Work by engineering Associate Professor Corinne Lengsfeld may help gene therapy reach its full potential. Photo: Matt Suby
Most kids with a cold will sniffle, whine and cough. But when 3-year-old Benjamin Ratico comes down with a bug, it can trigger a life-threatening asthma attack.
“He breathes rapidly and shallowly, and his skin sucks in to his ribs,” says his mother, Jennifer Ratico (attd. 1993-94), who also has asthma. “It’s frightening.”
When Ratico sees her son struggling for air — “It’s like an elephant is sitting on your chest,” she says — she outfits Benjamin with a facemask, measures a dose of Albuterol into an attached plastic cup, switches on an air compressor and has him inhale a medicated mist for 10 minutes. The treatment helps him breathe more easily.
More than 20 million Americans have asthma, and millions more suffer from emphysema, chronic bronchitis, cystic fibrosis or acute respiratory distress syndrome. Like Benjamin, many of those people use a nebulizer — a mist-generating device — to ingest medical aerosols to treat pulmonary diseases. Nebulizers also are a potential gene therapy delivery mechanism.
Unfortunately, nebulizers don’t work very well. In fact, most of the medication never even makes it into the lungs.
That’s something that DU engineering Associate Professor Corinne Lengsfeld is working to change. She and Thomas Anchordoquy, an associate professor of pharmaceutical biotechnology at the University of Colorado, aim to make nebulizers work more efficiently while delivering drugs with greater efficacy than currently possible. Their work may also help gene therapy finally achieve its full potential as a treatment for infectious diseases as well as genetically based diseases such as cancer.
DNA delivery
Lengsfeld — a mechanical engineer who teaches fluid dynamics, among other subjects — did her postdoctoral work in a biochemical laboratory specializing in pharmaceutical research. She began collaborating with Anchordoquy six years ago.
At that time, gene therapy research was stuck on a persistent problem: Because DNA “breaks” easily, Lengsfeld explains, it is very difficult to deliver it into the body with any sort of positive therapeutic effect. In fact, Lengsfeld notes, only 33 percent of gene-therapy DNA made it through the production process intact, and even less was being delivered to the target lung tissue.
“This was making a potentially fabulous cure not feasible for even the wealthiest people,” Lengsfeld says. “It wasn’t even feasible for clinical trials, because you couldn’t make enough of the drug to test it.
“The whole purpose of that work was that DNA therapeutic treatments were never going to be a commercial reality if you couldn’t get the costs under control,” she adds. “The biggest cost factor was waste, both during manufacturing and drug delivery.”
Pharmaceutical giants Amgen, Genentech and Pfizer asked Lengsfeld and Anchordoquy to help solve that problem by studying drug delivery systems, including nebulizers. Those conversations, in turn, led to several projects funded by $350,000 in National Science Foundation (NSF) grants.
Lengsfeld and Anchordoquy enlisted DU student Leslie Worden (BSCPE and BSME ’03, MS mechanical engineering ’05), CU pharmacy doctoral candidate Yvonne Lentz (who graduated in 2005) and an army of other student co-investigators to help to explain the lost or damaged DNA and devise ways to conserve it.
The most commonly used nebulizer types include jet nebulizers, which create aerosols by forcing compressed air through liquid medicine, and ultrasonic nebulizers, which use inaudible sound waves. Lengsfeld and DU student Ben Filas (BS biochemistry ’05) experimented with different brands of jet nebulizers to determine how well they atomized and delivered the asthma drug Salbutamol to the tissues lining a model lung. They blew a measured dose of the drug in a saline solution through each nebulizer under varying conditions — with and without an electrostatic charge, for instance — and measured the effects.
In all three brands tested, they found that most of the medicine stuck to the insides of the nebulizer and never made it into the lungs. They also discovered that humidifying the mixture reduced the amount of Salbutamol deposited in the nebulizer housing by half.
In addition to looking at traditional medications, the team also studied gene therapy delivery and found that cavitation was damaging the genetic material that actually made it into the lungs before it could be absorbed.
“Cavitation is the formation of vapor or bubbles in liquid during any fluid process that puts it in tension,” Lengsfeld explains. “The bubble could be stable, but if there’s oscillating tension and compression, it becomes unstable and collapses, creating a shock wave that annihilates the therapeutic.” As it turns out, the presence of DNA — the whole point of gene therapy — was triggering these destructive bubbles.
