Hans Herweijer

Background: To properly study gene expression in vivo, often long‐term expression is desired. Previous studies using plasmid DNA (pDNA) vectors have typically resulted in short‐term expression. Here, we evaluated combinations of the albumin promoter with different enhancers and untranslated regions for liver‐specific expression in mice.Methods: A series of pDNA secreted alkaline phosphatase (SEAP) reporter gene expression vectors was constructed using the albumin promoter and various other expression cassette elements. Each was evaluated for level and duration of SEAP expression in mice following hydrodynamic tail vein delivery.Results: Sustained liver expression was obtained from vectors combining the albumin promoter with an albumin 3′ untranslated region (3′UTR). The level of expression was increased by inclusion of enhancers and a 5′ intron. The optimal expression vector consisted of the albumin promoter combined with an α‐fetoprotein MERII enhancer, 5′ intron from the factor IX gene, and the 3′UTR from the albumin gene including intron 14. With this vector, SEAP reporter gene expression levels remained high for 1 year, at levels comparable to those obtained from the cytomegalovirus (CMV) promoter on day 1. Expression of human apolipoprotein E3 (hApoE) in ApoE knockout mice provided a dose‐dependent correction of their hypercholesterolemia.Conclusions: Liver‐specific sustained transgene expression can be obtained at very high levels from optimized pDNA vectors, without the use of integration systems. Such vectors will further facilitate biological studies of genes in vivo and may find application in gene therapy. Copyright © 2008 John Wiley & Sons, Ltd.

Therapeutic treatment with hu14.18-IL-2 immunocytokine (IC) or Flt3-L (FL) protein is initially effective at resolving established intradermal NXS2 neuroblastoma tumors in mice. However, many treated animals develop recurrent disease. We previously found that tumors recurring following natural killer (NK) mediated IC treatment show augmented MHC class I expression, while the tumors that recurred following T cell dependent Flt3-L treatment exhibited decreased MHC class I expression. We hypothesized that this divergent MHC modulation on recurrent tumors was due to therapy-specific immunoediting. We further postulated that combining IC and Flt3-L treatments might decrease the likelihood of recurrent disease by preventing MHC modulation as a mechanism for immune escape. We now report that combinatorial treatment of FL plus hu14.18-IL-2 IC provides greater antitumor benefit than treatment with either alone, suppressing development of recurrent disease. We administered FL by gene therapy using a clinically relevant approach: hydrodynamic limb vein (HLV) delivery of DNA for transgene expression by myofibers. Delivery of FL DNA by HLV injection in mice resulted in systemic expression of >10 ng/ml of FL in blood at day 3, and promoted up to a fourfold and tenfold increase in splenic NK and dendritic cells (DCs), respectively. Furthermore, the combination of FL gene therapy plus suboptimal IC treatment induced a greater expansion in the absolute number of splenic NK and DCs than achieved by individual component treatments. Mice that received combined FL gene therapy plus IC exhibited complete and durable resolution of established NXS2 tumors, and demonstrated protection from subsequent rechallenge with NXS2 tumor.

Expression of Becker-like and full-length human dystrophlns was stable for at least 6 months In mdx mouse muscle following Intramuscular plasmid DNA Injection. Intramuscular Injection of a single plasmld DNA encoding both luciferase and dystrophln resulted In stable luclferase expression for at least 2 months in mdx muscle, whereas Injection of plasmld DNA encoding only luclferase did not result In stable luclferase expression. These results suggest that expression of either full-length or Becker-like dystrophlns protects mdx mouse myoflbers from degeneration.