Researchers at Ohio State University and the University of Texas Southwestern Medical Center say they have developed a new gene therapy technique by transforming human cells into mass producers of tiny nano-sized particles full of genetic material that has the potential to reverse disease processes. Though the work was intended as a proof of concept, the experimental therapy slowed tumor growth and prolonged survival in mice with gliomas, which constitute about 80% of malignant brain tumors in humans.

The technique takes advantage of exosomes, fluid-filled sacs that cells release as a way to communicate with other cells. While exosomes are gaining ground as biologically friendly carriers of therapeutic materials—because there are a lot of them and they don’t prompt an immune response—the trick with gene therapy is finding a way to fit those comparatively large genetic instructions inside their tiny bodies on a scale that will have a therapeutic effect.

This new method (“Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation”) appears in Nature Biomedical Engineering and relies on patented technology that prompts donated human cells such as adult stem cells to spit out millions of exosomes that, after being collected and purified, function as nanocarriers containing a drug. When they are injected into the bloodstream, they know exactly where in the body to find their target, even if it’s in the brain.

“Exosomes are attractive as nucleic-acid carriers because of their favorable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides,” the investigators wrote.

“Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 10³-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic phosphatase and tensin homologue (PTEN)-deficient glioma mouse models, mRNA-containing exosomes restored tumor-suppressor function, enhanced inhibition of tumor growth and increased survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.”

“Think of them like Christmas gifts: The gift is inside a wrapped container that is postage- paid and ready to go,” said senior study author L. James Lee, PhD, professor emeritus of chemical and biomolecular engineering at Ohio State University. And they are gifts that keep on giving, Lee noted: “This is a Mother Nature–induced therapeutic nanoparticle.”

In 2017, Lee and colleagues made a regenerative medicine-related discovery called tissue nanotransfection (TNT). The technique uses a nanotechnology-based chip to deliver biological cargo directly into skin, an action that converts adult cells into any cell type of interest for treatment within a patient’s own body. By looking further into the mechanism behind TNT’s success, scientists in Lee’s lab discovered that exosomes were the secret to delivering regenerative goods to tissue far below the skin’s surface.

The technology was adapted in this study into a technique first author Zhaogang Yang, PhD, a former Ohio State postdoctoral researcher now at the University of Texas Southwestern Medical Center, termed cellular nanoporation.

The scientists placed about one million donated cells (such as mesenchymal cells collected from human fat) on a nano-engineered silicon wafer and used an electrical stimulus to inject synthetic DNA into the donor cells. As a result of this DNA force-feeding, as Lee described it, the cells need to eject unwanted material as part of DNA transcribed messenger RNA and repair holes that have been poked in their membranes.

“They kill two birds with one stone: They fix the leakage to the cell membrane and dump the garbage out,” Lee said. “The garbage bag they throw out is the exosome. What’s expelled from the cell is our drug.”

The electrical stimulation had a bonus effect of a thousand-fold increase of therapeutic genes in a large number of exosomes released by the cells, a sign that the technology is scalable to produce enough nanoparticles for use in humans.

Essential to any gene therapy, of course, is knowing what genes need to be delivered to fix a medical problem. For this work, the researchers chose to test the results on glioma brain tumors by delivering a gene called PTEN, a cancer-suppressor gene. Mutations of PTEN that turn off that suppression role can allow cancer cells to grow unchecked.

For Lee, founder of Ohio State’s Center for Affordable Nanoengineering of Polymeric Biomedical Devices, producing the gene is the easy part. The synthetic DNA force-fed to donor cells is copied into a new molecule consisting of messenger RNA, which contains the instructions needed to produce a specific protein. Each exosome bubble containing messenger RNA is transformed into a nanoparticle ready for transport, with no blood-brain barrier to worry about.

“The advantage of this is there is no toxicity, nothing to provoke an immune response,” said Lee, also a member of Ohio State’s Comprehensive Cancer Center. “Exosomes go almost everywhere in the body, including passing the blood-brain barrier. Most drugs can’t go to the brain.

“We don’t want the exosomes to go to the wrong place,” he explained. “They’re programmed not only to kill cancer cells, but to know where to go to find the cancer cells. You don’t want to kill the good guys.”

The testing in mice showed the labeled exosomes were far more likely to travel to the brain tumors and slow their growth compared to substances used as controls.

Because of exosomes’ safe access to the brain, Lee said, this drug-delivery system has promise for future applications in neurological diseases such as Alzheimer’s and Parkinson’s disease.

“Hopefully, one day this can be used for medical needs,” Lee said. “We’ve provided the method. If somebody knows what kind of gene combination can cure a certain disease but they need a therapy, here it is.”