Cupping Technique Has Standout Performance In DNA Vaccine Studies

By Deborah Borfitz

December 8, 2021 | The ultimate challenge of drug delivery—successfully getting a medicine into a cell—has several lines of pursuit when it comes to nucleic acid-based vaccines and the latest and greatest is an ancient cupping technique which, at least in rodents, is almost unfathomably good at driving immune responses. To say the Rutgers University research team was surprised by the finding would be an understatement.

It was what Jonathan Singer, Ph.D., associate professor in the department of mechanical and aerospace engineering, refers to as a “why the hell does it do that” moment prompting researchers to try every conceivable variation on dose amount and concentration, size of the suction cup, and suction pressure to see what they might not have considered. When used to deliver a SARS-CoV-2 DNA vaccine, the suction method generated an immune response roughly 100 times stronger than an injected vaccine alone.

Ironically, the finding totally upends recent misinformation by anti-vaxxers about how cupping could be used to remove COVID vaccine through the injection site, he says. Anyone attempting to undo their vaccine in this way would likely only be making it work better.

Results of the study, funded by GeneOne Life Science, Inc., recently published in Science Advances (DOI: 10.1126/sciadv.abj0611). The Korea-based biopharmaceutical company has licensed the technology for human clinical trials of a COVID vaccine. Phase I/IIa testing in Korea is expected to be completed next year, and work is already underway on design of a larger phase III study, says GeneOne Chief Medical Officer Joel Maslow, M.D., Ph.D.

The cupping device, called GeneDerm, is expected to add about a penny to the cost of the vaccine over the lifetime of the thermometer-size product, he notes. The clinically expensive part is the device’s replaceable cap, costing roughly 20 to 30 cents.

Much of the overall cost will be for manufacturing of the DNA. But, as was just demonstrated preclinically, a dose of 30 micrograms of the DNA-based COVID vaccine using GeneDerm was just as effective as 300 micrograms without it. “We know that we can have significant dose sparing with this device,” says Maslow, the first step in making the vaccine affordable in resource-challenged parts of the world.

VGXI, a wholly owned subsidiary of GeneOne, also has a 120,000-square-foot space for the manufacturing of DNA and RNA opening in Texas in the next few months, he adds, with more to follow. Currently, only a handful of companies in the world make DNA of sufficient quality for human vaccine purposes and “none make DNA to the quality of VGXI.”

Mild Suction

In addition to the efficacy of the GeneDerm device for vaccine delivery, it also came as a surprise that the cupping method had been tried previously, says Singer. A decade ago, a group in Japan used a suction device to deliver DNA to various internal organs of mice and, more recently, for cardiac gene transfer to their beating hearts.

But those were invasive procedures, unlike the approach of the Rutgers team. As GeneDerm is being used in clinical trials for the COVID-19 vaccine, the injection will be delivered via a shallow injection like the tuberculin test known as purified protein derivative (PPD), explains Professor Hao Lin, Ph.D., also in the department of mechanical and aerospace engineering. The device is then placed against the skin, applying “very slight pressure” at the injection site for 30 seconds. Before the device gets to market, researchers plan to have the suction time lowered to between 5 and 10 seconds, he adds.

As Maslow elaborates, the opening of the cap on the GeneDerm device creates a seal once pressed against the skin. A motor creates the suction pressure, which gets programmed in. “We are making it as failsafe as possible.”

Data in the Science Advances paper show how important it is to get the pressure right, says Singer, but also that there is potentially significant wiggle room—an ideal scenario in terms of device design. Good results can be had within a certain range, although the exact upper and lower thresholds for efficacy are still under investigation.

Initially, researchers thought they would need to damage cells a bit or cause an inflammatory response to stimulate vaccine uptake, Singer says. But it turned out the desired effect would occur even with moderate levels of suction routinely used for cosmetic purposes that is non-damaging to the cells.

Transport Problem

In a study led by a researcher in India and published in 2018 paper, the mechanisms for how purified nucleic acids get across cell-membrane barriers into the cytoplasm (RNA) and nucleus (DNA) of host cells—called transfection—was tied to alterations in membrane tensions, Singer says. That study was done on cells in a dish, and, since conditions in live tissue are very different, the Rutgers team still has not confirmed that the same mechanism is at work.

RNA is “extremely unstable” and when injected into the skin very rapidly gets chopped up by ribonuclease without protection—in the case of the mRNA COVID vaccines of Pfizer and Moderna, by being encapsulated with lipid nanoparticles, says Lin. The nanoparticles also enable the RNA cargo to get into the cell via chemical transfection.

In contrast, DNA is “very stable” and will stick around a while and not do anything, Lin continues. Unlike a small-molecule calcium ion weighing 40 grams per mole, DNA is a humungous molecule weighing about several million grams per mole, he notes, which is why it tends to stay in a small region once injected.

“DNAs and RNAs are like blueprints… of the virus” floating in the body and the cells act like Xerox machines, Lin uses as an analogy. If those blueprints are outside the cell, they don’t get copied. But if they break through the cell’s membrane, they can make copies of the virus’s bad-guy spike protein.