Lentz and Worden joined Lengsfeld and Anchordoquy to study the primary reason DNA degrades in jet-style nebulizers. Using computational fluid dynamics methods designed to describe visible events, they determined that submicroscopic shock waves with wavelengths shorter than the DNA molecules can rip the DNA to shreds. However, the DNA can safely “surf ” the surface of the shock waves with longer wavelengths. The study was published in the Journal of Aerosol Science.
“The three most remarkable outcomes were, first, that no one knew that nebulizers were degrading the drug delivery to the extent they do,” Lengsfeld says. “Everyone studying these drugs was using the worst two delivery methods: jet and ultrasonic nebulizers.” That discovery affected dozens of human and animal clinical studies under way worldwide.
“The second thing is that no one in protein or DNA therapeutics had considered cavitation as a problem, but cavitation occurs in every biotech processing system,” adds Lengsfeld, who credits Lentz with these two discoveries.
“The third thing was this idea that we don’t have to do process development by trial and error,” Lengsfeld continues. “We now know if you can keep your turbulent eddy size bigger than the DNA, it will never fragment.” Worden is credited with that discovery.
The studies from the team’s first research phase have been published, and the phones have been ringing with inquiries from biotech companies. Lentz recently joined Genentech, one of the world’s largest biotechnology companies, where she will continue her cavitation research.
But, the work published so far is less important than the work it inspired, Anchordoquy says. Thanks to a recent $500,000 NSF grant plus a $75,000 seed grant from the Keck Foundation, he and Lengsfeld are now building on their earlier work.
“We’re working on some very basic problems that have been largely ignored,” Anchordoquy says. “Some of the things we’ve reported would greatly improve gene delivery, especially by pulmonary means.
“We haven’t solved all the problems by any means,” he adds, “but in connection with other discoveries yet to come, we could move toward making genes a viable therapeutic alternative.”
Their current research involves protecting genes while they are en route to the targeted cells. An existing technique bonds a negatively charged gene to a larger, positively charged molecule — either a long molecule called a polymer or a fat molecule called a lipid. Although the coating helps protect the gene from shear damage, the strong chemical bond interferes with cells’ ability to absorb the therapeutic gene.
“If you’re a gene, it doesn’t do you any good to get to the outside of a cell. You have to get into the cell,” Anchordoquy says. He and Lengsfeld have patented a process for encapsulating the gene with several small particles — between one-tenth and one-one hundredth the size of the target cell — rather than a single large one.
“The gene is free-floating within the capsule instead of bound to the capsule,” Anchordoquy explains. “This technology could represent a significant breakthrough if we can get it to work in the way we deliver genes.” The gene capsules could be aerosolized or injected.
It’s a totally new approach that draws on manufacturing techniques from the food processing, perfume and pharmaceutical industries, Lengsfeld adds.
“Gene therapy has really kind of come to a halt because we can’t get the DNA to the cell efficiently enough to make a difference,” she says. If they are successful in revitalizing gene therapy, it could be applied to such diseases as cancer, hemophilia, cystic fibrosis and Severe Combined Immunodeficiency Syndrome, commonly known as the “bubble boy disease.”
“We’re trying not to keep what we’ve learned a secret,” Lengsfeld says. “The whole reason we started this in the first place is the companies said ‘solve this problem.’ Now we feel it’s important to push it out there.”
To that end, Lengsfeld and Anchordoquy have written a chapter on their discoveries for a forthcoming pharmaceutical biotechnology handbook, and Lengsfeld created a new advanced fluid dynamics course that incorporates their research.
And, as their uniquely qualified students enter graduate schools, university research and the biotech industry, their discoveries will continue to spread and inspire new research.
“What we’re doing is on the forefront of where education needs to go,” Anchordoquy says. “In the pharmaceutical industry you typically have a mix of device people who are engineers and biologists who understand the disease and the drugs. What you need is someone who speaks both languages.
“My pharmaceutical students are getting some engineering knowledge, and DU’s engineering students are getting some pharmaceutical knowledge,” he adds. “That’s exactly the kind of cross-training that’s needed.”