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Sp1-Dependent Regulation of the RTP801 Promoter and Its Application to Hypoxia-Inducible VEGF Plasmid for Ischemic Disease

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Abstract

Purpose. Gene therapy using vascular endothelial growth factor (VEGF) is a new potential treatment of ischemic disease. To be safe and effective, VEGF expression should be enhanced locally in ischemic tissue. In this study, we identified the cis-regulatory element for the hypoxia induction of the RTP801 promoter. In addition, pRTP801-VEGF was evaluated as a therapeutic plasmid in vitro.

Methods. The cis-regulatory element for hypoxia induction was identified by deletion and mutation analyses. Antisense oligonucleotide co-transfection assay was performed to evaluate the role of Sp1. pRTP801-VEGF was constructed by the insertion of the RTP801 promoter into the VEGF plasmid. The hypoxia-inducible expression of VEGF was evaluated by in vitro transfection assay.

Results. In luciferase assay, the region between -495 and -446 was responsible for the hypoxia-induced transcription. The mutation of the Sp1 site in this region reduced hypoxia-induced transcription. In addition, co-transfection with antisense Sp1 oligonucleotide suggests that hypoxia induction of the RTP801 promoter is mediated by Sp1. In vitro transfection showed that pRTP801-VEGF had higher VEGF expression than pEpo-SV-VEGF. In addition, VEGF expression by pRTP801-VEGF was induced under hypoxia.

Conclusions. With strong basal promoter activity and induction under hypoxia, pRTP801-VEGF may be useful for gene therapy for ischemic disease.

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Authors and Affiliations

  1. Clinical Research Center, College of Medicine, Inha University, Inchon, 400-711, Korea

    Minhyung Lee

  2. Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah, 84112, USA

    Malavosklish Bikram & Sung Wan Kim

  3. Department of Internal Medicine, College of Medicine, Kyung Hee University, Seoul, 130-701, Korea

    Seungjoon Oh

  4. Department of Surgery, Division of Cardiothoracic Surgery, University of Utah Health Sciences Center, Salt Lake City, Utah, 84112, USA

    David A. Bull

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  1. Minhyung Lee
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  2. Malavosklish Bikram
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  5. Sung Wan Kim
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Correspondence to Sung Wan Kim.

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Lee, M., Bikram, M., Oh, S. et al. Sp1-Dependent Regulation of the RTP801 Promoter and Its Application to Hypoxia-Inducible VEGF Plasmid for Ischemic Disease. Pharm Res 21, 736–741 (2004). https://doi.org/10.1023/B:PHAM.0000026421.09367.b3

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  • DOI: https://doi.org/10.1023/B:PHAM.0000026421.09367.b3

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