Expert Tips to Improve Plasmid Quality and Reliability

Plasmid DNA, a fundamental molecular biology tool, is increasingly critical to gene therapy approaches. Plasmids are commonly deployed as gene delivery vectors in vitro and in vivo, either directly or as starting materials for generation of viral and mRNA vectors. In addition to their role in research, plasmids have also become a crucial source material in the production of many biotherapeutic products, including recombinant protein drugs (e.g., antibodies), gene therapy vectors, and mRNA vaccines (e.g., Covid-19).

Due to the diversity of applications and uses, plasmids are highly customized reagents. Historically, researchers have typically designed and engineered their own plasmids in the laboratory or been gifted them by other researchers. To date, there is no systematic quality assessment of laboratory-generated plasmids despite their important role in life science and clinical research. This lack of consistent and rigorous quality control (QC) has recently been flagged as a major cause for concern for research and clinical applications dependent on the integrity of plasmid DNA.

The areas with the biggest potential impact are associated with plasmid design or sequence fidelity. If these elements are not well controlled and errors are incorporated then the planned study may not deliver the desired and expected outcome, ending or delaying an otherwise viable approach. A recent publication highlighted a concerning and high level of errors in laboratory-made plasmids. The authors are well-positioned to make an assessment of plasmid quality as they are a well-established cloning service provider, receiving DNA samples from academia and industry worldwide. The authors state that their analysis of submitted plasmids (2,521) identifies that nearly one-half carried errors and places doubt on the trustworthiness of these reagents.

Prevalence of errors in lab-made plasmids

Sequence errors were identified as a key area for concern. Even on a relatively simplistic analysis, based on restriction enzyme digestion profile, the authors flagged 15% of samples that failed to give the expected DNA fragment patterns—suggesting significant rearrangements or point mutations in restriction enzyme recognition sites.

Given their importance in therapeutic approaches, the authors paid particular attention to adeno-associated virus (AAV) vectors. They highlight that around 40% of transfer plasmids used in making AAV vectors, which are widely used in gene therapy, carried sequence mutations in internal tandem repeat (ITR) regions—integrity of ITR sequences is key for appropriate packaging of the target gene into the virus for delivery. Interestingly the sequence analysis revealed that the 5’ ITRs (30%) were far more prone to mutation compared to 3’ ITRs (4%).

The authors also go on to evaluate how flanking sequences affect the stability of ITRs and found that high GC content in the immediate flanking sequence contributed to ITR instability. It is not currently well understood how different types of mutant ITRs influence packaging efficiency, viral genome integrity, and intended biological functions. Clearly, compromised sequences in such key elements associated with gene therapy delivery vectors could ultimately mean that the intended approach does not appear to work and the project may be terminated.

From the analysis of 2,521 plasmids the authors also found that around 15% (384) had significant design errors that could impact function. These design errors were identified across the majority of plasmid component types, but most prevalent were incorrect choice or positioning of promoters, followed by inappropriate choice or design of open reading frames (ORFs). Their analysis also flagged poor understanding of some of the nuances of plasmid design, such as cargo capacity, sequence stability, or toxicity of the gene of interest. These elements can all be managed and controlled (e.g., through the use of low copy number plasmids, weak or inducible promoter selection, modified culture conditions, etc.) once the issue is recognized by the researcher.

Implications and mitigating the risks

This analysis clearly identifies that more robust processes and procedures are needed in research laboratories to routinely validate the structure and fidelity of plasmids. A valuable quick first check should be to routinely verify the expected restriction digestion pattern of plasmid DNA. Furthermore, checking plasmids for errors or mutations by sequencing should be a fundamental step for researchers where plasmid DNA is integral to the approach. The modest time and money invested in this simple analysis can avoid much more significant impacts if an incorrect plasmid is progressed.

The practice of open sharing of plasmid reagents between researchers also runs the risk that errors in plasmid design or sequence are propagated into new projects in the recipient laboratory. Again, a simple quality control step to ensure any plasmid reagents received into a laboratory are sequence-verified before any work is conducted mitigates the risk of committing time and resource to a strategy that is compromised from the outset.

The flaws in plasmid design identified also highlight the need for a set of standardized design principles that are implemented across research and therapeutic applications. Consistency in design and construction of plasmid vectors, feeding off the expertise and gained knowledge in the field, would reduce the frequency and likelihood of subsequent design errors.

Best experimental practices, including construction, propagation, and storage, and verification of structure and sequence integrity will help ensure reliability and functionality of plasmids across both research and clinical applications.

Key tips from these findings

• Incorporate routine checks (restriction digest profiling and sequencing) for key plasmids to ensure appropriate structure and fidelity.
• Robustly verify plasmid design and construction to minimize incorporation of flaws that may impact downstream applications and functionality.
• Encourage sharing of knowledge and expertise in plasmid design to maximize plasmid performance.
• Maintain good documentation and sequence data for plasmids and ensure this is readily shared with any recipients of the plasmid.