'Cookbook Recipes' Would Cure Disease With Nontoxic DNA Delivery Systems

As reported in the Aug. 9 issue of the Proceedings of the NationalAcademy of Sciences, researchers have now performed a careful,comprehensive study to see how negatively charged lipids stick tonegatively charged DNA and self-organize into structures.

"Many research groups have made concoctions with ingredients indifferent proportions and then assessed their effectiveness in genedelivery, but this is hard and requires a lot of intuition," saidGerard Wong, a professor of materials science and engineering, physics,and bioengineering at the University of Illinois at Urbana-Champaign,and corresponding author of the paper.

"By understanding some of the physics, we now have recipes forassembling delivery systems with different structures, which can haveintrinsically different, controllable DNA delivery efficiencies," Wongsaid. "We found that the same family of structures are generated formany different ions."

Gene therapy is one of the most promising strategies fordeveloping cures for many hereditary and acquired diseases. Protocolshave been approved for treating cancer, cystic fibrosis andneuromuscular disorders, for example, but delivering DNA to the properlocation and getting the right amount of DNA expression without killinginnocent cells has become the Achilles' heel in DNA delivery.

Positively charged (cationic) synthetic molecules will readilybond to negatively charged DNA molecules and have been used for DNAdelivery, but these cationic molecules are often toxic to cells, Wongsaid. An alternative is to use naturally occurring negatively charged(anionic) lipids that won't harm cells.

"The problem then becomes: 'How do you stick a negativelycharged lipid to a negatively charged DNA molecule?'" said Wong, whoalso is a researcher at the Beckman Institute for Advanced Science andTechnology. "One idea is to glue the lipids and DNA together withpositively charged ions like calcium."

Using synchrotron small angle X-ray scattering and confocalmicroscopy, Wong and his colleagues -- former Illinois graduate studentHongjun Liang (now a postdoctoral researcher at the University ofCalifornia at Santa Barbara) and theoretician Daniel Harries at theNational Institutes of Health -- investigated how differention-mediated interactions were expressed in self-assembled anioniclipid-DNA structures.

At low membrane charge densities, for example, anionic lipidsand DNA molecules self-assemble into structures with alternating layersof DNA and anionic membranes bound together by cations, Wong said. Athigh membrane charge densities, there is a surprise: The DNA isexpelled, leaving a stack of anionic membranes glued together bycations -- a feature that could prove useful in other controlled drugdelivery applications.

The researchers also produced inverted hexagonal structureswith encapsulated DNA. "First, the strands of DNA are coated withpositively charged ions," Wong said. "The strands are then wrapped withnegatively charged lipids and resemble tubes, which are then groupedinto hexagonal arrangements."

Utilizing naturally occurring anionic lipids instead ofcationic lipids "creates a whole hierarchy of interactions and a muchricher range of structures," Wong said, "which really opens up newpossibilities."

The U.S. Department of Energy and the National Science Foundation funded the work.