Helper-Independent sleeping beauty Transposon–Transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo

作者：Jacob Giehm Mikkelsen, Stephen R. Yant, Leonard Meuse, Zan Huang, Hui Xu, Mark A. Kay · 发表于：Molecular Therapy · 年份：2003 · DOI：10.1016/s1525-0016(03)00216-8 · 被引用次数：147 · 研究领域：Virus-based gene therapy research、RNA Interference and Gene Delivery、CRISPR and Genetic Engineering

Transposon-based vectors represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. Here, we report the development of helper-independent transposon-transposase (HITT) vectors, which contain on single plasmids (i) a Sleeping Beauty (SB) transposon containing the transgene and (ii) a SB transposase expression cassette. To obtain an optimal level of transposase expression from HITT vectors, we determined the relative strength of a panel of different promoters in mouse liver and used these promoters to drive transposase expression from injected HITT vectors carrying a human alpha(1)-antitrypsin (hAAT) expression cassette flanked by transposon inverted repeats. By correlating promoter strength with stabilized serum hAAT levels, a narrow expression window supporting high-level transposition in the liver was defined. Peak levels of long-term gene expression were obtained with promoters 30- to 40-fold less active than CMV in mouse liver, whereas reduced stable levels of hAAT were detected with both weaker and stronger promoters. Injected HITT vectors induced transposase-dependent insertion of transposon DNA into the genome of at least 5-6% of transfected hepatocytes, generating levels of persistent hAAT expression that were 2- to 4-fold higher than with an optimized two-plasmid approach. In addition, we show that HITT vectors carrying a human factor IX (hFIX)-containing transposon support (i) long-term hFIX expression i...