Abstract
BackgroundA key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR).ResultsHere, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer.ConclusionsIn this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches.
Highlights
A key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes
An upstream transcription unit regulated by the CMV immediate early promoter (CMV-Immediate early promoter (IEP)) and directed into the S/ Scaffold/matrix attachment region (MAR) is necessary for episomal replication of pEPI [10,13,14]
The alpha fetoprotein (AFP) promoter is more active in HUH7 hepatocellular carcinoma compared to HPGL promoter and Apolipoprotein E (APOE) enhancer Three liver specific promoters, alpha-fetoprotein promoter (AFP), haptoglobin promoter (HPGL) and the apolipoprotein E enhancer (APO E) were evaluated for their use as liver specific episomal vectors
Summary
A key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR). Sustained and tissue specific transgene expression are key issues for the development of successful gene therapy approaches. Tissue specificity can be achieved either by gene delivery systems which explicitly deliver the gene of interest into a distinct cell type or tissue [1] or by the use of tissue-specific expression cassettes [2]. Similar effects with pEPI vectors utilizing chemically inducible promoters could be shown [15]
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