Gene therapy holds a promise to treat a variety of genetic disorders including cancer, diabetes and many other inherited and acquired diseases. Successful clinical application of gene therapy requires the development of efficient tools for targeted delivery of nucleic acids into affected cells. Presently used virus-based DNA delivery systems produce serious immunological and oncological side effects. Lipid-based DNA delivery systems are widely viewed as a potentially safer alternative. The development of effective lipid-based DNA nanoparticles (lipoplexes) is limited by poor understanding of how DNA is packed into the lipid vesicles. This project aims at characterizing the assembly of lipoplexes using single DNA manipulations. Preliminary studies revealed a number of mechanistic details of lipid-DNA interaction that were not detected using more conventional, bulk methods. We plan to evaluate the idea that the interactions of lipids with DNA can be correlated with the efficiency of lipoplex assembly and cell transfection efficacy. Specific aims of the project are (i) Quantify deformation of single DNA by DOTAP, (ii) compare a series of lipids in their ability to compact DNA;(iii) correlate the single molecule data with the quality of assembled lipoplexes. The completion of this study will evaluate the idea that single molecule data can improve the assembly of lipoplexes and transfection efficiency. If true, this would make the assembly of DNA containing nanoparticles a much more predictable process and would lay grounds for more rational design of non-viral DNA delivery systems. This in turn may help develop lipoplexes suitable for clinical applications and thereby enable novel interventions in treating disease.

PUBLIC HEALTH RELEVANCE: Gene therapy could potentially be used to treat a variety of inherited and acquired ailments including cancer, diabetes, aging and heart and neurological disorders. The development of non-viral, lipid-based DNA delivery vectors could help design efficient gene therapies with reduced side effects. We plan to evaluate the idea that the design of such vectors can be greatly improved using single DNA nanomanipulation technique.

R21EB009238

2009-07-06

2012-06-30