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Alternative splicing as a novel of means of regulating the expression of therapeutic genes

Cancer Gene Therapy volume 9, pages 133–141 (2002)Cite this article

Abstract

In order to determine the potential of alternative splicing as a means of targeting the expression of therapeutic genes to tumor cells in vivo, a series of episomal plasmid-based “splice-activated gene expression” (pSAGE) vectors was generated, which contain minigene cassettes composed of various combinations of the three alternatively spliced exons present in the differentially expressed adhesion protein CD44R1 (v8, v9, and v10) with or without their corresponding intronic sequences, positioned in-frame between the CD44 leader sequence and a “leaderless” human liver/bone/kidney alkaline phosphatase (ALP) cDNA. Because both the v8–v9 and v9–v10 introns contain multiple in-frame stop codons, the expression and enzymatic activity of ALP are dependent upon the accurate removal of intronic sequences from the pre-mRNA transcripts encoded by these constructs. The various pSAGE constructs were introduced into CD44H-positive (T24) and CD44R1-positive (PC3) target cells by electroporation and transfectants selected in hygromycin B. ALP expression was determined by staining with the ALP substrate, BCIP/INT, and the transfected cells tested for their sensitivity to the inactive prodrug, etoposide phosphate. ALP-mediated dephosphorylation of etoposide phosphate generates the potent topoisomerase II inhibitor etoposide. The data obtained indicate that whereas the v8–v9 intron is spliced in both CD44H- and CD44R1-positive cells, the v9–v10 intron is efficiently and accurately removed only in CD44R1-positive cells. Furthermore, only CD44R1-positive cells were sensitized to etoposide phosphate when transfected with the v9–v10.ALP construct. These data emphasize the potential usefulness of alternative splicing as a novel means of targeting gene expression to tumor cells in vivo.

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Acknowledgements

We acknowledge the excellent technical assistance of Simon McCallum. This work was supported, in part, by Grant RO1 CA82296 from the National Institutes of Health.

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Authors and Affiliations

  1. Department of Radiation Oncology, UCLA Center for Health Sciences, Los Angeles, California, USA

    Gregory M Hayes, Carmine Carpenito, Shona T Dougherty & Graeme J Dougherty

  2. Angiogene Pharmaceuticals, The Magdalen Centre, The Oxford Science Park, Oxford, UK

    Peter D Davis

  3. Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, British Columbia, Canada

    Julie F Dirks

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  1. Gregory M Hayes
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  2. Carmine Carpenito
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  4. Shona T Dougherty
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Correspondence to Graeme J Dougherty.

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Hayes, G., Carpenito, C., Davis, P. et al. Alternative splicing as a novel of means of regulating the expression of therapeutic genes. Cancer Gene Ther 9, 133–141 (2002). https://doi.org/10.1038/sj.cgt.7700427

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