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Persistent hepatic expression of human apo A-I after transfer with a helper-virus independent adenoviral vector

Gene Therapy volume 9, pages 1520–1528 (2002)Cite this article

Abstract

Gene transfer with ‘gutted’ vectors is associated with persistent transgene expression and absence of hepatotoxicity, but the requirement of helper viruses hampers efficient production and leads to contamination of viral batches with these helper-viruses. In the present study, gene transfer with a helper-virus independent E1/E3/E4-deleted adenoviral vector induced persistent expression of human apo A-I (200 ± 16 mg/dl at day 35, 190 ± 15 mg/dl at 4 months, 170 ± 16 mg/dl at 6 months) and stable transgene DNA levels (3.5 ± 0.60 at day 35, 3.3 ± 0.39 at 4 months, 3.1 ± 0.47 mg/dl at 6 months) in C57BL/6 mice in the absence of significant toxicity. The vector contained the 1.5 kb human α1-antitrypsin promoter in front of the genomic human apo A-I sequence and four copies of the human apo E enhancer (hAAT.gA-I.4xapoE) and was deleted in E1, E3 and E4. Reintroduction of E4 ORF 3 and E4 ORF 4 in the viral backbone caused a more than four-fold decline of transgene DNA between day 35 and 4 months after transfer both in wild-type and in C57BL/6 SCID and C57BL/6 Rag-1−/− mice, indicating that the effect of E4 ORF 3 and E4 ORF 4 is independent of a cellular immune response against viral epitopes. Co-injection of an E1-deleted vector containing no expression cassette and the E1/E3/E4-deleted vector containing the hAAT.gA-I.4xapoE expression cassette indicated that E4 gene products destabilize transgene DNA in trans. Gene transfer with an E1/E3/E4-deleted vector containing only E4 ORF 3 and the hAAT.gA-I.4xapoE expression cassette was associated with transgene DNA decline, but not with hepatotoxicity, indicating that transgene DNA persistence and hepatotoxicity are dissociated processes. After transfer with E1/E3/E4-deleted vectors containing expression cassettes with a different promoter or a different position of the apo E enhancers, transgene DNA levels were less stable than after transfer with the vector containing hAAT.gA-I.4xapoE, indicating that the expression cassette is an important determinant of episomal stability. In conclusion, gene transfer with an E1/E3/E4-deleted vector containing the hAAT.gA-I.4xapoE expression cassette induces persistent expression of human apo A-I in the absence of hepatotoxicity. Transgene DNA turnover is independent of an adaptive cellular immune response against viral epitopes and of hepatotoxicity. E1/E3/E4-deleted vectors containing transgenes under control of the hAAT promoter in combination with four copies of the human apo E enhancer may be suitable for hepatocyte-specific overexpression of transgenes after gene transfer.

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Acknowledgements

Sophie Van Linthout is a Research Assistant of the Instituut voor Wetenschappelijk en Technisch Onderzoek-Vlaanderen. Bart De Geest is a Postdoctoral Fellow of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. We thank D Dreyer for expert assistance in adenoviral vector construction. W Fransen and Z Zhang are acknowledged for excellent technical assistance.

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Authors and Affiliations

  1. Center for Molecular and Vascular Biology, University of Leuven, Belgium

    S Van Linthout, D Collen & B De Geest

  2. Transgène SA, Strasbourg, France

    M Lusky

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Van Linthout, S., Lusky, M., Collen, D. et al. Persistent hepatic expression of human apo A-I after transfer with a helper-virus independent adenoviral vector. Gene Ther 9, 1520–1528 (2002). https://doi.org/10.1038/sj.gt.3301824

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