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Long-term phenotypic correction in factor IX knockout mice by using phiC31 integrase-mediated gene therapy

Gene Therapy volume 18, pages 842–848 (2011)Cite this article

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Abstract

Hemophilia B, a hereditary bleeding disorder caused by a deficiency of coagulation factor IX (FIX), is an excellent candidate for gene therapy. However, to date, success in hemophilia gene therapy clinical trials has been limited due to failure to achieve or sustain therapeutic levels of factor expression. The phiC31 integrase system efficiently integrates plasmid DNA carrying a transgene and an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, sustained transgene expression. A strategy utilizing plasmid DNA integrated with phiC31 integrase may offer a facile and safe alternative for sustained human FIX (hFIX) expression. Hydrodynamic tail vein injection was used for delivery of plasmids encoding phiC31 integrase and hFIX to the liver of FIX knockout mice. We demonstrated prolonged therapeutic levels of hFIX in this knockout mouse model of hemophilia B over a 6-month time course when phiC31 integrase was used. Additionally, we observed sustained FIX activity in plasma and phenotypic correction of bleeding after tail clip in phiC31-treated mice. In the livers that received integrase, we also demonstrated prolonged hFIX expression in hepatocytes by immunohistochemistry and documented sequence-specific genomic integration of the hFIX plasmid. These studies suggest the possibility that a similar approach in large animals and humans could lead to a simple and successful gene therapy for hemophilia.

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Acknowledgements

We would like to thank Kristen Newburn for assistance with blood draws and Dr Mark A Kay for providing mating pairs of FIX−/− mice. We are also very grateful to Alfonso Farruggio for help with plasmid constructions. This work was supported in part by NIH grant HL68012 to MPC. LEW was funded by PHS grant CA09302, awarded by the National Cancer Institute, DHHS. GH and PEM received support from the Hemophilia and Thrombosis Research Society and from Homecare for the Cure.

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Authors and Affiliations

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

    A Keravala, C L Chavez, L E Woodard & M P Calos

  2. Department of Pediatrics and Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA

    G Hu & P E Monahan

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  1. A Keravala
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  2. C L Chavez
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  3. G Hu
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  4. L E Woodard
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  5. P E Monahan
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  6. M P Calos
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Correspondence to M P Calos.

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MPC is an inventor on Stanford-owned patents covering φC31 integrase.

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Keravala, A., Chavez, C., Hu, G. et al. Long-term phenotypic correction in factor IX knockout mice by using phiC31 integrase-mediated gene therapy. Gene Ther 18, 842–848 (2011). https://doi.org/10.1038/gt.2011.31

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