Anionic lipids help gene therapy reach its target
up to its promise as a targeted, specific and well-tolerated
treatment approach.
The primary reason for this is that it has been difficult to deliver the gene to the required areas of the body safely, in sufficient quantities, and in a form stable enough for good expression of the protein for which it codes. Viruses have been used as delivery vectors but have fallen out of favour somewhat because of toxicity issues, and attention has turned to non-viral alternatives.
Now, a team of US researchers has developed a 'recipe book' of non-toxic, non-viral DNA delivery systems that aims to help in the design of genetic medicines that can be delivered safely and effectively.
The scientists, from the University of Illinois at Urbana-Champaign, collated data on how a class of molecules known as negatively charged lipids and DNA molecules bind to each other and self-organise into structures, in order to develop a protocol for selecting the ingredients for genetic medicines.
"Many research groups have made concoctions with ingredients in different proportions and then assessed their effectiveness in gene delivery, but this is hard and requires a lot of intuition," said Gerard Wong, a professor of materials science and engineering, physics, and bioengineering at the university.
"By understanding some of the physics, we now have recipes for assembling delivery systems with different structures, which can have intrinsically different, controllable DNA delivery efficiencies," he said. "We found that the same family of structures are generated for many different ions."
Positively charged (cationic) synthetic molecules will readily bind to negatively charged DNA molecules and have been used for DNA delivery, but these cationic molecules are often toxic to cells, Wong said. An alternative is to use naturally occurring negatively charged (anionic) lipids that do not harm cells.
"The problem then becomes: 'How do you stick a negatively charged lipid to a negatively charged DNA molecule?'" said Wong. "One idea is to glue the lipids and DNA together with positively charged ions like calcium."
Using synchrotron small angle X-ray scattering and confocal microscopy, Wong and his colleagues investigated how different ion-mediated interactions were expressed in self-assembled anionic lipid-DNA structures.
At low membrane charge densities, for example, anionic lipids and DNA molecules self-assemble into structures with alternating layers of DNA and anionic membranes bound together by cations, Wong said. At high membrane charge densities, there is a surprise: The DNA is expelled, leaving a stack of anionic membranes glued together by cations -- a feature that could prove useful in other controlled drug delivery applications.
The researchers also produced inverted hexagonal structures with encapsulated DNA. "First, the strands of DNA are coated with positively charged ions," Wong said. "The strands are then wrapped with negatively charged lipids and resemble tubes, which are then grouped into hexagonal arrangements."
Using naturally occurring anionic lipids instead of cationic lipids creates a richer range of structures which open up new delivery possibilities for DNA, concluded Wong.
While still in its infancy, a market research report published earlier this year by Kalorama has suggested that the the nucleic acid therapeutic market, including gene therapy, could in time achieve a value of over $210 billion (€161bn).
The work is reported in the 9 August issue of the Proceedings of the National Academy of Sciences.
