Serious bugs plague biology’s workhorse DNA tool

Plasmids (shown here in color transmission electron microscopy with different genes highlighted) are circular DNA structures used in biology laboratories.Author: Dr. Gopal Murti/Science Photo Library

Lab plasmids, the workhorses of modern biology, are in trouble. The researchers conducted a systematic evaluation of circular DNA structures, analyzing more than 2,500 plasmids made in labs and sent to a company that provides services such as packaging the structures inside viruses for use as gene therapy. The team found that nearly half of the plasmids had structural flaws, including errors in sequences crucial to expressing the therapeutic gene. The researchers published their findings on the preprint server bioRxiv last month, ahead of peer review.1.

The study sheds light on a “lack of knowledge” about how to properly control the quality of plasmids in the lab, says Hiroyuki Nakai, a geneticist at Oregon Health & Science University in Portland, who was not involved in the work. He already knew about problems with lab-made plasmids but was surprised by the frequency of errors found in the study. There are likely many papers published whose results cannot be reproduced because of flaws in plasmid design, he adds.

Wasted time

Plasmids are a popular tool in biology laboratories because bacteria, including the commonly used model organism E. coliuse the structures to store and exchange genes. This means that biologists can create designer plasmids containing different genes of interest and then induce E-coli to take them and make multiple copies.

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. For example, when Lahn was a professor at the University of Chicago, a graduate student in his lab spent six months trying to recreate two plasmids that had been reported in the scientific literature. “We didn’t think twice about the quality of the plasmids, but then the experiment failed” because the plasmids contained errors, he says.

Now at VectorBuilder, Lahn says he’s faced this problem all the time—so he decided to evaluate it systematically. When customers submit plasmids riddled with errors, “it ends up wasting a lot of time,” and the extra quality-control steps increase the cost of producing plasmids and packaging them into viruses, he says.

The VectorBuilder team’s analysis revealed a patchwork of errors in the more than 2,500 plasmids it evaluated. Some contained genes encoding proteins toxic to E-colimeaning they could slow or stop the growth of the organisms that biologists rely on to replicate their plasmids. Others, designed to be packaged into viruses, coded for proteins that were toxic to those viruses. And some contained repeated DNA sequences that could accumulate mutations inside the plasmids.

Error checking

The most common mistakes Lahn and his colleagues found were related to a key tool in gene therapy. The therapies are often packaged in adeno-associated viruses (AAVs), which are mostly harmless and can carry the treatment into cells. When creating plasmids for these AAVs, scientists place the therapeutic gene between sequences called ITRs, which play a key role in ensuring that the gene is packaged into the virus for delivery. In essence, these sequences send a biological signal to cells that says, “I belong to this virus.” But the team found that about 40% of the AAV plasmids in the study had mutations in the ITRs that could distort this important message. If the researchers were to use these poorly designed plasmids, their gene therapy might not work—and it might take a long time for scientists to figure out why.

Mark Kay, a pediatrician and geneticist at Stanford School of Medicine in California, has also seen firsthand how plasmid errors can delay lab projects. But he’s confident that scientists can spot and fix the mistakes. He says gene therapy researchers are familiar with the potential problems with ITRs and that the errors are unlikely to lead to problems in the clinical setting. That’s because regulatory agencies like the U.S. Food and Drug Administration have rigorous standards that require researchers to carefully analyze plasmids before using them in the clinic.

Nakai says that checking plasmids for errors by sequencing them can alert scientists to the problems highlighted in the study. Several 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 builders; it’s a painstaking, artisanal process, he says, but if it doesn’t work out, it can be a huge waste of time and money.

Another way for labs to avoid problems is to make plasmid sequences publicly available in open-access repositories, says Melina Fan, chief scientific officer of the nonprofit Addgene in Watertown, Mass. Addgene hosts one such repository, Fan says, and “sequences the deposited plasmids and makes the sequence data available on its website for the community to use.” Plasmid verification is important, she adds.

Lahn hopes his team’s analysis will alert researchers to the fact that these working lab tools are often taken for granted. “People don’t question the condition of the tool,” he says, but they should.