Laboratory-engineered plasmids, a workhorse of modern biology, have problems. Researchers conducted a systematic assessment of circular DNA structures by analyzing more than 2,500 plasmids made in laboratories and sent to a company that provides services such as packaging the structures within viruses so they can be used as gene therapies. The team found that nearly half of the plasmids had design errors, including errors in sequences critical to the expression of a therapeutic gene. The researchers published their results last month on the preprint server bioRxiv 1.

The study exposes "a lack of knowledge" about conducting proper quality control of plasmids in the laboratory, says Hiroyuki Nakai, a geneticist at Health & Science University in Oregon who was not involved in the work. He had already noticed problems with lab-made plasmids, but was surprised by the frequency of errors uncovered by the study. There are likely many scientific papers that have been published for which the results are not reproducible due to errors in plasmid design, he adds.

Wasted time

Plasmids are popular tools in biology laboratories, as bacteria, including the widely used model organismEscherichia coli, which use structures to store and exchange genes. This means that biologists can create designer plasmids that contain various genes of interest, and thenE.colipersuade them to record these and make many copies of them.

Bruce Lahn, chief scientist at VectorBuilder, a Chicago, Illinois-based company that provides gene delivery tools, says he and other biologists have noticed problems with plasmid quality for years. When Lahn was a professor at the University of Chicago, a graduate student in his laboratory spent six months reproducing two plasmids that had been reported in the scientific literature. “We didn't think about the quality of the plasmids, but then the experiment didn't work” because the plasmids contained errors, he says.

Now at VectorBuilder, Lahn says he sees the problem all the time - so he decided to systematically evaluate it. When customers submit faulty plasmids, "they waste a lot of time," and the extra steps in quality control increase the cost of producing the plasmids and packaging them into viruses, he says.

The VectorBuilder team's analysis revealed a hodgepodge of errors in the more than 2,500 plasmids evaluated. Some contained genes that encoded proteins responsible forE.coliwere toxic, meaning they could slow or stop the growth of the organisms that biologists rely on to replicate their plasmids. Others, intended for packaging into viruses, encoded proteins that were toxic to those viruses. And some contained repetitive DNA sequences that can accumulate mutations in plasmids.

Checking errors

The most common errors Lahn and his colleagues found were linked to a key gene therapy tool. Therapies are often packaged in adeno-associated viruses (AAVs), which are mostly harmless and can deliver treatments to cells. When creating the plasmids for these AAVs, researchers sandwich a therapeutic gene between sequences called ITRs, which play a critical role in ensuring the gene is packaged into the virus for delivery. These sequences essentially send a biological signal to cells that says “I belong in this virus.” However, the team found that about 40% of the AAV plasmids in the study had mutations in the ITR regions that could distort this important message. If researchers used these misdesigned plasmids, their gene therapy might not work — and it could take a long time for scientists to figure out why.

Mark Kay, a pediatrics and genetics specialist at Stanford School of Medicine in California, has also seen firsthand that plasmid errors can delay laboratory projects. However, he is confident that scientists can identify and correct these errors. He says gene therapy researchers are aware of possible ITR problems and that errors are unlikely in clinical settings. That's because regulatory agencies like the US Food and Drug Administration have strict standards that require researchers to carefully analyze their plasmids before using them in the clinic.

Nakai says checking plasmids for errors through sequencing could alert researchers to the problems highlighted in the study. Some companies, including Plasmidsaurus in Eugene, Oregon, and Elim Biopharmaceuticals in Hayward, California, offer plasmid sequencing for about $15.00 per sample, says Nakai, who has no financial interest in either company. He also recommends that new lab members spend time learning from experienced plasmid designers; It's a laborious, hand-crafted process, he says, but if you make a mistake it can waste a tremendous amount of time and money.

Another way for labs to avoid problems is to make their plasmid sequences publicly available in open-access repositories, says Melina Fan, chief scientific officer at the nonprofit Addgene in Watertown, Massachusetts. Addgene provides such a repository, says Fan, and it “sequences the deposited plasmids and shares the sequence data through the website for community use.” Checking plasmids is important, she adds.

Lahn hopes his team's analysis draws researchers' attention to the fact that these workhorse lab tools are often taken for granted. “The health of the tool is something people don’t think about,” he says, even though they should.