It is highly desirable to generate tissue-specific and persistently high-level transgene expression per genomic copy from gene therapy vectors. Such vectors can reduce the cost and preparation of the vectors and reduce possible host immune responses to the vector and potential toxicity. Many gene therapy vectors have failed to produce therapeutic levels of transgene because of inefficient promoters, loss of vector or gene expression from episomal vectors, or a silencing effect of integration sites on integrating vectors. Using in vivo screening of vectors incorporating many different combinations of gene regulatory sequences, liver-specific, high-expressing vectors to accommodate factor IX, factor VIII, and other genes for effective gene transfer have been established. Persistent and high levels of factor IX and factor VIII gene expression for treating hemophilia B and A, respectively, were achieved in mouse livers using hydrodynamics-based gene transfer of naked plasmid DNA incorporating these novel gene expression systems. Some other systems to prolong or stabilize the gene expression following gene transfer are also discussed.