Non-viral gene therapy generally uses DNA plasmids as vectors. These plasmids are typically produced in E-coli. The method is often the limiting factor in gene therapy implementation because of the cost and the inadequately small amounts of vectors produced. A novel and highly efficient method for the production of DNA vectors in insect cells has been developed by using only a single, non-structural gene of the adeno-associated virus (AAV) in the expression system, rather than the complete baculovirus-based AAV vector production system. Biochemical studies indicate that the vectors exist primarily as monomers and dimers and differ from the typical plasmid configuration. Using the AFM we showed that the vector monomers exist as linear intramolecular duplexes with closed ends. This was demonstrated by denaturing the DNA vectors and showing the existence of circular, closed chains of single-stranded DNA in the resulting solution. Duplexes and even higher oligomers form via electrostatic interactions between the closed ends of the monomers as shown by the abolition of intermolecular complexes under high salt conditions.
