By David N. Leff
In the expanding array of gene-therapy delivery vehicles, DNA is infected by virus, injected by needle, shot from guns. Would you believe genes squeezed from sponges?
These particular synthetic polymer matrices are designed to enhance gene therapy applied to tissue engineering, as in necrotic ulcers, skin trauma or periodontal disease. "Lost or deficient tissue function," stated chemical engineer David Mooney, "leads to millions of surgical procedures each year and a loss to the U.S. economy of hundreds of billions of dollars. Tissue engineering," he went on, "has emerged as a potential means of growing new tissues and organs to treat these patients."
Mooney, who is on the faculty of the University of Michigan at Ann Arbor, made these points in the June 1999 issue of Nature Biotechnology. The paper, of which he is senior author, is titled: "DNA delivery from polymer matrices for tissue engineering."
Its lead author is postdoctoral fellow Lonnie Shea, who this fall moves to Northwestern University in Evanston, Ill., as assistant professor of chemical engineering.
"Obviously people working in the field of tissue engineering," Shea told BioWorld Today, "are looking for carrier material generally considered to be biocompatible. This PLG polymer that we're using - the polylactide-coglycolide - has a long history. It's FDA-approved for biodegradable surgical sutures; therefore a lot of people are using this material for making matrix scaffolds.
"But what's really novel about this PLG," he continued, "is our processing approach to elaborate this porous, sponge-like material. It doesn't damage DNA, and would help get a sustained release from it."
Shea described that patent-pending procedure, from purchase of the starting material, in powder or pellet form, to insertion of the DNA-delivering carrier disc under the skin of laboratory rats.
"We take these polymer granules," he began, "and grind them down till we get small particles 100 microns in diameter - smaller than a grain of salt. Then we mix just enough of this polymer powder to make the disc itself, adding salt and our DNA plasmid of interest, freeze-dried from aqueous solution to a solid powder. Then we press this mixture into a disc 1.3 centimeters across and 3 millimeters thick. Using a jack, a plate and a die, we just smash it together, and the disc holds its shape.
"Next," Shea recounted, "comes the crucial gas-forming part of the process. We put the discs in a pressure vessel and turn up carbon dioxide (CO2) pressure till it's 800 pounds per square inch. We just let it sit there till the CO2 dissolves into the polymer. Once it's set long enough and reaches equilibrium, we release the pressure relatively quickly - in a matter of minutes.
"When we do that, the CO2 wants to come out of the polymer. Each individual PLG particle actually expands, and when it kind of collides with the other polymer particles they fuse together. The idea is that when the particles expand, they are able to incorporate the DNA, and trap it within the pores that are created."
Subtract Grain Of Salt, Gain A Pore
Shea continued: "But we still have salt in there. So when we leach that out, we leave behind these relatively big sponge-like pores - about 90 percent of the disc mass - within this material. All the granules have connected to form a 3-dimensional porous structure."
After extensive in vitro testing of their PLG- based gene-delivery carriers, Shea and his co-authors mounted an in vivo experiment to verify its action in a tissue-engineering situation. Their construct consisted of a DNA plasmid encoding platelet-derived growth factor (PDGF), a potent player in tissue repair.
"In these rat experiments," he recounted, "we made a small incision in the animals' back - underneath the dermis, sitting just above the rib cage, in with all the connective tissue that's just underneath the skin - and slipped the disc into that space. Then we sutured them back up."
"Tissues containing polymers releasing the PDGF plasmid," the co-authors' paper reported, "demonstrated an increase in granulation tissue and of vascularization." Moreover, in areas beyond the incision, edema showed that the gene was working its wound-healing effects well beyond the implant site. These indicator effects continued to increase for up to four weeks after insertion of the plasmid.
Control animals, which received the same treatment but with an irrelevant gene, showed no such effects. Then, to confirm that the polymer carrier was responsible for these sustained results, the team needle-injected a similar aliquot of PDGF-expressing plasmid directly into the subcutaneous pocket. At autopsy, these showed effects no better than those in the control rats.
"The enhanced blood-vessel formation," Shea observed, "is something that is being investigated clinically for various applications, one of them being tissue regeneration in periodontal disease. We're based in the dental school here at the University of Michigan," he pointed out, "so we're now setting up some collaborations with the people here, and getting different animal models where PDGF would be appropriate for growth factor gene delivery."
Rats Confirmed Proof Of Principle
"Another aspect of our PLG matrix that we're trying to characterize," Shea went on, "is how long would a single disc continue expressing protein. In the paper, we've showed results up to four weeks. So we took some similar types of discs, implanted them in rats, and are letting them go out to 20 weeks. Right now," he said, "we're about 14 weeks toward 20.
"We're also looking more at the muscle biology," Shea observed, "and trying to do things like in situ hybridization and histochemistry - actually seeing where the protein is being produced. Seeing if the gene is actually there."
He added, "One of the advantages of delivering DNA as opposed to delivering protein is DNA's stability. Proteins can denature, which means they lose their activity. DNA is much more stable, easier to work with in terms of this processing. Also, the effect of having it as a solid instead of a liquid is an advantage."
Mooney told BioWorld Today, "The University of Michigan has applied for patents covering this technology, and is considering licensing rights to industry."