Structural Studies of Novel DNA Vectors for Non-viral Gene Transfer

非病毒基因治疗一般使用DNA质粒作为载体。这些质粒通常是在大肠杆菌中产生的。该方法通常是基因治疗实施中的限制因素，因为成本和产生的载体数量不够少。在昆虫细胞中生产DNA载体的一种新的、高效的方法是在表达系统中仅使用腺相关病毒(AAV)的单个非结构基因，而不是整个基于杆状病毒的AAV载体生产系统。生化研究表明，载体主要以单体和二聚体的形式存在，与典型的质粒构型不同。利用原子力显微镜，我们证明了载体单体以末端闭合的线性分子内双链形式存在。这一点通过变性DNA载体和显示在所得到的溶液中存在环状的单链DNA闭链来证明。在高盐条件下，分子间络合物的废除表明，双链甚至更高的低聚物是通过单体的闭合末端之间的静电相互作用而形成的。

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.