DNA delivered to cells via electrical pulses was first explored for creating new vaccines and is now being tested in the lab to produce disease-fighting proteins.
Researchers have discovered that mouse muscle tissue zapped with electrical pulses can absorb synthetic DNA that instructs an antibody to neutralize the human immunodeficiency virus (HIV). The cells do this by following the instructions, producing the corresponding protein, and enhancing the protein’s activity.
Biologic pharmaceuticals - antibodies or proteins that treat autoimmune diseases such as ulcerative colitis and rheumatoid arthritis - are expensive to produce, transport, and store in freezers. Making therapeutic proteins, such as this HIV antibody, inside human cells could make biologic drugs accessible to more people, says David Weiner at The Wistar Institute, an independent nonprofit biomedical research institute.
The idea of using synthetic DNA to produce proteins inside cells started with efforts to make DNA-based vaccines in the early 1990s. Researchers first synthesized small loops of synthetic DNA called plasmids. These plasmids contained genetic instructions for proteins from HIV, tumors, or the flu virus. When cells used those instructions, they produced a foreign protein that stimulated an immune response.
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“We pretty much cured every disease known to mouse,” Weiner says. But the approach didn’t work well in people. Clinical trials of these early vaccines did not stimulate the immune system enough.
The main reason was that the plasmid didn’t enter the cells efficiently. One way to simplify plasmid delivery is to open pores in the outer membrane of cells and allow the DNA to pass through. This technique, called electroporation, involves zapping cells with nano- to millisecond pulses of electricity, which creates open spaces in the membrane that last for seconds to minutes before the membrane reseals.
To improve both delivery and expression of the plasmid, Weiner and his colleagues have simultaneously optimized both the genetic instructions and the machine that delivers the electrical pulses. In collaboration with companies that make electroporation machines for use on human skin or muscle, the researchers now have machines that sense the resistance building in a person’s skin and automatically adjust within milliseconds to deliver a consistent dose of electricity without arcing or burning. The electric pulses also change direction to help the negatively charged DNA move deeper into cells.
In the past few years, Weiner, his colleagues, and collaborators at pharmaceutical companies have tested DNA-based vaccines delivered by electroporation in preclinical and clinical trials. The researchers have developed plasmids that immunize mice against lethal pneumonia and a range of flu viruses. Last year they also tested a DNA vaccine for the Zika virus in phase 1 clinical trials.
Now Weiner and his colleagues are taking this concept of DNA delivery through electroporation beyond vaccines and into therapeutic protein production, such as the HIV-neutralizing antibody recently published in EbioMedicine.
This time the researchers used a trick new to DNA-encoded biologics. They included a second plasmid along with the instructions for the HIV-neutralizing antibody. The second plasmid specifies how to assemble an enzyme that adds a sulfate group to the antibody to make it fully active.
The researchers carefully designed the sequence of this plasmid so that both proteins would end up in the cell’s central distribution network. Once the cells make the HIV-neutralizing protein eCD4-Ig, its identity as an antibody means it naturally enters a pathway that collects, packages, and sorts molecules en route to various locations in the cell. The researchers added a sequence to the plasmid so that the final enzyme contained an antibody-like tail, which directed it to the same molecular sorting network in the cell.
The researchers delivered both plasmids to leg muscles of mice, using 1,000 times less plasmid for the modifying enzyme than for the therapeutic antibody. They measured peak concentrations of sulfated antibody at 80-100µg/mL in the blood. Although the level then declined, it remained above 3 µg/mL for 150 days. The researchers isolated the sulfated antibody from the mouse blood and found that it neutralized all 12 recombinant HIV pseudoviruses in a standardized panel used to test the efficiency of HIV vaccines.
The researchers now need to test the approach in larger animals, Weiner says. From the clinical trial success of DNA vaccines delivered by electroporation, he thinks DNA-based biologics have a strong foundation.
Melissae Fellet is an independent technology writer.
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