An added hurdle for DNA is that it needs a way to get past both cellular barriers to reach its destination—the nucleus. And cells will do everything they can to protect their control center where precious genetic information is stored, says Lin.

The original idea was to use suction at the vaccine injection site to break some of the microvessels in the skin, thereby bringing fluid to that region and helping the DNA disperse, he says. The cupping technique worked remarkably well, but for reasons that are not entirely clear.

Currently, the working hypothesis is that stressing a cell enlarges its surface—much like wrinkled skin that has been smoothed out, Lin explains. Relaxation of the skin after the stretching forms a natural, sac-like structure that ferries the DNA through the nuclear membrane.

Delivery Options

DNA vaccines have known advantages over RNA vaccines in terms of “manufacturability, stability, and reach,” says Singer. They also have notably lower cold chain requirements, which make them ideal for the developing world.

Several competing strategies have emerged for getting a DNA vaccine into the cell, he says. One is called electroporation, which uses high electric fields to aid in this transport but also causes damage and discomfort. Another is jet delivery of microdroplets.

RNA can also be contained within lipid nanoparticles, but that adds cost and steps to the procedure for making a vaccine and have been associated with many of the widely reported unpleasant side effects. On the other hand, Singer quickly adds, mRNA vaccines have demonstrated high efficacy in preventing severe COVID-19.

The Rutgers team was looking for a more affordable and user-friendly alternative delivery method that would also not cause tissue injury or unwanted side effects. They have been working with GeneOne on this front for the past five years.

During the phase I portion of the clinical trial in Korea, the DNA vaccine (GLS-5310) elicited no severe adverse effects in study participants and significantly stimulated the immune system, Maslow reports. The vaccine showed better efficacy and stronger T cell response—the key to long-term immunity—than all other nucleic acid vaccines based on outcomes data that have been publicly reported.

A phase I study of the GLS-5310 vaccine is expected to launch soon in the U.S.

The Pipeline

Two other coronavirus DNA vaccines are in advanced stages of clinical development, most notably the three-dose ZyCov-D vaccine of Zydus Cadila that recently received emergency use authorization in India, says Maslow. Another (the two-shot INO-4800) being developed by U.S.-based Inovio is in phase III testing.

The Zydus vaccine uses 2 mg per shot (6 mg total) and the INO-4800 2 mg per shot (4 mg total), he says. GLS-5310 is being studied at 1.2 mg per shot (2.4 mg total) with “no significant difference” seen between dosing at the 0.6 mg and 1.2 per shot level, “making future dose reduction very possible using suction.”

One of the big advantages of nucleic acid-based vaccines in general is the antigens are made by the people who are being vaccinated, he continues. The delivered RNA or DNA gets into cells where proteins get made that are then exported to provoke an immune response.

“The genes that encode those proteins can be easily swapped out… [so] designing a new vaccine is not time-intensive,” says Maslow. Other kinds of molecules can also be encoded for therapeutic purposes, including the gene that encodes for factor VIII in the treatment of hemophilia (one of the products in GeneOne’s pipeline).

The goal here is to replace the mutated or non-functional factor VIII in patients, he says. DNA molecules would be delivered (possibly self-delivered) as a means of preventing bleeding disorders. “Currently, factor VIII, even recombinant factor VIII, is very expensive and only given during significant bleeding times, and this is a product we are envisioning can be given prophylactically.”

Monoclonal antibodies are another nucleic acid product type. GeneOne has the lead when it comes to nucleic acid encoded hepatitis B monoclonal antibodies, Maslow says. Hepatitis B is common in many parts of Asia and the typical means of preventing disease transmission from mother to child in the U.S. and Europe—giving babies a hepatitis B vaccine and hepatitis B immune globulin (for mothers who are hepatitis B positive) shortly after birth—are often cost-prohibitive. In India, babies will only be given the vaccine unless their family can afford to pay cash for the immune globulin.

A DNA-encoded product would again be much less expensive as well as much more easily stored and administered, he says, and therefore might raise the standard of care across Asia closer to first-world standards. The same or similar products could also be used to prevent hepatitis B reactivation in people who get liver transplants from people who previously had the disease.

To Be Determined

GeneDerm is the first of potentially several devices to deliver DNA to cells is a way that is non-damaging to cells and exceedingly well-tolerated by patients, says Maslow. Following its good showing in the study with the GLS-5310 vaccine, it will next be tested with some of the company’s other DNA-based products.

On the RNA front, the limitations are not yet known, Maslow says, including whether the less reactogenic lipid nanoparticle formulations now under development will be as effective as the ones already in use. The pandemic has also only partially addressed the question of longer-term RNA manufacturing capacity.

Whether DNA or RNA proves to be the better platform for therapeutic purposes remains to be seen, he adds. Perhaps one approach or the other, or a combination of the two, will be needed in different scenarios.