Development of an efficient method of gene introduction to target cells is the key issue in treating genetic and acquired diseases by in vivo gene therapy. Although various nonviral approaches have been developed, any method needs to be optimized in terms of the target disease and transgene product. The most important information required is (i) target cell-specificity of gene transfer, (ii) efficiency, (iii) duration of transgene expression, and (iv) the number of transfected cells following in vivo application of a vector. These characteristics are determined by the properties of the vector used, as well as the route of its administration, biodistribution, interaction with biological components and the nature of the target cells. Cell-specific gene transfer can be achieved by controlling the tissue disposition of plasmid DNA (pDNA), although the interaction of the pDNA complex with biological components might limit the specificity. Various approaches have been reported to increase the efficiency of transgene expression, from cationic lipids/polymers to physical stimuli, but some of those are ineffective under in vivo conditions. The duration of transgene expression is a complex function involving variables including the cell type, transfection method, and plasmid construct. Immune response often reduces the level and duration of transgene expression. In addition, the number of transfected cells is important, especially in cases in which the therapeutic protein localizes within the target cells. Successful clinical application of nonviral gene delivery methods rely on the development of such methods optimized for a particular target disease.