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Site-specific genomic integration produces therapeutic Factor IX levels in mice

Nature Biotechnology volume 20, pages 1124–1128 (2002)Cite this article

Abstract

We used the integrase from phage φC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to ∼4 μg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.

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Figure 1: Integrase mediates increased and prolonged expression of hAAT in mouse liver.
Figure 2: Therapeutic levels of hFIX persist after partial hepatectomy.
Figure 3: Quantitation of extrachromosomal vectors and total vectors persisting in four mouse livers after partial hepatectomy.
Figure 4: Detection of integrase-mediated site-specific recombination at the DNA level.

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Acknowledgements

This work was supported by NIH grants HL68112 to M.P.C. and DK49022 to M.A.K.

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Authors and Affiliations

  1. Department of Genetics, Stanford University School of Medicine, Stanford, 94305, CA

    Eric C. Olivares, Roger P. Hollis, Thomas W. Chalberg & Michele P. Calos

  2. Department of Pediatrics, Stanford University School of Medicine, Stanford, 94305, CA

    Leonard Meuse & Mark A. Kay

Authors
  1. Eric C. Olivares
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  2. Roger P. Hollis
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  3. Thomas W. Chalberg
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  4. Leonard Meuse
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  5. Mark A. Kay
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  6. Michele P. Calos
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Correspondence to Michele P. Calos.

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The authors declare no competing financial interests.

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Olivares, E., Hollis, R., Chalberg, T. et al. Site-specific genomic integration produces therapeutic Factor IX levels in mice. Nat Biotechnol 20, 1124–1128 (2002). https://doi.org/10.1038/nbt753

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