Minicircle DNA: Can This Technology Change CAR-T Therapy For the Better?

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In biotechnology, plasmids are commonly used as vectors for the replication and cloning of recombinant DNA. For decades, scientists have been using plasmids for the development of therapies, the most recent one of which is the famous CAR-T therapy. But although many plasmid-based therapies are promising, they also come with problems.

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Dr. Martin Schleef, CEO of PlasmidFactory

Plasmids contain specific regions called the origin of replication, antibiotic-resistance elements and marker genes that are needed for stable maintenance and to amplify the plasmids in bacteria for large-scale production,” says Dr. Martin Schleef, CEO of PlasmidFactory. “These elements have different disadvantages, such as toxicity. They contain sequences called CpG motifs, which are known to induce immune responses in eukaryotic cells. So if plasmids are used for any therapeutic application, they can cause adverse effects in the human body.”

Another drawback of plasmids is the limited dose of DNA that can be inserted into cells before they react defensively or die. “The dose that you can apply always depends on the toxicity of a substance,” Schleef adds. “In the case of plasmids, there can be too much DNA from a certain moment onwards.”

Sidestepping the pitfalls of plasmids with minicircle DNA

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Plasmids have many disadvantages, including toxic elements and a limited amount of DNA that can be inserted into cells

In order to benefit from the advantages of plasmids and circumvent their disadvantages, Schleef and his team at PlasmidFactory have developed a proprietary technology for the production of so-called minicircle DNA.

Just like a plasmid, the minicircle DNA is circular, double-stranded, and supercoiled. However, unlike plasmids, it does not contain toxic or damaging DNA sequences. “We have found a way to get rid of these elements by reducing the plasmid so that it only contains the gene of interest with a small scar, which is a residual element that cannot be avoided during the manufacturing process,” Schleef explains.

Minicircle DNA comes with a number of advantages. Due to their small size, they are easily transfected into the nucleus of target cells; as there is no residual bacterial backbone, the danger of adverse immune responses and cell toxicity is eliminated; and the minicircle DNA remains in the cell for a longer amount of time improving a drug’s performance.

“By reducing the amount of DNA in the plasmid, the active molecules in treatments can be higher,” Schleef adds. “So the molarity of the product allows for the application of a lot more molecules, still stays under the toxicity level, and enables the expression of the gene of interest in the target cell.”

How minicircle DNA is created

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The process of creating minicircle DNA

The process for developing a minicircle starts with a parental plasmid into which the gene of interest is placed. “When a customer sends us a gene, we take it out of the original plasmid, plant it into our parental plasmid and grow it in bacterial cells,” explains Dr. Marco Schmeer, Project Manager at PlasmidFactory. “After growth, we proceed with a chemical induction that activates a recombinase. The recombinase forms a cis-recombination – cis meaning within the same molecule – of the plasmid, forming it into something that looks similar to an eight.”

Eventually, the eight breaks apart, forming two separate, supercoiled, smaller circles. One circle contains all the DNA of the plasmid that is not needed, including the selection marker, the origin of replication, and all of the unnecessary bacterial sequences. The other circle is the minicircle containing only the gene of interest and those elements needed for the regulation in the target cells.

“This product, the miniplasmid and the minicircle, is still in the bacterial cell,” Schmeer points out. “For the purpose of purification, we have a specially designed affinity chromatography, which can selectively distinguish between the molecules. It holds the minicircle in place, while we wash away all the molecules that are not needed. The minicircle is later diluted from the chromatography column, free of all other cellular contaminants, such as endotoxins or unwanted DNA.”

The resulting minicircle is helical and supercoiled, which positively influences gene expression. Another advantage from a pharmaceutical point of view is that the minicircle is monomeric, meaning that researchers can count on a homogeneous product.

The Sleeping Beauty system for CAR-T therapy

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Dr. Marco Schmeer, Project Manager at PlasmidFactory

The use of minicircles for the development of CAR-T therapy is a new and promising approach. Currently, the only approved technology that allows the genetic modification of T cells for the development of personalized CAR-T therapy uses lentiviral vectors. However, lentiviral vectors are known to integrate into parts of the target cells’ genome, which can cause mutagenesis and genotoxicity, triggering concerns about their safety. Furthermore, the high costs and strict regulatory guidelines prevent a fast clinical turnaround.

Another approach that allows the integration of genetic material into a cell’s genome is called the Sleeping Beauty transposon system. In this process, genes are randomly integrated into the genome, and as approximately 96% of the genome is non-coding, the risk of unwanted mutations or genotoxicity is much lower, raising the safety profile at the same time.

“As an alternative to lentiviral vectors, researchers have tried the Sleeping Beauty transposon system with regular plasmids,” Schleef explains. “They soon realized that especially for T cells, the plasmid’s DNA is toxic and a large proportion of T cells dies after taking up this DNA.”

With minicircles, on the other hand, the Sleeping Beauty transposon system works well because, unlike plasmids, the minicircles are free of harmful DNA. That is the result of a study conducted by PlasmidFactory’s main collaborators, the University of Würzburg in Germany.

Another advantage of minicircle DNA for the development of CAR-T therapy is the fact that the production is much faster and therefore more cost-effective than that of lentiviral vectors. Whereas lentiviral vectors require the production of three to four different plasmids, minicircle DNA can be transfected directly into T cells.

“In this instance, we only need two molecules for the transfection of the T cell,” Schleef says. “One is the minicircle containing the CAR gene, the other is the enzyme that places it into the genome of the T cell. This is an important aspect for people who are calculating the cost of such a therapy.”

Big plans for the future

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“With minicircle DNA we will be able to change CAR-T therapy” – Dr. Martin Schleef

“In the past few years, we have been working on upscaling the minicircle production,” Schleef explains. “At the beginning of the minicircle development, we were working in scales of 20 to 100 micrograms, which is not a lot. Now, we get requests from around the world for larger amounts, so we are already working with milligrams and are expecting to grow this. In general, our technology is easily scalable.”

Together with experts in the field of CAR-T therapy, the team at PlasmidFactory is also working on the production of higher quality grade minicircle DNA to meet the requirements of the majority of regulatory agencies.

“We have created a dedicated laboratory facility where only one type of minicircle DNA is produced at a time to prevent any potential contamination and the purification process is completely separate as well,” says Schleef. “The special purification procedure allows us to produce supercoiled minicircle monomers that meet the requirements of a homogenous, active, and highly qualitative product. With minicircle DNA we will be able to change CAR-T therapy.”

Do you work in the field of CAR-T therapies and want to learn more about minicircle DNA? Get in touch with the experts at PlasmidFactory today!

Images via Shutterstock & PlasmidFactory

Author: Larissa Warneck, Science Journalist at

Explore other topics: CAR-TGene therapy

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