future Directions
The promise of producing pDNA at much higher efficiencies presents exciting opportunities for researchers and manufacturers to push these boundaries further. The research proves that “directed evolution” can be used to produce useful refinements to bacterial strains. This, in turn, makes these enhanced strains naturally better at producing various kinds of diverse pDNA.
Li explained the approach taken by the research team, stating, “Essentially, we started with a type of E. coli that had already been modified to produce pDNA.” This crucial building block then freed them to focus on tailoring and tuning the bacteria itself for maximum productivity.
The researchers are optimistic for future applications of their new approach. They’re looking forward to more progress that will help processes become more efficient and affordable for biomedical manufacturing.
“pDNA is relatively easy to work with in the lab—it’s stable and easy to modify.” – Nathan Crook