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Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene

Nature Biotechnology volume 18, pages 1311–1314 (2000)Cite this article

Abstract

Persistent expression of a transgene at therapeutic levels is required for successful gene therapy, but many small vectors with heterologous promoters are prone to vector loss and transcriptional silencing. The delivery of genomic DNA would enable genes to be transferred as complete loci, including regulatory sequences, introns, and native promoter elements. These elements may be critical to ensure prolonged, regulated, and tissue-specific transgene expression. Many studies point to considerable advantages to be gained by using complete genomic loci in gene expression1,2,3. Large-insert vectors incorporating elements of the bacterial artificial chromosome (BAC) cloning system4, and the episomal maintenance mechanisms of Epstein–Barr virus (EBV), can shuttle between bacteria and mammalian cells, allowing large genomic loci to be manipulated conveniently5. We now demonstrate the potential utility of such vectors by stably correcting a human genetic deficiency in vitro. When the complete hypoxanthine phosphoribosyltransferase (HPRT) locus of 115 kilobases (kb) was introduced into deficient human cells, the transgene was both maintained as an episome and expressed stably for six months in rapidly dividing cell cultures. The results demonstrate for the first time that gene expression from an episomal genomic transgene can correct a cell culture disease phenotype for a prolonged period.

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Figure 1: (A) Vector map.
Figure 2: (A and B) Enzymatic activity of HPRT in HP10 transformants.
Figure 3: Fluorescent in situ hybridization analysis.

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Acknowledgements

We would like to thank Drs José Mejía and Zoia Larin for providing and analyzing PAC71G04, Dr. Jon Frampton for sharing his flow cytometery expertise, and Dr. Steve Hart for his assistance with the LID complex transfection. We thank the Wellcome Trust for support. R.W-M. is a Wellcome Trust Prize Fellow; H.K. is supported by the E.P. Abraham Trust and the Wellcome Trust.

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Author notes

  1. Richard Wade-Martins

    Present address: Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Room 6119, Building 149, 13th Street, Charlestown, MA, 02129

  2. Michael R. James

    Present address: Queensland Institute of Medical Research, 300 Herston Road, Q 4006, Brisbane, Australia

Authors and Affiliations

  1. Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom

    Richard Wade-Martins, Robert E. White & Michael R. James

  2. Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom

    Hiroshi Kimura & Peter R. Cook

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  1. Richard Wade-Martins
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Correspondence to Richard Wade-Martins or Michael R. James.

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Wade-Martins, R., White, R., Kimura, H. et al. Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene. Nat Biotechnol 18, 1311–1314 (2000). https://doi.org/10.1038/82444

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